D.T.M.T.

Protein Engineering

PDF 01- INTRODUCTION OF PROTEIN ENGINEERING.

• Large biomolecules
• Comprise one or more longer chain of amino acid
• Differ from one another according to sequence of amino acid.
• Fold to specific 3D structure.
• Functions:
▪ Act ac enzymes – metabolism
▪ Structural units – keratin
▪ Protective agent – antibody
▪ Cell recognition- proteins in cellular surface
▪ Regulate the gene expression – repressors and enhancers
• 20% of body mass
• Peptide- 50 or less amino acids
• Protein – more than 50 amino acids
• Bound by peptide bonds
• 4 common structures
• Produce by transcription and translation
• Codon – 3 nucleotides
• t RNA has anticodons
• 25000 genes in body
• 20000 genes- coded for proteins
• Make at least 20000 proteins ??? I think amino acids
• 21 amino acid – building blocks of proteins.
• Protein engineering: study the modifications of proteins with recombinant DNA
technology or chemical treatment that more suitable for particular function.
• Modifications are done through – insertion, deletion, inversion and substitution.
• Objectives:
Create superior enzymes
Produce enzyme in large quantities
Produce biological compounds superior to natural ones.
• Enzyme-strong with long life Rationale of protein engineering
• Should be work under pressure – like extreme pH
• Elimination of allosteric regulation.
• Improve kinetic properties.
• Increase thermostability
• Enhance the substrate and reaction specifity.
• Alteration in optimum pH.
• Soluble in organic solvents.
• Speedup the process.

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• Properties of proteins:
▪ Concentration
▪ Molecular weight
▪ Specific amino acids
▪ Amino acid composition
▪ N and C term amino acid
▪ 2D structure
▪ 3Dstructure with ligands Basic assumptions of protein engineering
• Many amino acid changes are silent mutants
• Basic structure of minor changes – lead the variations minor changes in basic structure
• Similar folding patterns-similar domain structures-different amino acid sequences- can
cause little or no homology.
• 3D structure of protein recognized by X-ray crystallography and NMR process.
• Methods of protein engineering: Mutagenesis – improve property of the proteins.
Recombinant DNA technology
• Applications:
• Food industry:
▪ Wheat gluten protein – studied by E coli and yeast expression systems.
▪ Protease, amylase, lipase – improved by rDNA technology.
▪ Deletion of native gene in extracellular protease – increase enzyme yield.
▪ Protease- low allergenic infant formulas, milk clotting and flavors.
▪ Protease – remove protein stains
▪ Improve protease features: high activity in alkaline pH,low temperature/ improve
stability at high temperature.
▪ Starch to bioethanol or functional ingredients like fructose, wine, glucose and
trehalose – using amylase liquefy and saccharify starch.
• Environmental applications:
▪ Eliminate environmental pollutanats such as organic pollutanats, phenols,
azodyes, organophosphorus, pesticides and polycyclic aromatic hydrocarbon can
be detoxified.
▪ Petroleum bio refining – new bio catalysts are required.(biodesulfurization,
denitrogenation, heavy metal removal, depolmerization)
• Medical applications:
▪ Cancer treatments produce novel antibodies-have ability to select antigens
specifically
▪ -also called modular protein engineering
▪ Pre targeted radioimmunotherapy
▪ Long circulating antibody
▪ Multifunctional an smart drug vehicles – identify and select targets for protein
based drug delivery.

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▪ Pharmacokinetic properties of antibodies

• Biopolymer production:
▪ Produce peptide based bio material called elastine like polypetides/silk like
polymers.
• Nanobiotechnology:
Produce amyloid fibrils-structural templates for nano wire construction.

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PDF 02 - UNDERSTANDING PROTEIN CONFORMATIONS.

• Protein confirmation- characteristics of proteins which have primary,secondary,tertiary and

quaternary structures.
• Important for: understanding biomolecular interactions.
• Hemoglobin shows – Allosteric behavior.
• Peptide bonds are form: between -NH2 group of Amino acid and -COOH group ofother amino
acid.
• 4 structures:
▪ Primary – peptide bonds – linear
▪ Secondary – alpha beta sheets- H bonds
▪ Tertiary – 3D- disulphide bonds- interaction between side chains
▪ Quaternary- have 2 or more amino acids bound together.
• 20 amino acids we have
• R side chain – occur distinctive properties of amino acids.
• Due to the side chains : have structural and chemical diversity between amino acids.

Polar Non-polar Positive charge Negative charge

Serine Glycine Lysin Aspartic acid
Threonine Alanine Arginine Glutamic acid
Thyrosin Valine Histidine
Asparagine Cysteine
Glutamine Proline
Leucine
Isoleucine
Tryptophan
Methionine
Phenylalanine

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• Non polar- no separation between charge

• Electrical charges are evenly distributed across the molecule.
• Dissolve in Non-polar solvents- organic solvents.
• Polar amino acids has -NH2 or -OH groups
• They can form H bonds.
• Polar amino acids-outside of the proteins – have hydrophilic properties of the side chain.
• Aliphatic AAs:
▪ Hydrophobic
▪ Located core of the proteins
▪ Shielded them from aqueous surroundings.
▪ Glycine – simplest and smallest
Not optically active
R group – single H atom
▪ Alanine – methyl group in side chain
▪ Valine – longer side chain
More hydrophobic.
▪ Leucine – similar to valine
Have another methyl group
▪ Isoleucine – similar to Leucine and valine
Side chains are slightly different
Two centers of Asymmetry
▪ Proline – side chain bound to carbon and amino acid group.

• Aromatic amino acid:

▪ Contain aromatic ring
▪ Highly hydrophobic
▪ Phenylalanine – phenyl attached R group
Hydrophobic
▪ Tyrosine – a hydroxyl group at the end of phenyl ring
Less hydrophobic than phenyl alanine
▪ Tryptophan – indole ring

Highly hydrophobic

• Sulphur containing amino acids:

Cysteine - contain -SH bonds
Very reactive
Can form H bonds.
Can form di-sulphide bridge
Quite hydrophobic.
Methionine - start codon
Has thioether linkage
Relatively unreactive
Highly hydrophobic side chain
• Hydrophilic amino acids:

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➢ Neutral:
▪ Not charged molecule
▪ Polar side chains with H Bonds.
▪ Hydrophilic.
▪ Serine- hydroxylated version of alanine.
Due to OH group – highly reactive and hydrophilic
From H Bonds.
▪ Threonine – highly reactive
Highly hydrophobic
Two centers of asymmetry.
▪ Asparagine – carboxylate side chain
Amide is uncharged
▪ Glutamine – amide derivative
➢ Acidic:
▪ Aspartate – negatively – carboxylate anion
▪ Glutamate - carboxylate group - negative
➢ Basic
• Primary structure:
▪ completely defined order of amino acids.
▪ Sequence unique for individual protein.
▪ All information for protein production – contain in primary structure.
• Secondary structure:
▪ Core – hydrophobic residues
▪ Shell – hydrophilic residues.
▪ Peptide bonds – polar
▪ It neutralized by H bonds in hydrophobic environments.
▪ Form regular secondary structure called secondary structure.
▪ 2 types 3D
1. Alpha helix
2. Beta sheets
• Alpha helices
▪ Rod like structure
▪ AAs are 1.5 A apart
▪ Peptide bonds are formed.
▪ Link every 4th amino acid
▪ Side chains- outward from the chain
▪ Curved or kinked
▪ 2 or more polypeptides – very long, very stable, alpha helical coiled structure.
▪ Ex: keratin, Myosin, epidermis, fibrin
▪ Because of coiled coils -those are fibrous protein.
• Beta sheets
▪ Form H bonds
▪ 3.5 A between adjacent residues
▪ Parallel or Anti parallel networks.

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• Motifs (Super secondary structure)

▪ Ex: beta hair pin motif – made by two adjacent antiparallel beta strands joined by small
loop.
▪ Beta- Alpha-Beta motif – connect 2 parallel beta sheets.
▪ Middle peptide chain bind with one corner of N termini of one PC and C termini of other
Pc.
▪ It shield the hydrophobic residues of beta strands.
• Domains:
▪ Several motifs packed together to form compact, local, semi-independent units called
domains.
▪ Also called proteins tertiary structure
▪ Self- stabilize
▪ Fold independently from rest
▪ 3D
▪ 50-250 length
▪ Fundamental units of tertiary structure.
▪ Contain individual hydrophobic core.
▪ Interior packing is much tighter
▪ Solid like core
▪ Fluid like surface
▪ Core residues- often conserved.
▪ Loop residues-less conserved.
• Domain classes:
▪ Alpha domain – domain core built from alpha helices.
▪ Beta domains – core – antiparallel beta sheets – usually two sheets packed against each
other.
▪ Ex: Greek key motif
▪ Alpha + beta domains – structure is overlap with other 3 classes.
▪ Alpha/beta domains – parallel beta sheets surrounded by amphipathic alpha helixes.
• Structural classification of proteins (SCOP)
▪ Show evolutionary relationships between proteins
• Tertiary structure:
▪ 3D
▪ Amino acids in primary structure are interact together via charge to charge,
hydrophobic, disulfide and form tertiary structure.
▪ It is responsible for how regional structures are put together in space.
▪ Normally anchored in the membrane by hydrophobic alpha helices

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▪ Salt bridges – increase the stability of tertiary structure.

▪ Hydrophobic interactions, hydrogens bonds, salt bridges are non covalent interations.
▪ Dislphide bonds - covalent.
▪ Form after protein folded.
▪ Tertiary -Native
▪ When disrupted the tertiary structure: it is irreversibly denatured.
• Quaternary:
▪ More than one polypeptides involved
▪ Bonds between them,
hydrophobic bonds
hydrogen bonds
van der waals interactions
salt bonds
occasionally di--sulfide bonds
▪ ex: hemoglobin
polymerase
ribosome
antibodies
ion channels
• Denaturation:

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▪ Lose it shape
▪ Reasons-
Changes of pH
Increased temperature
Exposure to UV light
Protonation amino acid residues
High salt concentration
• Tertial structure is essential to its biological function.

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PDF 03 - PROTEIN COMPOSITION AND STRUCTURE

Ionization of Amino acids

• A Zwitterion – functional group one has positive charge.

Another functional group has negative charge.
Whole molecule net charge is negative.
• cIEF – separate protein or peptide from their mixture.
Based on their iso electric point
• Procedure - voltage applied to anolyte and catholyte tanks.
Establish pH gradient through the capillary.
Proteins are applied.
Separated based on their net charge.
Become immobilize when pH is equal to their iso electric point.
• Less disulphide bonds – because of DDT, beta mercaptoethanol.
• S-S to-SH – reducing by beta Mercaptoethanol
• Peptide bond are formed between -NH2 and -COOH.
• 2 protein categories based on composition – simple protein.
Conjugated proteins
• Conjugated proteins – phosphoprotein
Glycoprotein
Metalloprotein
Nucleoprotein
Chromoprotein
Lipoprotein
Flavoprotein
• Simple proteins- only amino acids
Fibrous or globular
Ex: Actine, Myosin, Insulin, Keratin, Collagen
Found n Blood plasma, salivary glands, immunoglobulins, Human
chronic gonadotropins
• Globulin 4 types – alpha 1
Alpha 2
Beta
Gamma
• Alpha 01 – transport lipid, thyroxin, corticosteroids.
• Alpha 02 – transport lipids and copper ions.
• Beta- transport lipids and irons
Act as a protective agent
• Gamma – immunoglobulin functions
• Hemoglobin contain - alpha 1 and alpha 2

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Beta 1 and beta 2

Made by chromosome 16 and 11
• Albumin – open flexible structure
High solubility
Higher tyrosine fluorescence intensity
Higher content of unordered secondary structure fraction
Lower content of aromatic and hydrophobic amino acids
Made in liver
Keep fluid in ur body stream
Carry various substances including hormones, vitamins, and enzymes.
Low albumin level indicate problem of liver or kidney.
• Globulin – closed non-flexible structure.
Lower solubility
Lowe foam capacity
Lower tyrosine fluorescence intensity
Lower content or unordered secondary structure fraction
Higher content of aromatic and hydrophobic amino acids
• Conjugated proteins - complex
One or more non amino acid components.
They tightly or loosely bound with amino acid.
Non protein partis called prosthetic groups.
Prosthetic groups may be – metal ion, carbohydrate, lipids,
phosphoric acid, nucleic acid and FAD.
PG essential for biological function of their group
Globular in shape
Soluble in water
Ex: more enzymes
• Phosphoproteins- Casein in milk
Vitellin of egg yolk
• Glycoprotein – Most of membrane proteins
Mucin
Involve many physiological functions called immunity.
Serve to be therapeutic and preventative targets.
• Flavoproteins – PG is FAD or FMN.
Protein in electron transport system removal of radicals contributing to
oxidative stress
Photosynthesis
DNA repair
5-10% has FAD vi covalent link
• Nucleoproteins – proteins in chromosomes

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Structural proteins of ribosomes

Histone
Protamine
Telomeres – Nucleoprotein

At the end of eukaryotic chromosomes

Protect linear chromosomes against damage by endogenous nucleases.

Act against recognition of erroneous recognition as unpaired chromosomal break.

• Chromoproteins – PG is pigment or chrome.

Hemoglobin-hem factor-iron containing molecule- makes oxygenated
blood appear red
Phytochrome
Cytochrome
• Lipoproteins – Membrane proteins
Carry cholesterol through your blood stream
2 types of cholesterol – LDL bad
HDL good
Primary function transport hydrophobic lipid molecule in water
• Metalloproteins – PG is metal ions
Nitrate reductase.
There may be 3000 zinc binding protein domains
Catalyzing with high efficacy
Catalyzing photosynthesis, respiration, water oxidation, signal
transduction, complex chemical transformation.
• Structure of casein – Mostly serine and Threonine.
Made by 3 similar proteins which differ from molecular weight and
amount of phosphorus they contain
Made by alpha casein, beta casein and k-casein
• Protein classification – fibrous
Globular
Intermediate
• Fibrous proteins – linear
Secondary structure
Do not have tertiary structures
Tough and strong
Insoluble in water
Long parallel polypeptide chains are crosslinked with regular intervals

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• Globular proteins- spherical of globular

Tightly folded
Tertiary structure
Soft the FB
Readily soluble
Ex: enzymes, antibodies, some hormones, Insulin, hemoglobin, DNA
polymerase, RNA polymerase

• Intermediate proteins- linear and globular

Short
Soluble in water
Blood clotting proteins
Fibrinogen

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PDF 04 - METHODS FOR PREDICTING CONFORMATIONS OF PROTEINS FROM SEQUENCE DATA

• Characterization of new protein is done by determining primary structure.

• Procedure:
Hydrolyze
Amino acids are separated by chromatography.
Quantified.
• It five amino acid composition – Not sequence
• For sequencing – one amino acid is removed by one at a time
• For that:
EDMAN-degradation
Mass Spectrometry.
• EDMAN degradation:
▪ Determine primary structure of protein.
▪ 80-95% efficiency
▪ Up to 50 amino acids can be sequenced.
▪ Larger proteins should be split.
▪ Use different proteases.
▪ Create overlapping reagents.
▪ Put together.
▪ Digestion is done if N terminus protein is blocked by chemical
modification.
▪ Phenyl Iso Thio Cynate bound to N terminus in alkaline condition.
▪ Conjugated N terminal AA with PITC is cleaved as a thiazolinone under
acidic condition.
▪ PITA-AA converted to PTH-AA most stable form.
▪ Identified by chromatography.
▪ Remaining protein can go directly to the next cycle.
• Like N terminal sequencing: C terminal sequencing also can be done.
• Both ends sequencing is much easier.
• For find sequencing of unknown proteins:
▪ Like recombinant proteins
▪ In-vitro synthesized molecules
▪ Engineered antibodies
▪ De novo protein sequencing is used
▪ Based on mass spectrophotometry
• In mass spectrophotometry:
▪ Cut proteins to small polypeptide fragments.
▪ Separate them using high performance liquid chromatography.
▪ Determine the sequence through 2 stages of mass spectrometry.
▪ High accuracy

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▪ Do not need data base comparison.

• MALDI-TOF:
▪ Use to ionize large organic molecules like DNA, proteins, peptide, sugar,
polymers.
▪ Used to material that tend to be fragile and fragmentation while conventional
ionization method.
▪ Principle: disperse analytes in matrix molecule and from crystals
▪ Irradiate crystal by laser.
▪ Energy absorb by matrix molecule.
▪ Accumulate rapidly generated heat
▪ Sublimate the matrix crystals
▪ Enter molecules to gas phase.
▪ Generate singly-charged ions
▪ Ions are detected by time of flight detector.
▪ Fast, convenient, can do 100 spots at same time.
▪ Identify pure or simple proteins.
▪ Lower cost.
• ESI-MS:
▪ High voltage is applied to liquid and create aerosols.
▪ Useful for ionize macro molecules
▪ Because they tend to fragmentize while ionization.
▪ Generate highly charged ions rather than fragmented ions.
▪ Reduce mass/charge ratio.
▪ Greatly expand the molecular weight analyzing range.
▪ True molecular mass can be calculated via charge ration and charge number.
▪ 10-100 proteins can be identified at same time.
▪ Low concentrated samples also can be detected.
▪ Can analyze many more type of samples.
• While mass spectrophotometry:
▪ Avoid various containations.
▪ Avoid repeated freezing and thawing.
▪ Avoid keratin contamination while preparing and cutting gel
▪ Protein Coomassie and blue staining can be used to general process.
▪ Silver stained process is irreversible.
▪ So can not use for glutaraldehyde.
▪ It can affect MS results.
• Procedure of ESI-MS,
▪ Start with mixture of proteins.
▪ Digest proteins using enzyme.
▪ Digested proteins are separated from several chromatography methods.

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▪ Peptides are ionized.

▪ Sampled into MS.
▪ Charged peptides are separated via mass//charge in MS.
▪ Abundant peptide cations selccted for fragmentation via tandem MS.
• Tandem mass spectrophotometry:
▪ Different compounds with same m/z ratio, cannot be identified by normal MS.
▪ That’s why tandem MS is used.
▪ All ions with given m/z ratio are fed into a collision gas.
▪ Collisions break the sample molecules.
▪ Sample is disintegrated to fragments.
▪ They fed in to second MS.
▪ Use for forensic purposes.
▪ Use for inborn screening of inherited diseases.
▪ Blood or urine sample of babies are analyzed to find out unusual accumulation of
metabolites.
▪ Toxic substances can be identified and quantified.

• LC/MS/MS:
▪ Used to identify and quantify unusual compound in biological fluid.
▪ Firstly, sample is separated on chromatography.
▪ Then mass of the compounds are determined by the MS.
▪ Finally, fingerprint spectrum of each compound of interest is determined.
▪ These data can identify compounds.
• De novo protein sequencing:
▪ Multiple enzyme digestion
▪ Orbitrap Fusion Lumos Mass Spectrometer Analysis. -accurately distinguish AAs.
▪ MS data analysis.
▪ Reverse sequence conformation by MassAnalyzer – verification of sequencing results.
• sequenced proteins identifications: western blotting technique.
▪ Specificity and sensitivity
▪ 100% accuracy
▪ Accuracy determined by- comparing results of western blot with predicted molecular
weight of the identified protein.

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• Protein identification:
▪ EDMAN
▪ Peptide mapping – MS
▪ De novo protein sequencing-

• Gene sequencing:
▪ Much easier than protein sequencing.
▪ Can predict structure of known sequenced proteins.

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• Structure prediction Methods:

▪ Homology modeling - using PDB
▪ Fold recognition – same folds are available.
▪ Ab initio modeling – construct model using energy functions.
• Energy minimizing methods are used to protein prediction:
• Predict folding, secondary, tertiary, quaternary structures.
• Homology modeling:
▪ Most reliable
▪ Well established
▪ 3D structure is found
▪ If various in sequence, but there are some similarities between structures like
folding patterns.
▪ BLAST is used to search homologous proteins.
▪ Can found similar templates also.
• The twilight zone:
Compare all known structured proteins using sequence comparison.
Up of line, 90% confident with alignment, length, sequence identity
Under the line, twilight zone- donno similar or not.

• Fold recognition:
▪ Identify proteins have similar tertiary structure.

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▪ Can used as a template for unknown protein structure.

▪ Structurally similar proteins are found
▪ Useful to find structures in query proteins, special have low sequence level
identity with other proteins with known structures.
• Ab initio modelling:
▪ Computational method
▪ Used laws of physics along with protein fragments of construct model using the
law of physics.
▪ If homology modeling is not reliable, this can be used.
▪ Can identify given sequence even no similarity sequences are not found.
• SCOP: used to structure classification of proteins.

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• Rosetta algorithm:
▪ De novo method
▪ Short fragments of known proteins are assembled by Monte Carlo strategy
▪ Yield native like protein conformations.

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PDF 05 - Structure functional relationships for protein design

• Structure determining methods:
➢ X-ray crystallography
➢ NMR spectroscopy
➢ Electron microscopy
X-ray crystallography

• Protein – purified and crystallized

• Then subjected to intense beam of x-rays
• Characteristics- pattern of spots
• According to that, determine the distribution of electrons in the proteins.
• Result- map the electron density
• Determine the location of each atom
• PDB – two types of data for crystal structures.
• Provide detailed atomic information.
• Show every atomic detail of ligands, inhibitors, ions, other molecules that are
incorporated into crystals.
• Difficult
• Ex: determine structures of rigid proteins
• Other than rigid proteins, difficult to do this on flexible proteins.
• Flexible proteins are invisible in x-ray crystallography – bcz their e density is smeared
over a large space.
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NMR spectroscopy

• Determine the structure of proteins

D.T.M.T. Protein engineering

• Purify proteins
• Placed in a strong magnetic field
• Probed with radio waves
• Characterize the local confirmation of atoms that are bonded together.
• Helps to build a model of the proteins
• That show location of each atom
• Limited to small or medium proteins
• Large proteins- overlap peaks in NMR spectra
• Provide information on proteins in solution
• Suitable for flexible proteins also
3D electron microscopy

• Determine 3D structure of large macromolecules assemblies

• Commonly used technique – many thousand of different single particles preserved in a
thin layer of non-crystalline ice(Cryo-EM)
• Show molecules in myriad different orientation
• CAT scanning of yields – show 3D density map
• Sufficient number of single particles . 3DEM map interpreted . elctron density map
interpreted
• Very similar to x-ray crystallography
• Ex: built beta-galactosidase
- Provide atomic method
- EMDataBank – entry EMD-2984
- PDB entry – 5a1a
Integrative modeling

• Combine information from variety of methods

• Study particular aspect of system
• Create an overall picture of the assembly
• Ex: combine spectroscopic and chemical crosslinking data – identify distance between
components in an assembly - low resolution electron microscopic data – give
information on overall shape of complex
• New methods to structure determination and form integrative modeling – small angle
solution scattering, forster resonance energy transfer, chemical crosslinking, mass
spectrometry, electron paramagnetic resonance spectroscopy.
• Integrative models can take - regions of coarse grained beads that represent multiple
atoms
• different kinds of experiments provide information at different levels of resolution
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• Ex: structure of the Nuclear pore complex – from budding yeast determining using data
from chemical cross linking, small angle solution scattering and electron microscopic
environment.
• The
• NPC is an eight fold symmetric assembly
• consisting of 552 copies of 32 different proteins
• belonging to the nucleoporin family

• Function of proteins:
Enzymes
- Hormones
- Signaling molecules
- Antibodies
- Fluid balance
- Acid-base balance
- Channels and pumps
- Transport functions
- Structure and mechanics
Post translational modifications of proteins

• Human genome has 23000 genes

• 3mRNA per gene
• Produce 10 different proteins from each mRNA.
• Post translational modifications – modify proteins
• This modification can be increased by environmental stimuli.
• Ex: exposure to hormones
• Names of post translational modifictaions:
- Hydroxylation
- Glycosylation
- Disulfide bond
- Ubiquitination
- Lipidation
- SUMOlylation
- Phosphorylation
- Acetylation
- Methylation
Glycosylation

• Enzymatic transfer of oligosaccharide trees to proteins

• Bound with -OH group of Ser and Thr( O linked)
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-acid amide group of Asn( N linked)

• Cytosolic proteins are rarely glycosylated
• Ex: in bacteria – occur in periplasm
• Required for proper protein folding
• Glycosylation inhibitors are used as antiviral drugs
• Act as address labels – intracellular transport of proteins between compartments.
• Act as recognition sites for cell -cell interactions
As immunological determinant
Glycation(non-enzymatic glycosylation)

• Glycosylation sugars are transferred to proteins

• Covalently attachment of sugar to protein and lipid.
• Sugars – glucose, fructose and their derivatives
• Spontaneous reaction between sugar aldehyde and amino acids groups in protein,
nucleic acids and lipids
• Velocity of glycation – depend on the concentration of glucose in the blood
• Ex: concentration of glycation haemoglobin(HbA1c) depend on the average blood
glucose concentration
Disulfide Bond formation

• Oxidation of -SH groups of 2 cysteine residues – form covalent bonds

• Tripeptide glutathione as reducing agent
• Not happen in the reducing environment of the cytosol – but inER
• Protein disulfide isomerase – responsible for take right disulfide bond formation
Proteolysis

• Involve the activation of pro-enzyme

• Digestive enzymes – secreted as inactive presursors
• So, they can not harm the cell secreting them
• When going inside the intestine, remove part of the enzyme blocking the active site.
• Cascade of proteolytic enzymes – increase blood clotting and complement system
• Prohormones have similar manner
• Proteins no longer needed can be inactivated by proteolysis.
• Proteolysis – remove signal peptides
• Signal peptides- cleaved off by matrix protease
• There the signal peptide is cleaved off by matrix protease,
• exposing a second signal directing the protein’s export into the intermembrane space
through a different transporter.
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Hydroxylation
• Hydroxylation occurs - pro and Lys residues.
• Regulated by – pro-hydroxylation. – hypoxia induced transcription factors
• Cells can survive – under low oxygen supply for a long time
Phosphorylation/Dephosphorylation

• Transfer of phosphoryl groups from ATP to hydroxy groups Ser, Thr, Tyr .
• Important to reversible regulation of enzyme activity
• Transfer is catalyzed by protein kinase
• Removal by protein phosphates.
• 1/3 proteins – undergo regulatory phosphorylation/ dephosphorylation cycles.
Acetylation/Deacetylation

• Transfer acetyl group from acetyl CoA to amino acids

• Removal by protein deacetylases
• Used for regulation of the enzymatic activity
• Human acetylome – 1750 proteins
• DNA binding proteins – regulated by acetylation
• Ex: acetylated Lys – much less likely to be protonated
• Affect the binding of transcription factors
• Ex: example, the glycolytic activity of Glyceraldehyde 3 phosphate dehydrogenase
(GAPDH) is increased by acetylation
gluconeogenesis is stimulated by deacetylation.
Methylation/Demethylation

• Transfer methyl group from S-adenosyl methionine on to proteins

• Transfer can be to carboxyl groups, forming methyl esters.
• Mark damaged proteins for destruction, but also in signal cascades of unknown function.
Addition/removal of Hydrophobic tails

• Activate some enzymes

• Transferase make possible drug targets
• Addition of
- palmitoyl-(fatty acid) groups to internal Cys or Ser
- myristoyl-(fatty acid) groups to N terminal Gly
- farnesyl or geranylgeranyl (isoprenoid) groups to C terminal Cys , converts
- cytosolic enzymes to membrane bound (cytosolic leaflet).

S-Nitrosylation
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• occurs in Cys-residues
• basic or acidic residue- because of acid-base catalysis
• serves as an additional pathway for NO regulation besides cGMP dependent kinases.

ADP-Ribosylation

• addition of ADP-ribose moieties

• used by some bacterial toxins to inactivate cellular proteins
• starting point of patho-mechanisms

deamidation

• removal of acid amide group from Gln or Asn

• followed by racemization(formation of d-amino acids)

AMPylation(Adenylylation)

• performed from ATP on to critical Thr hydroxy groups of Rho GTPases

• This results in depolymerization of the actin cytoskeleton, affected cells round up.
Transfer of peptides

• Ubiquitin – highly conserved 76 AA

• Major function – selectively label proteins for degradation.
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PDF 06 – STRUCTURAL FRAMEWORKS SUITABLE FOR ENGINEERING PROTEINS

Structural framework – hold or support a theory of research study
Protein engineering – recombinant DNA technology to modify AA
For designing new enzymes with modified or enhanced properties.

Protein engineering is done by recombinant DNA technology and high throughput

screening techniques.
Broadly classified, - rational design and directed evolution
Rational design – create improved protein molecules based on the 3D structure
- Relationship between structure and function, which develop over the
years as part of the protein sciences.
Directed evolution
Mimics the process of natural selection to steer proteins or nucleic acids toward a used-
defined goal.
Consist of subjecting a gene to iterative round of mutagenesis, selection and
amplification
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Performed in vivo or in vitro

Most successful in 3 areas:

- Improve protein stability in higher temperatures and harsh solvents
- Improve binding affinity of therapeutic antibodies and denovo-
designed enzymes.
- Altering substrate specificity of existing enzymes.
Generation of a mutant protein library
Libraries with favorable ratio - can act as effective starting points
Most common methods – error-prone PCR and DNA shuffling methods
Error-prone PCR – introduce random mutations into defined segment of DNA
- That too long to chemically synthesized as a degenerate sequence
DNA shifting method
1. Preparation of genes to be shuffled
2. Fragmentation with DNase 1
3. Reassembly by thermocycling in the presence of a DNA polymerase
4. Amplification of reassembled products by a conventional PCR
D.T.M.T. PROTEIN ENGINEERING

site directed mutagenesis – start PCR

- Use a primer with known mutant
- Process

Success story of cytochrome P450 engineering

D.T.M.T. PROTEIN ENGINEERING

Enzyme – part of drug metabolism pathway in mammals

Genetic tuning of p450 -generate the new chemistry and expansion of the enzymes
reaction space into a new biosynthetic pathway
Functionality of p450 – bacillus megaterium - transformed from fatty acid hydroxylation
to alkane degradation using directed evolution.
Computational prediction is done – process is coupled with directed evolution
methodologies.
Directed evolution cont.
Generate thousand variants
Ex: produce pure isomers of pharmaceutical and proteins for critical life-sciences
applications
Rational design - effective only when the structure and/or folding mechanism of the
proteins of interest are well established
Direct evolution involved – random mutagenesis and selection
Achieve desired properties of proteins
The advantages of rational and directed evolution have been combined together, as
localized/region specific random mutations to design proteins, often known as semi
rational protein engineering.
D.T.M.T. PROTEIN ENGINEERING
D.T.M.T. PROTEIN ENGINEERING
D.T.M.T. PROTEIN ENGINEERING

Rational design-
- Protein structure determining
- Computational aided design
- Planning mutanat
- Site directed mutageneis
- Transformation
- Protein expression
- Protein purification
- characterization
- Mutant enzyme
Directed evolution-
- Random mutageneis
- Library of mutated genes
- Transformation
- Clones
- Protein expression
- Mutant enzymes
Prerequisites of DE
- The availability of the genes encoding the enzymes of interest,
D.T.M.T. PROTEIN ENGINEERING

- A suitable expression system,

- An effective method to create mutant libraries, and
- Suitable screening or selection system
Protein expression is done under - Escherichia coli, Saccharomyces cerevisiae, or Bacillus
subtilis
Assay method
Fasts and accurate
targeted identification of desired biocatalysts out of libraries comprising 10 4 10 6
mutants
screening and selection are the principles
photometric assay
several types
- phage display
- ribosome display
- fluorescence activated cell sorting are used to screen within mutant
libraries.
st
1 category – random mutagenesis method
- Use various forms of error prone PCR/ physical and chemical
mutagens/ mutator strains/some forms of insertion an deletion
mutagenesis
- Generate random amino acid substitutions throughout the proteins.
nd
2 category – do not directly create the new sequence diversity
- Similar to DNA shuffling method
- Working on modified PCR protocol
- Based on various nucleases or generation of synthetic
oligonucleotides.
3rd category – use structural information and targets a controlled level of randomization
of specific positions within the DNA sequence.
3 strategies for producing sequence libraries for directed evolution –
1. Random mutageneis
2. Targeted mutagenesis
3. Recombination
D.T.M.T. PROTEIN ENGINEERING
D.T.M.T. PROTEIN ENGINEERING
D.T.M.T. Protein Engineering

Lecture 07 – protein function prediction

• Protein structure prediction helps to – assign biological and biochemical roles of proteins.
• Bioinformatic tools are used.
• Before that, function prediction was studied poorly and it was done based on genomic
sequence data.
• Protein function prediction methods-
- Sequencing
- Phylogenetics
- Genomics
- Structure
- Protein interactions
- Text mining
- Gene expression
• Function prediction is more difficult in the post-genomic era.
• There are several different proteins that newly introduced, and techniques should be
improved for finding their functions.
• Gene bank database size is increasing year to year with newly developed techniques.
• In early – Homology relationship is done for predicting function.
• If new protein is more different from previous ones – homology relationship- not effective.
• For more accuracy – computational techniques are required.
• So, machine learning approaches are currently applied.
• Some proteins have no definite function.
• Traditional function prediction method: homology modeling
• Identify evolutionary relationships between new and previous proteins
• Find known protein similarities that could suggest a common evolutionary origin between
them.
• Functional annotation transfer – homology based transfer – name of the method
• Silico - in or on a computer : done or produced by using computer software or simulation.
in silico predictions.
• In silico methods provide a platform for screening the activity of potential therapeutics
against the molecular targets.
• Protein function = molecular functions of proteins
- Gene regulation
- Transport of material
- Catalysis of biochemicals reactions
• Amino acid mutations – produce nonfunctional proteins
• Gene Ontology (GO) is a major bioinformatics initiative to unify the representation of gene
and gene product attributes across all species
• Gene ontology mostly used for finding,( gene ontology classification)
D.T.M.T. Protein Engineering

- Molecular function
- Biological process
- Cellular location
• There is a graph I can not understand.
• DeepGOplus:
- Understand functions of proteins > understand biological processes on a molecular
level.
- In this method – based on deep learning and sequence similarity.
- Accurate
- Fast prediction
- Can predict any function of any proteins.
• PANNZER:
- Next generation sequencing cause explosion of gene catalogs for novel genomes,
transcriptomes and metagenomes which are functionally uncharacterized.
- Produce 100000 protein sequences at once.
- Provide both gene ontology annotations and free text description prediction.
• Some segments – important to determine functions of proteins.
• Motifs and domains are extracted from functionally related proteins.
• Domain – fold in to stable structure independently on the rest of the sequence.
• Proteins considered to be related often share the same domain(s).
• Motifs are very short stretches of amino acid sequences that potentially encode the
function of proteins.
• They frequently locate in the domains.
• Contain important functional region such as catalytic sites and binding sites.
• According to the sequence, we can find,
- Protein length
- Amino acid composition
- Physio chemical properties
• Help to predict ,
- protein cellular location
- distinguish members and non-members of functional classes such as enzymes.
- Membrane proteins
- DNA/RNA binding proteins
• Comparison of genomes – helps to predict the function.
• Predict the function of unknown protein,
- Gene neighborhood
- Gene fusion
- Phylogenetic profiles
• Gene neighborhood method
D.T.M.T. Protein Engineering

• Proteins whose corresponding genes are co-located on the chromosome in multiple

genomes – functionally associated.
• Gene fusion-based approaches
• Pair or sets of genes in one genome, that are merged to form a single gene in another
genome – expected to share same function.
• Phylogenetic profiles
• Vector that attest the presence or absences of a homologous in a given genome.
• Most popular- amino acid sequencing

• BLAST
• Measure similarities between pairs of sequences.
• Find homology relationships.
• But homology – not always functional identity
• Structure > much more informative than AA sequence
• Structure information in new proteins – present in PDB
• 3D structure is much better than sequencing
• Intermolecular interaction
• Biochemical function – performed in protein surface.
• characterization and identification of similarities within protein surface regions as
invaluable resource > investigate protein functions.
• Expression measurements
• Which genes active under certain conditions
• Active genes produce proteins.
• Co-expressed genes involve to similar cellular functions.
• Annotate unknown genes that co-expressed with known gene.
• Protein-protein interaction
D.T.M.T. Protein Engineering

• Interact proteins with other proteins and constitute valuable data to predict protein
function.
• Proteins close each other that interact with other proteins – possessing similar functions.
• Several methods to study protein-protein interactions:
- Co-immunoprecipitation
➢ Determine whether 2 target proteins that bound in the body.
➢ Identify new role of particular protein
➢ Isolate and obtain the interaction protein complex in its natural state.
- Yeast two hybrid system
- Fluorescence resonance energy transfer technology
- Biomolecular fluorescence complementation.
• For structure prediction, there are 3 pipelines.
➢ Structure function data base – predict by structure – ligand binding prediction
➢ Sequence function data base- predict by sequence
➢ PPI function data base- predict by PPI
• Take a explanation on 25 page image (mRNA sequence analysis)

• 2th slide- can not understand.

D.T.M.T. Protein Engineering

•
• According to this graph, for function prediction,
- Most accurate method is protein interactions.
- Most easiest method is text mining.
Bioinformatics tools in protein engineering

Tool Link Applications

1 SCOP: https://scop.mrc-lmb.cam.ac.uk/ Browse structural classes
Structural and protein types
Classification
of Proteins
2 Uniprot https://www.uniprot.org/ protein sequence and
functional information
search peptides
3 PDB: Protein https://www.rcsb.org/ Search structural motifs
Data Bank Homology modelling
3D visualization
4 Ramachandr https://swift.cmbi.umcn.nl/server
an plot s/html/ramaplot.html
builder
5 PEAK https://www.bioinfor.com/peaks- De novo protein MS data
software studio/ analysis
6 NCBI blastp https://blast.ncbi.nlm.nih.gov/Blas Similarity search for
t.cgi?PAGE=Proteins proteins
7 Molecules in https://www.molsoft.com/icm_bro Molecule Visualization, Fully
silico wser.html Interactive 3D Slides in
sotware PowerPoint and Web,
Publication Quality Images,
Display Ligand Binding
Pocket Surfaces, Hydrogen
Bond Display, Measure
Distances and Angles
8 RoseTTAFold https://robetta.bakerlab.org/ RoseTTAFold is a “three-
softaware track” neural network,
meaning it simultaneously
considers patterns in protein
sequences, how a protein’s
amino acids interact with
one another, and a protein’s
possible three-dimensional
structure.
9 The http://elm.eu.org/ annotation and detection of
Eukaryotic eukaryotic linear motifs
Linear Motif (ELMs)
resource for
Functional
Sites in
Proteins
1 Prosite https://www.expasy.org/resource PROSITE consists of
0 s/prosite documentation entries
describing protein domains,
families and functional sites
as well as associated
 patterns and profiles to
identify them
1 DeepGOWeb https://deepgo.cbrc.kaust.edu.sa/d predicting protein functions
1 Protein eepgo/ based on protein sequence
Function
prediction
webserver
1 PANNZER http://ekhidna2.biocenter.helsinki. facility of next-generation
2 fi/sanspanz/ sequencing has led to an
explosion of gene catalogs
for novel genomes,
transcriptomes and
metagenomes, which are
functionally uncharacterized
1 GROMACS http://www.gromacs.org/
3
1 Propka http://propka.ki.ku.dk/
4
1 ProDy http://prody.csb.pitt.edu/
5
1 Bio3D http://thegrantlab.org/bio3d/inde
6 x.php
1 VMD http://www.ks.uiuc.edu/Research
7 /vmd/
1 Pymol https://www.pymol.org/
8
Predicting protein function from sequence and structure
Funtion prediction tools
Data bases in function prediction
D.T.M.T. Protein engineering

Lecture 08- Computational protein design

• Designs are defined by a requested structure, function and working environment.

• The target size of the CPD

- Anything from a small region to a full protein
- How small of a region is still regarded in the frame of CPD is an open question as a
single residue site directed mutagenesis is commonly regarded as protein
engineering rather than design.
- design of a binding site or designing a protein with altered specificity may include
very few amino acids.
• The target identity of CPD
- Target function, structure and biophysical properties.
- Each of these end point dictate a different approach to the design scheme.
• Level of rationality of CPD
- I can not understand what is saying

Objectives

• Protein folding or inverse folding problems

-entropic hydrophobic effect is long known
D.T.M.T. Protein engineering

- when inverse folding, which amino acid sequences fold into a known 3D structure is
the problem.
• Specificity
- Design specific interactions ( protein – protein / protein – ligand)
- Focus to find specificity
- Add similar templates to examine the target affinity in respect to a background of
unwanted interactions.
• Stability and extremophilicity
- Designed proteins act on hostile environment
- They act in biotechnology industry as a fermenter.
- CPD provide a unique method to study veery determinants underlying the requested
extremophile traits.
• Synthetic biology
- Natural proteins were optimized according to the need of organisms and the
constraints of the evolutionary process.
• Negative design rule
- Can not understand
Structural Levels of CPD: Design Target and Design Building Block
• De novo CPD
- Classical one
- Totally new fold or function are pursued.
- Ex: Beta doublet beta sandwich design
• Core stabilization
- Driving force for folding of soluble proteins – entropic hydrophobic effect
- It collapse the hydrophobic protein core – maintain the disorder of the aqueous
solvent- around hydrophilic amino acids of the protein
- Hydrophobic effect – result molten globule – later optimized for enthalpic
contribution of specific interactions and packing.
- As this process often not optimized for the stability.
- Most CPD depend on core stabilization
- Others approach to stabilization include most unstable parts of proteins.
• Solubilization of protein solvent interface
- Ex: genetic disease- sickle cell anemia- include hydrophobic patch on the surface of
the hemoglobin beta sub unit – single glu > val mutation
- Maintain solubility > avoid aggregation
• Symmetry
- Complexity - largely trimmed by adding symmetry to the structural design
- Can be done symmetric proteins such as beta propeller proteins, for coiled coils,
crystallographic symmetry
• Binding sites
D.T.M.T. Protein engineering

- Heart of the proteins

- Binding sites include many different case-studies such as,
Changing the bound metal-as done for ferritin, de novo
designed metal binding or nonbiological cofactor binding proteins, and enzymes
• Protein-protein interactions
- Stable or transient interaction between spatial patches of amino acids that are on
the surface accessible part of the protein
- Essential to design new PPI incorporating unique features of the antibody such as the
hyper-variable loops.
• Dynamics
- Bh%$353345#$#%$%^
• Membrane protein and other unique protein groups
-aiooo man danne ne appa mewa. Wikara
• Unsuccessful protein designs are removes without any investigation.
Lecture 05 - Structure-functional relationships for protein design

1. How to predict protein function from sequence and structure?

•

2. What are the methods to predict the structure of proteins?

• X-ray crystallography
• NMR spectroscopy
• Electron microscopy

3. Describe the X-ray crystallography method.

• The protein is purified and crystallized, then subjected to an intense beam of X-rays.
• The proteins in the crystal diffract the X-ray beam into one or another characteristic pattern
of spots
• Then it will be analyzed to determine the distribution of electrons in the protein.
• The resulting map of the electron density is then interpreted to determine the location of each
atom.
• PDB archive contains two types of data for crystal structures.
o The coordinate files include atomic positions for the final model of the structure
o The data files include the structure factors (the intensity and phase of the X-ray spots
in the diffraction pattern) from the structure determination.
Advantages;
• Can provide very detailed atomic information, showing every atom in a protein or nucleic acid,
along with atomic details of ligands, inhibitors, ions, and other molecules that are incorporated
into the crystal.
• X-ray crystallography is an excellent method for determining the structures of rigid proteins that
form nice, ordered crystals.

Limitations;
• The process of crystallization is difficult and can impose limitations on the types of proteins that
may be studied by this method.
• Flexible proteins, on the other hand, are far more difficult to study by this method because
crystallography relies on having, many molecules aligned in exactly the same orientation, like a
repeated pattern in wallpaper. Flexible portions of protein will often be invisible in
crystallographic electron density maps, since their electron density will be smeared over a large
space.

4. Describe the NMR spectroscopy.

• The protein is purified, placed in a strong magnetic field, and then probed with radio waves.

H.T.B.M. 30
 • A distinctive set of observed resonances may be analyzed to give a list of atomic nuclei that are
close to one another, and to characterize the local conformation of atoms that are bonded
together.
• This list is then used to build a model of the protein that shows the location of each atom.
• A typical NMR structure will include an ensemble of protein structures, all of which are consistent
with the observed list of experimental restraints.
• The structures in this ensemble will be very similar to each other in regions with strong restraints,
and very different in less constrained portions of the chain.
• Areas with fewer restraints are the flexible parts of the molecule, and thus do not give a strong
signal in the experiment.

Limitations;
• Limited to small or medium proteins, since large proteins present problems with overlapping
peaks in the NMR spectra.

Advantages;
• It provides information on proteins in solution, this is the premier method for studying the atomic
structures of flexible proteins.

5. Describe the 3D Electron Microscopy (3DEM).

• Used to determine 3D structures of large macromolecular assemblies.
• A beam of electrons and a system of electron lenses is used to image the biomolecule directly.
• Several tricks are required to obtain a 3D structure from 2D projection images produced by
transmission electron microscopes.
o Most commonly used technique involves imaging of many thousands of different single
particles preserved in a thin layer of non-crystalline ice (cryo-EM).
o These views show the molecule in myriad different orientations, a computational
approach that is similar to that used for computerized axial tomography or CAT scans in
medicine will yield a 3D mass density map.
• With a sufficient number of single particles, the 3DEM maps can then be
interpreted by fitting an atomic model of the macromolecule into the map.
• In a restricted number of cases, electron diffraction from 2D or 3D crystals
or helical assemblies of biomolecules can be used determine 3D structures
with an electron microscope using an approach very similar to that of X-ray
crystallography.
• Example - Cryo-EM map of beta-galactosidase was built from over 90,000
images of the molecule frozen in ice

6. What is Integrative Modeling?

• Idea is to combine information from a variety of methods, each for studying a particular aspect of
the system, to create an overall picture of the assembly.

H.T.B.M. 31
 • Eg: Combining spectroscopic or chemical crosslinking data that identify distances between
components in an assembly, with low resolution electron microscopy data that give information
on the overall shape of a complex, has become an effective strategy in integrative modeling.
• As different kinds of experiments provide information at different levels of resolution, a key
aspect of integrative modeling is that the resulting structural models do not always comprise of
atomic coordinates and can contain regions of coarse-grained beads that represent multiple
atoms.

7. What are the techniques used in integrative modeling of proteins?

• X-ray crystallography
• NMR spectroscopy
• Electron microscopy
• Experimental methods
o Small angle solution scattering
o Forster resonance energy transfer
o Chemical crosslinking
o Mass spectrometry
o Electron paramagnetic resonance spectroscopy
o Other biophysical techniques

8. What are the examples for integrative modeling?

• The structure of the nuclear pore complex (NPC) from budding yeast determined using data from
chemical crosslinking, small angle solution scattering and electron microscopy experiments.

9. What are different function branches of proteins?

H.T.B.M. 32
 10. How the same amino acid sequence having protein has different functions?
• Different folding patterns
• Post translational modifications
• Transcriptional modifications

11. What are post translational modifications?

• Posttranslational modification produces 10 different protein species per mRNA

12. Explain the posttranslational modifications of proteins.

Glycosylation • Glycosylation is the process of enzymatic transfer of oligosaccharide (sugar)
trees to proteins.
• Affixed to OH- groups of Ser/Thr (O-linked) or acid amide group of Asn (N-
linked)
• Addition occurs in the ER and the golgi-apparatus to the extracellular domain
of membrane proteins and to secreted proteins.
• In bacteria, glycosylation occurs in the periplasm.
• Required for: proper protein folding and intracellular transport of proteins
• Sugar tress act as recognition sites for cell-cell interactions
• Glycosylation inhibitors → antiviral drugs

Glycation • Glycation (sometimes called non-enzymatic glycosylation) is the covalent

attachment of a sugar to a protein or lipid. Typical sugars that participate in
glycation are glucose, fructose, and their derivatives.
• A spontaneous reaction between sugar aldehydes and amino groups in
proteins, nucleic acids, and lipids.
• Velocity of glycation depends on conc of glucose in blood
Medical application: In diabetics
• Conc of glycated Hb depends on avg blood glucose conc during lifespan
of RBC

H.T.B.M. 33
 Disulfide Bond • Oxidation of SH- groups of 2 cysteine residues leads to formation of covalent
Formation bond
• Reducing agent (in cell) – tripeptide glutathione
• Occurs in ER (oxidizing environment)
• Protein disulfide isomerase ensures correct
Cys resides undergoes this
Proteolysis • Activation of proenzymes, eg: in digestive system, & prohormones, eg: insulin
• Inactivate proteins when not needed, eg: cyclins in cell cycle
• Remove signal peptides
Hydroxylation • C-H oxidises to C-OH
• Occurs on Pro & Lys residues
• Proteins regulated by pro-hydroxylation are hypoxia induced transcription
factors
• Then, cell can survive in low oxygen supply for longer time
Phosphorylation • Transfer of phosphoryl groups from ATP to hydroxy groups of Ser, Thr & Tyr
Dephosphorylation • Important for reversible regulation of enzyme activity
• Transfer→protein kinases
• Removal→protein phosphatases
Acetylation • Transfer of acetyl groups from acetyl-CoA onto amino group of Lys
Deacetylation • Transfer→protein acetylases
• Removal→protein deacetylases
• Regulated DNA binding proteins
• Affect binding of transcription factors
• Regulate activity of metabolic enzymes
• Eg: glycolytic activity of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
is increased by acetylation; gluconeogenesis is stimulated by deacetylation
Methylation • Transfer of methyl group from S-adenosyl methionine onto proteins
Demethylation • Can transfer to carboxyl groups, forming methyl esters. Used to mark damaged
proteins for destruction
Addition/Removal • Addition of palmitoyl- (fatty acid) groups to internal Cys or Sermyristoyl- (fatty
of Hydrophobic acid) groups to N-terminal Glyfarnesyl groups to C-terminal Cys,
Tails • Activate some enzyme
• Application: anticancer drugs
S-Nitrosylation • Attachment of a nitrogen monoxide group to the thiol side chain of cysteine
• Cys must be between basic & acidic residue
ADP-Ribosylation • Used by some bacterial toxins, eg: Vibrio cholerae, to inactivate cellular
proteins
Deamination • Removal of acid amid group from Gln/Asn, forming Glu & Asp
AMPylation • Adenosine monophosphate (AMP) molecule is covalently attached to the amino
(Adenylation) acid side chain of a protein.

H.T.B.M. 34
Lecture 6
Protein Engineering
Structural Frameworks Suitable for Rational Design
Engineering Protein
• Uses detailed knowledge about structure &
Definitions function of protein
• Combine protein engineering approaches

Protein engineering employs recombinant → protein variants → wide range of desired

DNA technology to modify amino acid properties (biodegradability,
sequences, for designing new enzymes or biocompatibility and biofunctionality)
proteins with modified or enhanced properties Eg:
• Scaffolds → tissue engineering

Classify Protein engineering: • Thermostable enzymes → industrial

1. Rational design – create improved protein biocatalysis

molecules based on 3D structure &
relationship between structure & function Steps:
1. Target gene/genes
Effective when: 2. Determine structure (eg: x-ray
Structure/folding mechanism is well- crystallography, NMR)
established 3. Choose suitable protein scaffold
Bcs of limit → directed evolution method - Need prior knowledge about structure
of protein/homologous protein
2. De novo design (automated) – screen 4. Identify important residues for modification
library of a.a sequences for compatibility - Computer models identify hot spots
with desired target design. Includes 5. Residues specifically mutated by site-
computational design from scratch directed mutagenesis
6. Transformation into cells
3. Directed evolution 7. Protein expression
Random mutagenesis & selection 8. Protein purification
Limits → screening of randomly evolved 9. Screening of mutants
proteins 10. Characterization of mutants

First protein design was Result → Modified protein variant

done rationally based on manual analysis of
existing protein sequences, amino acid charges Alter function by:
and desired structural geometry • Single-point mutation
Pioneered by: • Exchange elements in secondary structure
Gutte et. al. – rationally designed 70 amino • Exchange domains
acid peptide analog of ribonuclease S w/ • Generate fusion proteins
enzyme activity via solid phase synthesis

Hashani Hewagama
Biotech-1
• Isolate protein from natural source in large 3. DNA shuffling (mixes and matches pieces of
scale → not affordable successful variants to produce better
• Then, genetically engineered proteins are results)
developed with homogeneity ➔ Used to produce better results
• Eg: 4. Transformation into cells
− Combine silk-like proteins & elastin- 5. Protein expression
like proteins to form silk-elastin-like- 6. Screening & selection
proteins ➔ Stability, selectivity, affinity, activity

Advantage Advantage
• Inexpensive • No prior structural knowledge needed

• Technically easy → site-directed • No need to predict what effect a mutation

mutagenesis methods are well-developed will have

• Can identify undiscovered protein

Drawback sequences having novel functions

• Detailed structural knowledge often

unavailable Drawback
• Difficult to predict effect of mutations since • Require high-throughput screening – not

structural info only provides static picture feasible for all proteins
of protein structure • Larger amounts of recombinant DNA must

• Saturation mutagenesis → Less screening be mutated

efforts ➔ Requires automation by expensive
robot equipment
• Products screened for desirable traits

➔ Not easy to screen all desired activities

Directed Evolution

Steps: Prior conditions:

1. Random mutagenesis is applied on wild- 1. Availability of the genes encoding the
type parent gene enzymes of interest
- By error-prone PCR/site saturation 2. Suitable expression system
mutagenesis 3. Effective method to create mutant libraries,
- OR recombination of multiple 4. Suitable screening or selection system.
homologous genes
2. Selection method used to select variants Further:
having desired traits • Altered gene cloned into plasmid
− Mutant w/ slight improvement is • Expressed in host (eg: Escherichia coli,
mutated again for higher activity Saccharomyces cerevisiae, Bacillus subtilis)
− Several generations later,

improvement becomes significant Semi-Rational Protein Engineering

Hashani Hewagama
Biotech-1
• Combine advantages of rational & directed Directed evolution
evolution to design proteins Assay Methods
• Uses information about protein sequence,
structure & function together w/ predictive Enable:
algorithms • Fast, accurate, targeted identification of
• Uses directed evolution to create random desired biocatalysts out of libraries
diversity comprising of numerous mutants
• 2 approaches:
Steps: 1. Screening –
1. Target amino acids which most likely Methods to screen in mutant libraries:
influence protein function are identified − Phage display (fusion of protein of
2. Key amino acid residues are mutated interest & bacteriophage coat protein)
- By saturation mutagenesis − Ribosome display (fusion of
3. Libraries of mutant proteins more likely to peptide/protein and its mRNA
have enhanced properties are created − Fluorescence-activated cell sorting
- Library is smaller, but high quality (FACS)

De Novo Designing 2. Selection

• Using computer methodology: can make

Produce Sequence Libraries – 3 Strategies
new proteins with sequence unrelated to
those in nature 1. Random mutagenesis –
• Copying of DNA sequence is deliberately
Steps: disturbed
1. Requires design inputs • Includes:
2. In silico analysis − Error-prone PCR
3. Sequence & structure prediction − Physical/chemical mutagens
4. Synthetic gene design − Mutator strains
5. Cloning & expression − Insertion/deletion mutagenesis
6. Screening • Finally, → generate random a.a
7. Designed protein substitutions throughout protein

Advantage 2. Targeted mutagenesis

• High stability
• Using structural info, target controlled
• Robust starting points for creating new
level of randomization to specific positions
functions in DNA sequence
• Involve:

Hashani Hewagama
Biotech-1
 - Direct synthesis of DNA molecule
mixture

3. Recombination
• Combine existing sequence diversity in
new ways
• Based on use of:
− Modified PCR protocol
− Various nucleases OR
− Generation of synthetic
oligonucleotides

EXTRA Scaffolds: Regulators of signaling

pathways
1. Interact/bind to members of signaling
pathway
2. Tether them to complexes
3. Regulate signal transduction
4. Help localize pathway components to
specific areas of cell, eg: nucleus, Golgi,
cytoplasm

Hashani Hewagama
Biotech-1
Lecture 07

fProtein Engineering Gene Ontology – 3 aspects of protein

Protein Function Prediction functions
1. Molecular function
• No. of newly identified proteins increasing 2. Biological process
with advance in techniques 3. Cellular location
• In order to characterize their functions,
computational methods have been proposed • Each ontology represented as a graph
- Vertices → protein function terms
Earlier: - Edges → define relationship between
• Homology rontoelationships were studied them
to link known functions to new proteins
• This fails when new protein is considerably • Some segments are not whole sequence, but
different from known ones are important to determine function
• Method exists to encode segments into
Functional annotation/homology-based features (eg: motifs/domains), build
transfer: predictive models then predict function
• Predict function by identifying evolutionary
relationships between new protein and new Domain – part of protein sequence which can
protein by searching for sequence fold into a stable structure independently of
similarities that may suggest common the rest of the sequence
evolutionary origin between them
❖ Related proteins can share same domain
Issue:
• Lack of sequence similarity in public Motifs – very short stretches of amino acid
databases to new/query protein sequences that potentially encode the function
of proteins
Currently:
• Machine learning approached are applied ❖ Usually locate into domains
❖ Can contain important functional regions
(eg: catalytic sites & binding sites)
Protein Function

Refers to: Protein Function – A.A Sequence

• Molecular functions of a protein such as:
gene regulation, transport of materials, Can get:
catalysis of biochemical reactions, etc. 1. Protein length
2. A.A composition
3. Physicochemical properties
Protein Function - GO
Predict:

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Biotech-1
• Protein cellular location • These genes produce proteins which
• Distinguish members & non-members of perform give function
functional classes eg: enzymes, membrane • Methods followed to use this hypothesis to
proteins annotate unknown genes that co-express
with known genes
Use methods to explore:
• Local sequence similarities Protein-Protein Interaction:
• 3D structures • Proteins accomplish function by interacting
• Genomic context with other proteins
• Co-expression data • Function determined from protein
• Protein-protein interactions interaction networks

Examples: BLAST ❖ Assumption: proteins close to each other in

• Measure similarities between pairs of an interaction network have high
sequences probability of possessing similar functions
• To find homology relationships
Protein Function – Compare Genomes
Issue:
• Sequence homology not always related to Gene Neighborhood Methods
functional identity Hypothesis:
• Proteins whose corresponding genes are co-
located on the chromosomes in multiple
Protein Function – Protein Structure
genomes
• Check public databases eg: PDB ➔ Functionally associated
• Perform local/global structural comparisons
Gene Fusion-Based Approaches
• Not whole structure, but substructures eg:
Hypothesis:
3D motifs/folds
• Pairs/sets of genes in one genome, that are
• Substructures are associated with specific
merged to form single gene in another
functions
genome
➔ Share same function
Intermolecular Interactions:
• Lead to certain biochemical functions
Phylogenetic Profiles
• Explore similarities within protein surface
• Vector of bits that prove presence/absence
regions
of homologous in given genome.
Similar profiling → proteins evolved together
Expression Measurements:
→ function conservation
• Indicate which genes are active under
certain conditions
Gene Fusion
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❖ If
proteins A, B hare homologous to 2
domains in C, A & B predicted to have
interaction

Protein Function – Template-Based

• The workflow of COFACTOR for template-

based function predictions
Structure, sequence-function database & PPI
database
➔ Derive GO models

Structure-function database
➔ Derive enzyme commission & ligand-
binding predictions

Protein Function – Examples

Databases:
Sequence: UNIPROT, NCBI, SWISS-PROT
Interaction Network: DIP, MINT, STRING
Structure: SCOP,PDB
Genome: Reactome, KEGG Pathway

Computational Tools
InterPro, COFACTOR, SIFTER, PANTHER

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Lecture 08 - Computational protein design

1. What is computational protein design?

• Computational protein design (CPD), includes computer-aided engineering for partial or full de
novo designs of proteins of interest.
• Designs are defined by a requested structure, function, or working environment.

2. What are the broader structural bioinformatics and computational biophysics field and included
in computational protein designing?
1] Integration of knowledge-based and energy-based methods
2] Hierarchical designated approach towards local, regional, and global motifs and the
integration of high- and low-resolution design schemes that fit each such region
3] Systematic differential approaches towards different protein regions,
4] Identification of key hot-spot residues and the relative effect of remote regions,
5] Assessment of shape-complementarity, electrostatics and solvation effects,
6] Integration of thermal plasticity and functional dynamics,
7] Negative design,
8] Systematic integration of experimental approaches,
9] Objective cross-assessment of methods, and
10] Successful ranking of potential designs.

3. What are the objetives of computational protein design?

• Protein folding or the inverse folding problem –
• The entropic hydrophobic effect underlying protein folding is long known.
• The details of protein folding are still not fully elucidated.
• The inverse protein folding problem, the problem of finding which amino acid sequences
fold into a known 3D structure is in essence the holy grail of protein design.
• Specificity –
• The design of specific interactions (protein–protein or protein–ligand) is related to the
application of negative design rules.
• A priori focus the design efforts on regions that determine specificity, or, alternatively,
add similar templates (decoys or related molecules) to examine the target affinity in
respect to a background of unwanted interactions.
• Stability and extremophilicity –
o Our body invest energy in maintaining a mesophilic mild environment for proteins
including narrow range of temperature, salt concentrations, pH etc.
o designed proteins are often expected to function in hostile environments whether these
are fermenters in the biotechnology industry where protein yield is a goal or whether
these are synthetic biology applications e.g. bio- detergents.
o CPD approach provides a unique method to study the very determinants underlying the
requested extremophile trait.
• Synthetic biology –
o Natural proteins were optimized according to the need of organisms and the constraints
of the evolutionary process,
o e.g. not enabling large leaps at a time and not focusing on traits that don’t affect organism
survivability.
• Negative design rules –
o CPD offers a focused path to study negative design rules which are often overlooked due
to methodological challenges in studying them.
o In answering the question “how do things not work in the wrong direction?”
o therapeutic index (TI) combines the positive effect of manipulating the requested target
with the negative side-effects, generally expressed by the lethal-dose (LD) which is

H.T.B.M. 1
 usually due to lack of specificity and/or is due to toxicity of the drug or metabolites or
degradation products thereof.

4. What are the Structural Levels of CPD: Design Target and Design Building Block
• De novo CPD
o The most classical CPD procedure is the so called de novo design where a totally new fold
and/or function are pursued,
o e.g. as is the case for the beta-doublet beta sandwich design.
• Core stabilization
o The driving force for folding of soluble proteins is the entropic hydrophobic effect in
which the collapse of the hydrophobic protein core maintains the disorder of the aqueous
solvent around the solvent accessible hydrophilic amino acids of the protein.
o However, the hydrophobic effect results in a molten globule which is later optimized for
enthalpic contributions of specific interactions and packing.
o As this process is often not optimized for stability, the design of better protein cores is a
long- standing approach within CPD.
o In general, most CPD attempts thus far included a component of core stabilization. Other
approaches to stabilization include targeting the most unstable parts of the protein,
o e.g. loops.
• Solubilization of protein–solvent interface
o One of the most classic examples of genetic diseases, sickle-cell anemia, includes a
hydrophobic patch on the surface of the hemoglobin β-subunit following a single Glu ! Val
mutation.
o As such, maintaining the solubility of the protein may assist in avoiding aggregation.
o Likewise, membrane proteins were solubilized to allow for the study of the membrane
protein within an aqueous milieu as well as in order to study the basic features of
membrane pro
• Symmetry
o The complexity of the CPD process can be largely trimmed by adding symmetry to the
structural design.
o This can be done for symmetric proteins such as beta-propeller proteins, for coiled coils,
or crystallographic symmetry.
• Binding site
o The binding site is literally the heart of the protein and usually requires special care which
is different from the general approaches to other CPD regions.
o These range from quantum-mechanics optimization to grafting an existing site to a de
novo designed template.
o For example, a binding site CPD includes many different case-studies such as changing the
bound metal, e.g. as done for ferritin, de novo designed metal-binding or nonbiological
cofactor binding proteins, and enzymes.
• Protein–protein interactions (PPI)
o While a binding site is often specifically designed for a non-amino acid moiety, protein–
protein interaction CPD include the stable or transient interaction between spatial
patches of amino acids that are on the surface- accessible part of the protein.
o It is essentially the design of new PPI incorporating unique features of the antibody such
as the hyper-variable loops.
• Dynamics
o Proteins are often regarded as XYZ coordinates of frozen structures with a global
minimum energy conformation (GMEC) structure represented within PDB files.
o However, proteins are four-dimensional machines (space and time dimensions) with
intrinsic local flexibility and global dynamics.
o A ligand-controlled conformational switch, provide an example to CPD focusing on such
functional dynamics which must be a major focus of any CPD involving dynamic function.

H.T.B.M. 2
 • Membrane proteins and other “unique” protein groups
o As presented in this book, many protein families have designated CPD schemes which
harness family-specific parameterization.

5. What happen to proteins that fail in computational protein design?

• “Proteins from failed computational design efforts are typically discarded without comment or
investigation into the cause of failure. This situation is unfortunate, because valuable information
is lost when successful designs are reported. Without detailed computational and/or
experimental analysis of failed designs, flaws in the design procedure cannot be identified and
remedied.”

6. Compare two ways of protein designing.

7. How proteins are designed: from computer models to artificial intelligence What is structural
framework in research?

H.T.B.M. 3
H.T.B.M. 4
Lecture 09

Protein Engineering • Solution nuclear magnetic resonance –

Protein Dynamics & Evolution of Novel capture protein motions on broad
Protein functions timescales
• Fluorescence spectroscopy
Protein Dynamics - Issue
Hierarchy of Principal Motions in Protein
Difficult to obtain info bcs: Dynamics
• Local atomic positions in proteins
P
constantly change 1. Bond vibrations (fs-ps)
• Different conformational substrates over 2. Side-chain rotations (ps-ns)
time 3. Backbone fluctuation (ns)
• 4. Loop motion/gating (ns-ms)
Protein Dynamics – Importance 5. Ligand binding/unbinding events (>100 ns)
6. Collective domain movement (>µs)
Understand:
• Protein folding
Hierarchy of Protein Sequence, Structure,
• Disease-causing misfolding of proteins
Dynamics & Function
• Biological function of proteins

• Signal transduction

Protein Dynamics – Computer Simulation

Study:
• Biomolecules & their dynamics in detail

• At high temporal and spatial resolution

Protein Dynamics

• A highly complex phenomenon comprising

numerous contributions from motions with
different mechanisms of action and
happening with diverse timescales and Protein Dynamics – Molecular Dynamics
amplitudes that highly depend on the
system and the local environment. • Thanks to availability of:
• High quality 3D protein structures
Experimental techniques to study protein • Using: NMR, X-ray, cryogenic electron
dynamics: microscope (Cryo-EM), homology modelling

Hashani Hewagama
Biotech-1
 ➔ Computational biophysical methods are
used to study protein motions at atomic
resolution

MD (Molecular Dynamics) – golden

standard to investigate conformational
behaviour of a protein

Limitations of MD simulations:
• Accuracy of force fields used to calculate

interatomic interactions
• Quality of traditionally applied force fields is
limited by :
− numerous approximations (like lack of
particular interaction types)
− neglect of electronic polarizability

Evolution of Novel Protein Functions

Protein evolution describes:

• Changes over time in protein shape, function
and composition
• Understand rate & causes of protein evolution
using quantitative analysis & experimentation

Proposed mechanisms to generate new

genes:
• Gene duplication
• Transposable element protein domestication
• Lateral gene transfer
• Gene fusion
• Gene fission
• De novo origination

Hashani Hewagama
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Lecture 10 - Methods and tools used in protein engineering
1. What are the Methods and tools used in protein engineering?
• Rational design • Peptidomimetics
• Site directed mutagenesis • Phage display technology
• Evolutionary methods/ directed • Cell surface display technology
evolution • Flow cytometry/ cell sorting
• Random mutagenesis • Cell-free translation systems
• DNA shuffling • Designed divergent evolution
• Molecular dynamics • Stimulus responsive peptide systems
• Homology modeling • Mechanical engineering of elastomeric
• MolCraft in vitro protein evolution proteins
systems • Engineering extracellular matrix
• Computational methods variants
• Receptor based QSAR methods • Traceless Staudinger ligation
• NMR • De novo enzyme engineering
• X-ray crystallography • mRNA display

2. Compare and contrast different protein designing methods.

Directed Semi-rational Rational approach
Generates a vast pool of Approach generates a small pool of specific Relies on well-defined
mutated proteins through proteins through nonrandom processes. knowledge of the
random DNA manipulation. This involves a combination of in silico protein’s 3D structure
predictive tools, protein sequence, and function and
structure, and functional information. incorporates precise,
specific properties,
such as substrate
specificity, pH, or
temperature stability,
to the protein.

H.T.B.M. 5
 3. What re the mechanisms of action of therapeutic antibodies?

4. What are the steps of developing a therapeutic antibody?

5. What is MACSima Imaging platform?

H.T.B.M. 6
 6. What are the steps in developing monoclonal antibody?

Lecture 11 – Biochemical assays

1. What is a biochemical assay?

• A biochemical assay is a technique that detects or quantifies the activity of a biological molecule
or substance analytically.
• It is an in vitro process.

2. What are the examples for biochemical assays?

• Colorimetric assay and fluorometric assay
• Multiple techniques such as ELISA and western blotting are also complex biochemical assays for
the quantification of metabolic activity and measurement of the functional behavior of
biomolecules such as proteins, enzymes, and other small molecules.

3. For what are these assays used?

• These types of assays are used for the identification of protein-DNA, protein-RNA, and protein-
protein interactions.

4. What is Colorimetric protein assay?

• This assay is based on a dye-metal complex that binds to proteins in an acidic solution. Upon
binding, the reddish dye-metal complex changes to green, resulting in an absorbance that is
measured at 660 nm and normalized at 750 nm. Advantages: Room temperature stability of the
assay reagent.
• A colorimetric assay is a technique that determines the concentration of colored compounds in a
solution.
• It leads to a color change due to an enzymatic or chemical reaction between reagents and analytes.
• They use colorimeters or spectrophotometers. Colorimeters are instruments that characterize
colored samples in order to provide an objective measure of color characteristics. A
spectrophotometer is a device that measures light intensity as a function of the color or
wavelength of the light.

5. What is the key difference between colorimetric assay and fluorometric assay?
• The key difference between colorimetric and fluorometric assay is that colorimetric assay determines
the concentration of colored compounds in a solution while fluorometric assay determines the kinetic
mechanism of a solution.

6. How Does a Colorimetric Assay Work?

H.T.B.M. 7
 • In a colorimetric assay, a plate is prepared with a particular antibody bound to wells. Then the
sample is added. This may allow the sample to bind to the antibody.
• Then a detection antibody and a substrate are added to the wells to react with the detection probe.
• Stop solution is added at the end before the reading. An empty well called the blank is left without
the sample. In the colorimetric assay, the darker the color, the greater the analyte concentration.
Usually, only one wavelength is needed to take the reading. But if there is a reference
measurement, two or more wavelengths are used.

7. What is a Fluorometric Assay?

• The fluorometric assay is a technique that determines the kinetic mechanism of enzyme reactions.
A fluorometric assay takes place with a formation of a fluorescent product from a nonfluorescent
substrate or vice versa.
• This assay also makes use of fluorescence resonance energy transfer (FRET) where, the
enzymatic reaction changes the position of two fluorophores in the substrate thus, changing the
fluorescence intensity.
• Fluorometric assays are generally much more sensitive than other assays. The diagnostic enzyme
estimations in tissue, cell, or fluid samples of patients increase due to its higher sensitivity.

8. How Does a Fluorometric Assay Work?

• In a fluorometric assay, a substrate is added to the sample in the plate, and the fluorescence
response is taken using the plate reader.
• Here, each cell is measured separately.
• Opaque plates are used in fluorometric assays. This reduces the scattering of light.
• Two wavelengths are necessary for this type of assay. One wavelength is to detect the excitation
and the other wavelength is for the emission.

9. What are the Similarities Between Colorimetric and Fluorometric Assay?

• Colorimetric assay and fluorometric assay are two types of biochemical assays.
• Both assays are carried out for medical diagnostics.
• These assays involve an enzymatic reaction.
• Both assays involve a substrate and analytes.

10. What is the Difference Between Colorimetric and Fluorometric Assay?

• A colorimetric assay is a technique that determines the concentration of colored compounds in a
solution, while fluorometric assay is a technique that determines the kinetic mechanism of
enzyme reactions.
• Moreover, the fluorometric assays are more sensitive than the colorimetric assays; thus,
fluorometric assays have the ability to detect more analytes.
• Furthermore, fluorometric assays require two wavelengths, while colorimetric assays are carried
out with one wavelength.

11. What is fluorescence?

• Fluorescence is the emission of light by a substance that has absorbed light or other
electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a
longer wavelength, and therefore a lower photon energy, than the absorbed radiation.

H.T.B.M. 8
 12. Compare and contrast colorimetric and fluoroaromatic assays.

H.T.B.M. 9
Lecture 12 - Functional Analysis and application of protein engineering

1. What are the different steps of protein analysis?

2. List down the advantages and disadvantages of different Protein expression systems.
• Solubility, functionality, speed, and yield are often the most important factors to consider when
choosing an expression system.

H.T.B.M. 10
 3. What are Analytical protein microarrays and what are the steps involved?
• Analytical protein microarrays contain. antibodies with
high affinity and specificity that are spotted on an appropriate
surface.
• These chips are used for proteome profiling and
monitoring proteome expression pattern and clinical diagnostics.
• all of the proteins in two biological samples that one would
like to compare are labeled with distinguishable markers (e.g.,
labeled with red or green fluorescent dyes).
• Then, protein samples are mixed, and this mixture is used
to incubate the array.
• Spots that show up as green or red have an excess of
protein from one sample over the other.
• Spots that appear yellow approximate an equal amount of
protein from each sample.
• This experimental procedure is analogous to gene
expression profiling with DNA microarrays, and it allows the effect
of various physiological stimuli or genetic alterations on proteins
levels to be examined simultaneously.
• In analytical protein array, different types of ligands,
including antibodies, antigens, DNA or RNA, aptamers,
carbohydrates, or small molecules, with high affinity and specificity,
are spotted onto a derivatized surface.

4. What are Functional protein microarrays and what are the steps involved?
• Functional protein microarrays require native proteins or peptides that are individually purified
or synthesized from cDNAs by in vitro transcription/translation or other high-throughput
techniques, after which they are spotted onto a suitable surface to form the functional protein
microarrays.
• These chips are used to probe protein activities, binding properties, and post-translational
modifications. In the example shown, proteins are labeled with distinguishable markers (e.g.,
labeled with green fluorescent dye). The spots that “light up” are candidate binding partners.

H.T.B.M. 11
 • With the proper detection method, functional protein microarrays can be
used to identify the substrates of enzymes of interest. This class of chips is
particularly useful in drug and drug-target identification and in unraveling
biological networks.

Functional Analysis of Engineered and Mutant Enzymes

• The basic goal of protein engineering is the creation of altered forms of a
known enzyme
• catalyst that exhibits one or more of the following properties:
• An increased catalytic function relative to the parent enzyme.
• An altered substrate specificity or stereospecificity, such that the
engineered protein is capable of catalyzing the conversion of substrates
differing from the specific substrate of its parent form.
• An increased stability to the environment that is required for it to catalyze
the specific function required.
• An analysis of all of these desired properties of the engineered protein
requires detailed studies of its catalytic properties, which involve the
delineation of the steady - state kinetic behavior of the mutant protein.

5. What is the importance of enzyme engineering?

6. What is Steady - State Kinetics

• Steady-state kinetics applies whenever the concentration of the substrate is well above that of
the enzyme, so that the rate of change of substrate concentration greatly exceeds the rate of
change of the concentration of any enzyme form.
• The basic equation describing enzyme catalysis is the Michaelis – Menten equation:
𝑣0 𝑘𝑐𝑎𝑡 [𝑆]
=
[𝐸] 𝐾𝑚 + [𝑆]
kcat (units of time − 1) is the enzyme turnover number, which is defined as the maximum number of substrate molecules
converted to products per active site per unit of time (with units of a fi rst - order rate constant). Km (units of concentration) is
defi ned as the concentration of substrate where half the maximal activity ( kcat ) is observed. The term vo defi nes the initial,
zero - order rate of the reaction (with units of concentration of product - time − 1), the value of which depends hyperbolically
on the substrate concentration.

H.T.B.M. 12
 7. What are the requirements for the valid application of the Michaelis – Menten equation ?
• [S] must be significantly larger than [E] (ideally at least 100 - fold).
• The steady - state assumption holds (where the concentration of ES is constant over the course
of the assay) and the rate of product formation is linear with time.
• The initial rate of the reaction is such where the concentration of substrate does not materially
change over the course of the assay and the concentration of product is small enough, so that no
enzyme inhibition or reverse reaction occurs.

8. What are the potential applications of enzyme engineering?

9. What are Characteristics that one might have to change in a predictable manner in protein
engineering or enzyme engineering to get the desired function?

10. What is the application of PE in biological therapeutics?

• Applications of targeted drug neutralization
• Stimulus- responsive engineered protein prodrugs
• Emerging multicomponent smart drug systems (e.g., antibody-drug conjugates, responsive
engineered zymogens, prospective biochemical logic smart drug systems, drug buffers, and
network medicine applications)

H.T.B.M. 13
Simplified schematic of using protein engineering to redesign an enzyme’s substrate specificity
highlighting knowledge-based mutagenesis, computational protein design, and directed evolution.

H.T.B.M. 14