Theory and Examples

of Application of Protein Engineering

Basics of Protein Structure
20 Bricks - a world of possibilities!

Hydrophobicity Table
20 Bricks - a world of possibilities, or not?!
20 Bricks - a world of possibilities, or not?!

Helices have their own dipole moment! - N terminal (+) C- terminal (-)
20 Bricks - a world of possibilities, or not?!

Tossi A, Sandri L, Giangaspero A. Amphipathic,

alpha-helical antimicrobial peptides. Biopolymers.
2000;55(1):4-30. doi:
10.1002/1097-0282(2000)55:1<4.

Giménez-Andrés M, Čopič A, Antonny B. The

Many Faces of Amphipathic Helices.
Biomolecules. 2018 Jul 5;8(3):45. doi:
10.3390/biom8030045.
20 Bricks - a world of possibilities, or not?!
20 Bricks - a world of possibilities, or not?!
20 Bricks - a world of possibilities, or not?!
20 Bricks - a world of possibilities, or not?!

Close corner Splayed corner A supersecondary structure is a compact

three-dimensional protein structure of several
adjacent elements of a secondary structure that is
smaller than a protein domain or a subunit.

Helix Hairpin Beta Hairpin

Loop -> 2-16 residues

Orthogonal packing of B-sheets.

Intestinal Fatty Acid-Binding protein.
Aligned packing of B-sheets.
Nuclear pore complex protein A Greek key motif A Beta meander mofit
Nup98-Nup96 - Homo sapiens
 20 Bricks - a world of possibilities, or not?!

PLASTOCYANIN FROM THE PHOTOSYNTHETIC

PROKARYOTE, PROCHLOROTHRIX
HOLLANDICA

Rossmann Fold
A Greek key motif Β-α-β motif
20 Bricks - a world of possibilities, or not?!

Β-barrel - GFP

Nomenclature
Basics of Protein Folding
Before we engineer a protein - first it has to fold!

Levinthal's paradox

In 1969, Cyrus Levinthal noted that, because of the

very large number of degrees of freedom in an
unfolded polypeptide chain, the molecule has an
astronomical number of possible conformations.
An estimate of 10300 was made in one of his papers
(often incorrectly cited as the 1968 paper). For
example, a polypeptide of 100 residues will have
99 peptide bonds, and therefore 198 different phi
and psi bond angles. If each of these bond angles
can be in one of three stable conformations, the
protein may misfold into a maximum of 3198
different conformations (including any possible
folding redundancy). Therefore, if a protein were to
attain its correctly folded configuration by
sequentially sampling all the possible conformations,
it would require a time longer than the age of the
universe to arrive at its correct native
conformation. This is true even if conformations are
sampled at rapid (nanosecond or picosecond) rates.
The "paradox" is that most small proteins fold
spontaneously on a millisecond or even microsecond
time scale. The solution to this paradox has been
established by computational approaches to protein
structure prediction.
Before we engineer a protein - first it has to fold!

When the protein is born? – 11s to growth up!

Before we engineer a protein - first it has to fold!

Protein folding scheme Entropy funnel

Before we engineer a protein - first it has to fold!

Major driving force of protein folding is burying and clustering

hydrophobic side chains to minimize their contact with water:
“hydrophobic effect”

Compact structure -> minimize area of hydrophobic surface exposed to

solvent
buried H-bonds are paired

Globular proteins -> hydrophobic core and polar/charged groups outer layer

Packing density = 75% for native proteins, 70=78% for crystals! ->
reason why protein crystal structures are so useful in protein structure
determination and rational design in protein engineering!
 Before we engineer a protein - first it has to fold!
Inclusion
bodies

DNA -> mRNA -> Protein primary structure -> Folded protein Active form

Inactive
Theory ✔ form
Host
Practice ?
(Environment)

- rapid growth - best standardization

- high protein titer - expensive
production - only for screening (HT)
- inexpensive medium Escherichia
- conditions affect expression
- well known organism coli
and activity
- well defined genetics - only high-throughput
- many toolkits - modification of macromolecule
available In vitro
environment
expression
 Before we engineer a protein - first it has to fold!

4 2

4
5 5

1 - Protein tag and periplasm transport

2 - Chaperon aided folding (and/or transport)
3 - Correct folding
4 – Proteolysis
5 – Aggregation – IB formation
 Before we engineer a protein - first it has to fold!

Disulfite Bridges formation in E. coli periplasm Dsb system

Mizrachi D, Robinson MP, Ren G, Ke N, Berkmen M, DeLisa MP. A water-soluble DsbB variant that catalyzes disulfide-bond formation in vivo. Nat Chem Biol.
2017 Sep;13(9):1022-1028. doi: 10.1038/nchembio.2409.
Improving Protein Production
by Protein Engineering
Directed Evolution Application

YC2 – S83L, V257I, S285P

YC3 – K241T, S285P, D293G

Food Biotechnology 2020,, 34, 42-61.

 Site Directed Mutagenesis

Biotechnol Lett (2008) 30:717–722.

 Expression vs Activity

Chemistry & Biology (2015) 22:1406–1414; Nature Biotechnology (2005) 23:102 – 107.
 Expression vs Activity

OD600 Enzyme assay

SplitGFP RFU Activity/RFU

Chemistry & Biology (2015) 22:1406–1414

 Expression vs Activity

Chemistry & Biology (2015) 22:1406–1414.

 Expression vs Activity

Chemistry & Biology (2015) 22:1406–1414.

 Expression vs Activity

NATURE COMMUNICATIONS | (2021) 12:6093

 Expression vs Activity

NATURE COMMUNICATIONS | (2021) 12:6093

Samuel Wagner From Biogenesis to Over expression of Membrane Proteins in Escherichia coli
 Expression vs Activity

Ori:
-pBBR1
ColE1

Sensor:
GFP-ASV t1/2 110 min
GFP-mut3 – fast folding t1/2 1day

PARP1 soluble protein

BRCA1 insoluble

NATURE COMMUNICATIONS | (2021) 12:6093

 Expression vs Activity

chymotrypsin inhibitor 2 (CI2)

NATURE COMMUNICATIONS | (2021) 12:6093

Engineering Protein Stability
Engineering Protein Stability -> What is Stability?

Thermodynamic stability of proteins represents

the free energy difference between the folded and
unfolded protein states. This free energy
difference is very sensitive to temperature, hence
a change in temperature may result in unfolding or
denaturation. Protein denaturation may result in
loss of function, and loss of native state.

The free energy of stabilization of soluble globular

proteins typically does not exceed 50 kJ/mol.
Taking into consideration the large number of
hydrogen bonds that take place for the
stabilization of secondary structures, and the
stabilization of the inner core through hydrophobic
interactions, the free energy of stabilization
emerges as small difference between large
numbers.

Simplest model of Kinetic Stability

Native <-> Unfolded --> Inactive

A typical protein stability curve plotted using

the GibbsHelmholtz equation
 What to consider before the start of protein engineering campaign?

STEFAN LUTZ; UWE THEO BORNSCHEUER. Protein Engineering Handbook, Volume 3. Weinheim: Wiley-VCH, 2012. ISBN 9783527331239.
Engineering Protein Stability - Loop
 Loop Grafting

1st gen - 8000 clones

2nd gen - 2000 clones
3rd gen - 3000 clones
4th gen - 2000 clones
5th gen - 2000 clones
∑ - 17000 clones
about 1200 96 well plates
:)

What is the
outcome? How it was done/measured?

Protein engineering, 1999, 12, 47-53.

 Loop Grafting

166 118
What has
changed?
194
161
218
76

181
14

Protein engineering, 1999, 12, 47-53.

 Loop Grafting

B-factor - The term B-factor, sometimes called the

Debye–Waller factor, temperature factor, or atomic displacement
parameter, is used in protein crystallography to describe the
attenuation of X-ray or neutron scattering caused by thermal
motion.

J. Biol. Chem. (2019) 294(48) 18398 –18407.

 Loop Grafting

J. Biol. Chem. (2019) 294(48) 18398 –18407.

 Loop Grafting

J. Biol. Chem. (2019) 294(48) 18398 –18407.

 Loop Walking Method

Burkholderia cepacia lipase, commercialized as lipase PS (LPS), is one of the most useful biocatalysts.

Scientifc Reports | (2021) 11:11883.

 Loop Walking Method

L7

200 clones tested for each loop

Termostability plots for the twelve libraries with random mutations in each loop region: (a) L1, (b) L2, (c) L3, (d) L4, (e) L5, (f) L6, (g) L7, (h) L8, (i) L9, (j) L10,
(k) L11, and (l) L12. Relative activity without heat treatment (horizontal axis) and relative residual activity afer heat treatment at 60 °C for 30 min (vertical axis)
are based on the wild-type enzyme (blue square).

Scientifc Reports | (2021) 11:11883.

 Loop Walking Method

Scientifc Reports | (2021) 11:11883.

 Loop Walking Method

Scientifc Reports | (2021) 11:11883.

Engineering Protein Stability - Proline
 Proline Insertion – why is it so special?

Ramachandran plots of all terminally capped amino acids simulated with different force fields.
Represented is the logarithm of the {Φ-ψ}-pairs counts on a 1°-grid.

Protein engineering 2002, 15, 29-33.

 Proline Insertion

77
77

113

T4 lysozyme – red Gly What to replace?

Proc Natl Acad Sci U S A. 1987, 84, 6663-7.

 Proline Insertion

60

93

82
A82P

T4 lysozyme

Proc Natl Acad Sci U S A. 1987, 84, 6663-7.

 Proline Insertion

Proc Natl Acad Sci U S A. 1987, 84, 6663-7.

 Proline Insertion

Protein engineering 2002, 15, 29-33.

 Proline Insertion

Protein engineering 2002, 15, 29-33.

 Proline Insertion

Tb – hypertermophilic
EH – termophilic
CB - mesophilic.

PROTEINS: Structure, Function, and Bioinformatics 66:196–204 (2007).

Linkers
 Linker Insertion

Adv Drug Deliv Rev. 2013 October 15; 65(10): 1357–1369.

 Linker Insertion

S3 S2 S1 S H A1 A2
A3

Appl Microbiol Biotechnol. 2008 Jun;79(4):579-87.

 Linker Insertion

Appl Microbiol Biotechnol. 2008 Jun;79(4):579-87.

 Linker Insertion

Appl Microbiol Biotechnol. 2008 Jun;79(4):579-87.

 Linker Insertion

Appl Microbiol Biotechnol. 2008 Jun;79(4):579-87.

Domain Swapping
 Domain Swapping

XylC XylC (20-244)

green - signal peptide, green - N-terminal part,
blue - hydrolytic domain (H11), blue - xylopentose (2+3),
red - cellulose binding domain (CBD), red - C-terminal part,
AlphaFold PDB-3WP6

Int. J. Mol. Sci. 2022, 23(1), 463.

 Domain Swapping

CDBFV-1 -xylanase CDBFV

from Neocallimastix patriciarum

Linker (moderately flexible) -

PEVLPPLPKESRISEGEAVVVG

C2 - carbohydrate binding
submodule C2 from
hyperthermophilic CBM9_1-2

Int. J. Mol. Sci. 2022, 23(1), 463.

 Domain Swapping

(a) Effect of temperature on enzyme

activity: the enzymatic activity was
determined at different temperatures
of 10.0–80.0 °C and pH 5.5.
(b) Temperature stability at 60.0 °C
(c) Temperature stability at 65.0 °C
(d) Temperature stability at 70.0 °C

Int. J. Mol. Sci. 2022, 23(1), 463;

 Domain Swapping

Red represents the linker; blue represents the C2; green represents the CDBFV.
(a) Three-position structure diagram of the best chimera CDBFV-C2.
(b) Illustration of the newly formed hydrogen bonding interaction between CDBFV and linker in the best chimera CDBFV-C2.
(c) Illustration of the newly formed hydrogen bonding interaction between CDBFV and C2 in the best chimera CDBFV-C2.

Int. J. Mol. Sci. 2022, 23(1), 463.

Disulfide Bonds
Site Directed Mutagenesis - Cysteine - Disulfide Bonds

❑ 2.05 Å in length,
❑ strong bond ~40% weaker than C-C or C-H,
❑ sensitive to red-ox conditions,
❑ in prokaryotes formed in periplasm,
❑ may form the nucleus of a hydrophobic core of the
folded protein (protected from nucleophiles).

THREE-DIMENSIONAL STRUCTURE
OF INTERLEUKIN 8 IN SOLUTION
 Site Directed Mutagenesis - Disulfide Bridges

T4 Lysozyme Red I3-A97 Green I9-L164 Blue T21-T142

Nature 342, 291–293 (1989).

 Site Directed Mutagenesis - Disulfide Bridges

Nature 342, 291–293 (1989).

 Site Directed Mutagenesis -
Disulfide Bridges

Thermolysin from Bacillus thermoproteolyticus

J Biol Chem. 1997, 272, 11152-11156.

 Site Directed Mutagenesis -
Disulfide Bridges

99 99

323 323

Crystal Structure of Cel5A (EG2) from Hypocrea jecorina (Trichoderma reesei)

Green Cystein and –S-S- bridges, red planned Aa exchange

C99V and C323H

International Journal of Biological Macromolecules, 120, 2018, 1572-1580.
 Site Directed Mutagenesis -
Disulfide Bridges

99 323

99 323

Blue - Crystal Structure of Cel5A from Trichoderma reesei - mesophilic [C92-S-S-C99 + C273-S-S-323C]

Green - Crystal Structure of Cel5A Thermoascus aurantiacus - thermophilic [81I x 88A + 254T x 300I]

International Journal of Biological Macromolecules, 120, 2018, 1572-1580.

 Site Directed Mutagenesis -
Disulfide Bridges

International Journal of Biological Macromolecules, 120, 2018, 1572-1580.

 Site Directed Mutagenesis -
Disulfide Bridges

International Journal of Biological Macromolecules, 120, 2018, 1572-1580.

 Site Directed Mutagenesis -
Disulfide Bridges

International Journal of Biological Macromolecules, 120, 2018, 1572-1580.

 Site Directed Mutagenesis -
Disulfide Bridges

International Journal of Biological Macromolecules, 120, 2018, 1572-1580.

Salt Bridges
 Site Directed Mutagenesis - Salt Bridges

Glyceraldehyde 3-P Dehydrogenase from

Escherichia Coli

Glyceraldehyde 3-P Dehydrogenase from

hyperthermophilic Thermotoga martima

STEFAN LUTZ; UWE THEO BORNSCHEUER. Protein Engineering Handbook, Volume 3. Weinheim: Wiley-VCH, 2012. ISBN 9783527331239.
 Site Directed Mutagenesis - Salt Bridges

PLoS ONE 10(9):e0137113.

 Site Directed Mutagenesis - Salt Bridges

Early estimate (end of 90’) on protein folding stability that the removal of a hydrogen
bond generally lowers protein stability by 0.5 to 2.0 kcal/mol; estimates for ion pair
stabilization range from 0.4 to 1.0 kcal/mol.

Therefore, stabilization of about 0.5 kcal/mol is required per 1⁰C rise in melting
temperature.
Thus, for each 10⁰C rise in protein operational temperature, a mean total of about 13
hydrogen/salt bonds are required.

Salt bridges have been proposed to play a crucial role in promoting hyperthermostability in proteins, yet they appear
to make little contribution to protein stability at room temperature. The latter point has been rationalized previously
on the basis that the association of two charged molecules to form a salt bridge incurs a substantial desolvation
penalty, which is seldom completely compensated by favourable interactions within the salt bridge and with the rest of
the protein.

The hydration free energies of charged side-chains are more adversely affected by increasing temperature than are the
hydration free energies of hydrophobic side-chains of identical size and shape (isosteres). Direct consequence of the
temperature dependence of the hydration free energies is that at high temperatures the desolvation penalty for
formation of a salt bridge is markedly reduced in magnitude. As a result, the argument that relative to hydrophobic
isosteres, salt bridges destabilise proteins, may no longer be true at high temperatures.

PLoS ONE 10(9):e0137113.

 Site Directed Mutagenesis -
Salt Bridges
Some bioinformatic stuff - design and
The 1,4-α-glucan branching enzyme from “feeding” the model that indicate
Geobacillus thermoglucosidans STB02 possible sites for salt bridge formation

Computational and Structural Biotechnology Journal 17 (2019) 895–903.

 Site Directed Mutagenesis - Salt Bridges

The 1,4-α-glucan branching enzyme from Geobacillus thermoglucosidans STB02

Food Chemistry 316 (2020) 126348.

 Site Directed Mutagenesis - Salt Bridges

The 1,4-α-glucan branching enzyme from Geobacillus thermoglucosidans STB02

Food Chemistry 316 (2020) 126348.

 Site Directed Mutagenesis -
Salt Bridges

The 1,4-α-glucan branching enzyme from Geobacillus thermoglucosidans STB02

571

231

339

Food Chemistry 316 (2020) 126348.

 Site Directed Mutagenesis Salt Bridges

The 1,4-α-glucan branching enzyme from Geobacillus thermoglucosidans STB02

How it was done/measured?

Food Chemistry 316 (2020) 126348.
 Site Directed Mutagenesis - Salt Bridges Summary

1. The potential salt bridges (salt bridges that will be formed after introducing mutants) should include at least one
of endogenously charged residues in order to decrease the blindness of design;
2. The residues involving in potential salt bridges, including endogenous and mutant residues, should locate in the
same secondary structures;
3. When sequence separation of two residues is 3 or 4, select the residues that locate in helical structures; when
sequence separation of two residues is 2, the residues locating in beta sheet is preferred;
4. The sequence separation of residues in potential salt bridges is <5;
5. The Cα distance between residues is between 4 Å and 14 Å. Simultaneously, the distance between N+ and O–
atoms of the side chains in potential salt bridges is in the range of from 3.0 Å to 4 Å.
6. The above criterions are apparent properties of salt bridges, which should be strictly qualified when designing
salt bridges; the inherent characteristics of salt bridges determine their contribution to the stability of enzymes.
In our selection criteria, the residues in salt bridges should meet the condition that, at least, one part of
potential salt bridges is conserved. The conserved residues are defined as their conservation was above 70% by
using aligning corresponding sequences with ClustalW2 (https://www.ebi.ac.uk/Tool/msa/clustalw2/).
Fin