Achievement of Protein
Thermostability
byAmino Acid Substitution
2018/6/30 M1 Majima Sohei

Contents of Today’s seminar• Introduction• Study on hyperthermophilic enzymes tounderstand the origin of thermostability• Acquiring thermostability by amino acidsubstitution• Summary2Introduction
New type of synthesis:
combination of chemocatalyst and biocatalyst

Bornscheuer et al. Nature Catalysis, 2018, 1, 123

Introduction
New type of synthesis:
combination of chemocatalyst and biocatalyst

Denaturation in organic
solvent, high temperature

Bornscheuer et al. Nature Catalysis, 2018, 1, 124

Introduction
Organic reactions in aqueous media
NHK reaction
Micskei et al. Tetrahedron Lett. 2001, 42, 7711

Heck reaction

Suzuki-Miyaura coupling*CTAB = C16H32NMe3Br, amphiphilic molecule
Bhattacharya et al. Tetrahedron Lett. 2005, 46, 3557

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Introduction
Examples of the reactions

combination of Suzuki-Miyaura coupling andenzymatic stereoselective reduction of ketone✔Cool down, pH adjustment is necessary
Burda et al, Angew. Chem. Int. Ed. 2008, 47, 9551

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Introduction
Examples of the reactions

combination of Ru-catalyzed olefin isomerization andenzymatic stereoselective reduction of ketone.
✔Cool down is necessary
Sabin, et al. Angew. Chem. Int. Ed. 2016, 55, 8691

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Introduction
New type of synthesis:
combination of chemocatalyst and biocatalyst

Thermostability isimportant for one-pot
reaction
Bornscheuer et al. Nature Catalysis, 2018, 1, 12 8

Contents of Today’s seminar• Introduction• Study on hyperthermophilic enzymes tounderstand the origin of thermostability• Acquiring thermostability by amino acidsubstitution• Summary9What are Hyperthermophiles?
Three domains of Life

Hyperthermophiles,
Haloarchaea,Methanogen …
https://openoregonstate.pressbooks.pub/microbiology/chapter/archaea/
Woese et al, Proc. Natl. Acad. Sci. USA 87 (1990)

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What are Hyperthemophiles?
Hyperthermophiles live under extremely hotconditions (>80 ℃)

Hydrothermal ventin deep sea.
https://www.brh.co.jp/communication/shinka/201
5/post_000022.html
Zeikus et al. MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS. 2001, 65, 1

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Application of Hyperthemophiles

The DNA polymerase of thisarchaea is used for PCR.

Pyrococcus furiosus
Optimum growth temperature: 100 ℃

Why are these proteins stable to heat ???
https://alchetron.com/Pyrococcus-furiosus
https://rechtsmedizin.med.uni-rostock.de/arbeitsbereiche/forensische-genetik/

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The origin of protein stability

thermophilicmesophilic

Comparison of homolog proteins

Stability comes from several factors such as;
Hydrophobic interactions
Solvent Accessible Surface Area
Ion pairs
Hydrogen bondCation-π interaction
Covalent bond and so on …

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Hydrophobic interactions
One of the most important factor
Study on Adenylate Kinase(AK)AKthe , AKjan : thermophilic AKvol: mesophilicamino acid sequence

Philips et al, J. Mol. Biol. 2003, 330, 1087 14

Hydrophobic interactions
Comparison of sequence

S19I

T188I

Philips et al, J. Mol. Biol. 2003, 330, 1087 15

Hydrophobic interactions

N-terminal and C-terminal is stabilized by hydrophobicinteraction.

Philips et al, J. Mol. Biol. 2003, 330, 1087

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Solvent Accessible Surface Area
Study on O6-methylguanidine-DNAmethyltransferase

Kai, et al. J. Mol. Biol. 1999, 292, 707

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Ion pairs
Study on O6-methylguanidine-DNAmethyltransferase✔ Intrahelix/Interhelix ion pairs stabilize protein.
Kai, et al. J. Mol. Biol. 1999, 292, 707

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Ion pairs
Study on adenylate kinases

Important for loop-stabilization

Philips et al, J. Mol. Biol. 2003, 330, 1087

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Contents of Today’s seminar• Introduction• Study on hyperthermophilic enzymes tounderstand the origin of thermostability• Acquiring thermostability by amino acidsubstitution• Summary20

Improvement of stability by substitution

Bc-Csp: thermophilic
Bc-CspB: mesophilic(Csp:cold shock protein)

20.6 ℃

Arg … electrostatic interaction
Leu … decreases the polarity
around this residue to favor
intramolecular hydrogen bondSchmid, et al. Nat. Struct. Mol. Biol. 2000, 7, 380

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Improvement of stability by substitution
Two methods
Direct Evolution Method
Rational Design Method22

Direct evolution method
Cause evolution by “artificial selection”

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Direct evolution method
Study on subtilisin E (peptidase)t1/2… time that 50% of activity is lost at a fixed temperature
T50 … temperature that 50% of activity is lost during a fixed time
Zhao et al. Protein engineering. 1999, 12, 47 24

Direct evolution method
Study on subtilisin E (peptidase)

Activity increased.
Zhao et al. Protein engineering. 1999, 12, 47

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Direct evolution method
Study on subtilisin E (peptidase)
Analysis of mutation

Zhao et al. Protein engineering. 1999, 12, 47

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Direct evolution method✔ Mutation at loop position.
✔ Mutated amino acids
exist on the surface.
Zhao et al. Protein engineering. 1999, 12, 47

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Rational Design Method
Computer-based estimation of stability

Efficient screening isachieved.

✔ Substitution candidates areestimated by calculation.

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Rational Design Method
Study on L-rhamnosidase (r-Rha1)
Identified from Aspergillus niger (コウジカビ）

Increasing thermostability by substituting Lysto Arg. (constructing new hydrogen bondsand cation-π interactions)García et al. Archives of Biochemistry and Biophysics. 2012, 528, 118Ni, et al. Int. J. Biol. Macromol. 2018, 112, 1429

Rational Design Method
Study on L-rhamnosidase (r-Rha1)
Software for 3D structure … Modeller 9.15
for minimum energy … Minimization protocol
for site mutation … Mutation Energy protocol

García et al. Archives of Biochemistry and Biophysics. 2012, 528, 118Ni, et al. Int. J. Biol. Macromol. 2018, 112, 1430

Rational Design Method
The selected mutant positions as a result ofcalculation

Ni, et al. Int. J. Biol. Macromol. 2018, 112, 1431

Rational Design Method
Stability and activity of single mutantsNi, et al. Int. J. Biol. Macromol. 2018, 112, 1432

Rational Design Method
Stability and activity of single/double mutantsNi, et al. Int. J. Biol. Macromol. 2018, 112, 1433

Rational Design Method
Analysis
For destabilized mutants
Hydrophobic interactions may be hindered.
For K406R-K440R and K406R-K573R
No extra intramolecular interactions, compared tosingle mutants. May be due to the compactness ofthe protein.
Ni, et al. Int. J. Biol. Macromol. 2018, 112, 14 34

Rational Design Method
Study on Candida rugose lipases (CRL)• Lipase can catalyze hydrolysis andesterification.

• Starts inactivation at 45 ℃
Xu, Yan, et al. RSC Adv. 2018, 8, 194835

Study on the protein stability
Three prediction algorithms were used.
• FoldX … estimates Gibbs energy of protein folding
from empirical data.
• Rosetta ddg_monomer … predicts mutation-induced
stability change.
• I-Mutant3.0 … predicts stability of mutants fromdatabase
Xu, Yan, et al. RSC Adv. 2018, 8, 194836

Study on the protein stability
Result of estimation

Glu66… important for the activity
Asp457… at loop structure.
Xu, Yan, et al. RSC Adv. 2018, 8, 1948

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Results✔ Asp457Phe was most effective✔ Higher stability to acidic/alkaline condition✔ Higher optimal temperature achieved
Xu, Yan, et al. RSC Adv. 2018, 8, 1948

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Comparison of kinetic parameters✔ Kinetic parameters (Km, kcat, kcat/Km) of themutant was inferior to WT, but it’s negligible.
✔ What is the origin of stability???
Xu, Yan, et al. RSC Adv. 2018, 8, 194839

Analysis
Protein models were constructed by SWISS.
WTMutant✔ aromatic interaction
network and hydrophobicinteraction✔ Hydrophobic
interaction increases pH
tolerance, for it
decreases SASA (solventaccessible surface area).
Xu, Yan, et al. RSC Adv. 2018, 8, 1948

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Summary• The origin of protein thermostability is derived
from several factors.

• Thermostability can be increased by the single
or several substitution of amino acids.
• Thermostability can be increased by artificial
amino acid substitution.

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Appendix
Deamidation of proteins
These reactions areprohibited so far asprotein is folded
correctly.

Hensel et al. Biochem. Soc. Symp.
1992, 58, 127
Zeikus et al. MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS. 2001, 65, 142