Protein stability engineering insights revealed by domain-wide comprehensive mutagenesis


Abstract

The accurate prediction of protein stability upon sequence mutation is an important but unsolved challenge in protein engineering. Large mutational datasets are required to train computational predictors, but traditional methods for collecting stability data are either low-throughput or measure protein stability indirectly. Here, we develop an automated method to generate thermodynamic stability data for nearly every single mutant in a small 56-residue protein. Analysis reveals that most single mutants have a neutral effect on stability, mutational sensitivity is largely governed by residue burial, and unexpectedly, hydrophobics are the best tolerated amino acid type. Testing popular stability prediction algorithms against our data shows that all perform moderately, and combinations of algorithms are better at identifying the best variants in the single mutant landscape. We find that strategies to extract stabilities from high-throughput fitness data such as deep mutational scanning are promising and may be applicable toward training future stability prediction tools.

Submission Details

ID: gwoS2haU3

Submitter: Connie Wang

Submission Date: Oct. 9, 2018, 3:38 p.m.

Version: 1

Publication Details
Nisthal, A; Wang, C.Y.; Ary, M.L.; Mayo, S.L. (2018) Protein stability engineering insights revealed by domain-wide comprehensive mutagenesis; unpublished work from Mayo Lab, Caltech group
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2KN4 2010-08-18 The structure of the RRM domain of SC35
2KLK 2009-10-06 Solution structure of GB1 A34F mutant with RDC and SAXS
1Q10 2003-10-14 Ensemble of 40 Structures of the Dimeric Mutant of the B1 Domain of Streptococcal Protein G
2I2Y 2006-12-12 Solution structure of the RRM of SRp20 bound to the RNA CAUC
2GB1 1993-04-15 A NOVEL, HIGHLY STABLE FOLD OF THE IMMUNOGLOBULIN BINDING DOMAIN OF STREPTOCOCCAL PROTEIN G
2KHU 2010-02-23 Solution Structure of the Ubiquitin-Binding Motif of Human Polymerase Iota
2KBT 2009-02-03 Attachment of an NMR-invisible solubility enhancement tag (INSET) using a sortase-mediated protein ligation method
2JU6 2007-12-04 Solid-State Protein Structure Determination with Proton-Detected Triple Resonance 3D Magic-Angle Spinning NMR Spectroscopy
2KWD 2010-06-02 Supramolecular Protein Structure Determination by Site-Specific Long-Range Intermolecular Solid State NMR Spectroscopy
2N9L 2016-12-28 1H, 13C, and 15N Chemical Shift Assignments for in-cell GB1
2JSV 2008-04-15 Dipole tensor-based refinement for atomic-resolution structure determination of a nanocrystalline protein by solid-state NMR spectroscopy
2MBB 2014-06-04 Solution Structure of the human Polymerase iota UBM1-Ubiquitin Complex
1GB4 1998-07-22 HYPERTHERMOPHILIC VARIANT OF THE B1 DOMAIN FROM STREPTOCOCCAL PROTEIN G, NMR, 47 STRUCTURES
2K0P 2009-03-03 Determination of a Protein Structure in the Solid State from NMR Chemical Shifts
1MPE 2002-10-30 Ensemble of 20 structures of the tetrameric mutant of the B1 domain of streptococcal protein G
2PLP 2007-05-08 Ultra high resolution backbone conformation of protein GB1 from residual dipolar couplings alone
3GB1 1999-06-23 STRUCTURES OF B1 DOMAIN OF STREPTOCOCCAL PROTEIN G
2J52 2007-09-25 Solution Structure of GB1 domain Protein G and low and high pressure.
5UCF 2017-08-23 Solution NMR-derived model of the minor species of DANCER-2, a dynamic and natively folded pentamutant of the B1 domain of streptococcal protein G (GB1)
1ZXH 2005-11-08 G311 mutant protein
1GJS 2001-08-09 Solution structure of the Albumin binding domain of Streptococcal Protein G
2NMQ 2006-11-21 Simultaneous determination of protein structure and dynamics using rdcs
1PN5 2003-10-07 NMR structure of the NALP1 Pyrin domain (PYD)
2KHW 2010-02-23 Solution Structure of the human Polymerase iota UBM2-Ubiquitin Complex
1FCL 2001-09-19 DELTA1.5: A COMPUTATIONALLY DESIGNED CORE VARIANT OF THE B1 DOMAIN OF STREPTOCOCCAL PROTEIN G
1LE3 2002-04-24 NMR Structure of Tryptophan Zipper 4: A Stable Beta-Hairpin Peptide Based on the C-terminal Hairpin of the B1 Domain of Protein G
1GJT 2001-08-09 Solution structure of the Albumin binding domain of Streptococcal Protein G
2LGI 2011-10-26 Atomic Resolution Protein Structures using NMR Chemical Shift Tensors
2N9K 2016-12-28 1H, 13C, and 15N Chemical Shift Assignments for in vitro GB1
1FD6 2001-09-19 DELTA0: A COMPUTATIONALLY DESIGNED CORE VARIANT OF THE B1 DOMAIN OF STREPTOCOCCAL PROTEIN G
6HKA 2019-03-27 The solution structure of the micelle-associated FATC domain of the human protein kinase ataxia telangiectasia mutated (ATM)
2KQ4 2009-11-17 Atomic resolution protein structure determination by three-dimensional transferred echo double resonance solid-state nuclear magnetic resonance spectroscopy
1GB1 1993-04-15 A NOVEL, HIGHLY STABLE FOLD OF THE IMMUNOGLOBULIN BINDING DOMAIN OF STREPTOCOCCAL PROTEIN G
2RPV 2009-09-15 Solution Structure of GB1 with LBT probe
5UB0 2017-08-23 Solution NMR Structure of NERD-C, a natively folded tetramutant of the B1 domain of streptococcal protein G (GB1)
2IGH 1994-01-31 DETERMINATION OF THE SOLUTION STRUCTURES OF DOMAINS II AND III OF PROTEIN G FROM STREPTOCOCCUS BY 1H NMR
2N7J 2015-10-14 Sidechain chi1 distribution in B3 domain of protein G from extensive sets of residual dipolar couplings
5UBS 2017-08-23 Solution NMR Structure of NERD-S, a natively folded pentamutant of the B1 domain of streptococcal protein G (GB1) with a solvent-exposed Trp43
2J53 2007-09-25 Solution Structure of GB1 domain Protein G and low and high pressure.
2IGG 1994-01-31 DETERMINATION OF THE SOLUTION STRUCTURES OF DOMAINS II AND III OF PROTEIN G FROM STREPTOCOCCUS BY 1H NMR
2RMM 2007-12-04 Solution structure of GB1 A34F mutant
1P7E 2003-08-05 GB3 solution structure obtained by refinement of X-ray structure with dipolar couplings
2LUM 2012-08-29 Three-State Ensemble obtained from eNOEs of the Third Immunoglobulin Binding Domain of Protein G (GB3)
5UCE 2017-08-23 Solution NMR structure of the major species of DANCER-2, a dynamic and natively folded pentamutant of the B1 domain of streptococcal protein G (GB1)
1P7F 2003-08-05 GB3 solution structure obtained by refinement of X-ray structure with dipolar couplings
3FIL 2009-08-18 0.88 Structural and energetic determinants for hyperstable variants of GB1 obtained from in-vitro evolution
2QMT 2007-12-25 1.05 Crystal Polymorphism of Protein GB1 Examined by Solid-state NMR and X-ray Diffraction
2IGD 1998-07-29 1.1 ANISOTROPIC STRUCTURE OF PROTEIN G IGG-BINDING DOMAIN III AT 1.1 ANGSTROM RESOLUTION
1IGD 1994-11-01 1.1 THE THIRD IGG-BINDING DOMAIN FROM STREPTOCOCCAL PROTEIN G: AN ANALYSIS BY X-RAY CRYSTALLOGRAPHY OF THE STRUCTURE ALONE AND IN A COMPLEX WITH FAB
6CHE 2019-07-10 1.1 Selenomethionine mutant (A34Sem) of protein GB1 examined by X-ray diffraction
6CPZ 2019-07-10 1.12 Selenomethionine mutant (I6Sem) of protein GB1 examined by X-ray diffraction
2GI9 2006-04-25 1.14 Backbone Conformational Constraints in a Microcrystalline U-15N-Labeled Protein by 3D Dipolar-Shift Solid-State NMR Spectroscopy
3MP9 2011-02-23 1.2 Structure of Streptococcal protein G B1 domain at pH 3.0
6CTE 2019-07-10 1.2 77Se-NMR probes the protein environment of selenomethionine
6CNE 2019-07-10 1.2 Selenomethionine mutant (L5Sem) of protein GB1 examined by X-ray diffraction
6C9O 2019-07-10 1.2 Selenomethionine mutant (V29Sem) of protein GB1 examined by X-ray diffraction
6NLA 2019-01-23 1.34 Crystal structure of de novo designed metal-controlled dimer of B1 immunoglobulin-binding domain of Streptococcal Protein G (L12H, E15V, T16L, T18I, V29H, Y33H, N37L)-zinc
2ON8 2007-12-04 1.35 Gbeta1 stabilization by in vitro evolution and computational design
6NL7 2019-01-23 1.4 Crystal structure of B1 immunoglobulin-binding domain of Streptococcal Protein G (T16F, T18A, V21H, T25H, K28Y, V29I, K31R, Q32A, Y33L, N35K, D36A, N37Q)
6NL6 2019-01-23 1.4 Crystal structure of mutant B1 immunoglobulin-binding domain of Streptococcal Protein G (T16F, T18A, V21E, T25L, K28Y, V29I, K31R, Q32H, Y33L, N35K, D36H, N37Q)
6NL8 2019-01-23 1.5 Crystal structure of de novo designed metal-controlled dimer of mutant B1 immunoglobulin-binding domain of Streptococcal Protein G (L12H, T16L, V29H, Y33H, N37L)-zinc
5BMH 2016-04-06 1.6 Nitroxide Spin Labels in Protein GB1: T44 Mutant, Crystal Form B
1PGX 1992-07-15 1.66 THE 1.66 ANGSTROMS X-RAY STRUCTURE OF THE B2 IMMUNOGLOBULIN-BINDING DOMAIN OF STREPTOCOCCAL PROTEIN G AND COMPARISON TO THE NMR STRUCTURE OF THE B1 DOMAIN
6NL9 2019-01-23 1.7 Crystal structure of de novo designed metal-controlled dimer of mutant B1 immunoglobulin-binding domain of Streptococcal Protein G (L12H, T16L, V29H, Y33H, N37L)-apo
2ONQ 2008-01-08 1.7 Gbeta1 stabilization by in vitro evolution and computational design
4OZB 2014-07-16 1.8 Backbone Modifications in the Protein GB1 Helix: beta-ACPC24, beta-3-Lys28, beta-3-Lys31, beta-ACPC35
5HG2 2016-02-24 1.8 Backbone Modifications in the Protein GB1 Helix: beta-3-Ala24, beta-3-Lys28, beta-3-Lys31, beta-2-Asn35
1PGB 1994-04-30 1.92 TWO CRYSTAL STRUCTURES OF THE B1 IMMUNOGLOBULIN-BINDING DOMAIN OF STREPTOCCOCAL PROTEIN G AND COMPARISON WITH NMR
1QKZ 2000-02-06 1.95 Fab fragment (MN14C11.6) in complex with a peptide antigen derived from Neisseria meningitidis P1.7 serosubtype antigen and domain II from Streptococcal protein G
4KGS 2013-09-04 1.95 Backbone Modifications in the Protein GB1 Loops: beta-3-Val21, beta-3-Asp40
5HFY 2016-02-24 1.95 Backbone Modifications in the Protein GB1 Helix: beta-2-Ala24, beta-3-Lys28, beta-3-Lys31, beta-3-Asn35
4KGR 2013-09-04 2.0 Backbone Modifications in the Protein GB1 Helix: beta-3-Ala24, beta-3-Lys28, beta-3-Lys31, beta-3-Asn35
3V3X 2012-08-29 2.0 Nitroxide Spin Labels in Protein GB1: N8/K28 Double Mutant
4KGT 2013-09-04 2.0 Backbone Modifications in the Protein GB1 Turns: Aib10, D-Pro47
1EM7 2002-05-08 2.0 HELIX VARIANT OF THE B1 DOMAIN FROM STREPTOCOCCAL PROTEIN G
1PGA 1994-04-30 2.07 TWO CRYSTAL STRUCTURES OF THE B1 IMMUNOGLOBULIN-BINDING DOMAIN OF STREPTOCOCCAL PROTEIN G AND COMPARISON WITH NMR
5HI1 2016-02-24 2.15 Backbone Modifications in the Protein GB1 Helix: Aib24, beta-3-Lys28, beta-3-Lys31, Aib35
4OZA 2014-07-16 2.2 Backbone Modifications in the Protein GB1 Helix: beta-3-Ala24, beta-3-Lys28, beta-3-Gln32, beta-3-Asp36
4WH4 2015-08-05 2.2 Protein GB1 Quadruple Mutant I6H/N8H/K28H/Q32H
1UWX 2005-06-15 2.2 P1.2 serosubtype antigen derived from N. meningitidis PorA in complex with Fab fragment
5BMG 2016-04-06 2.2 Nitroxide Spin Labels in Protein GB1: E15 Mutant
4OZC 2014-07-16 2.3 Backbone Modifications in the Protein GB1 Helix and Loops: beta-ACPC21, beta-ACPC24, beta-3-Lys28, beta-3-Lys31, beta-ACPC35, beta-ACPC40
6NLB 2019-01-23 2.3 Crystal structure of de novo designed metal-controlled dimer of mutant B1 immunoglobulin-binding domain of Streptococcal Protein G (L12H, E15V, T16L, T18I, V29H, Y33H, N37L)-apo
5BMI 2016-04-06 2.5 Nitroxide Spin Labels in Protein GB1: T44 Mutant, Crystal Form A
1MVK 2002-10-30 2.5 X-ray structure of the tetrameric mutant of the B1 domain of streptococcal protein G
1IGC 1995-06-03 2.6 IGG1 FAB FRAGMENT (MOPC21) COMPLEX WITH DOMAIN III OF PROTEIN G FROM STREPTOCOCCUS
3UI3 2012-02-08 2.8 Structural and Biochemical Characterization of HP0315 from Helicobacter pylori as a VapD Protein with an Endoribonuclease Activity
1FCC 1995-04-20 3.2 CRYSTAL STRUCTURE OF THE C2 FRAGMENT OF STREPTOCOCCAL PROTEIN G IN COMPLEX WITH THE FC DOMAIN OF HUMAN IGG
5LDE 2017-10-04 3.38 Crystal structure of a vFLIP-IKKgamma stapled peptide dimer

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Immunoglobulin G-binding protein G P19909 SPG2_STRSG
100.0 Immunoglobulin G-binding protein G P06654 SPG1_STRSG