Robustness and evolvability in the functional anatomy of a PER-ARNT-SIM (PAS) domain.


Abstract

The robustness of proteins against point mutations implies that only a small subset of residues determines functional properties. We test this prediction using photoactive yellow protein (PYP), a 125-residue prototype of the PER-ARNT-SIM (PAS) domain superfamily of signaling proteins. PAS domains are defined by a small number of conserved residues of unknown function. We report high-throughput biophysical measurements on a complete Ala scan set of purified PYP mutants. The dataset of 1,193 values on active site properties, functional kinetics, stability, and production level reveals that 124 mutants retain the characteristic photocycle of PYP, but that the majority of substitutions significantly alter functional properties. Only 35% of substitutions that strongly affect function are located at the active site. Unexpectedly, most PAS-conserved residues are required for maintaining protein production. PAS domain activation often involves conformational changes in α-helices linked to the PAS core. However, the mechanism of transmission and kinetic regulation of allosteric structural changes from the PAS domain to these helices is not clear. The Ala scan data reveal interactions governing allosteric switching in PYP. The photocycle kinetics is significantly altered by substitutions at 58 positions and spans a 3,000-fold range. Nine residues that dock the N-terminal α-helices of PYP to its PAS core regulate signaling kinetics. Ile39 and Asn43 are identified as part of a mechanism for regulating allosteric switching that is conserved among PAS domains. These results show that PYP combines robustness with a high degree of evolvability and imply production level as an important factor in protein evolution.

Submission Details

ID: GdG9j3sb

Submitter: Shu-Ching Ou

Submission Date: Aug. 1, 2018, 3:27 p.m.

Version: 1

Publication Details
Philip AF;Kumauchi M;Hoff WD,Proc Natl Acad Sci U S A (2010) Robustness and evolvability in the functional anatomy of a PER-ARNT-SIM (PAS) domain. PMID:20889915
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
2KX6 2011-06-15 Signaling state of Photoactive Yellow Protein
1XFN 2005-08-16 NMR structure of the ground state of the photoactive yellow protein lacking the N-terminal part
1XFQ 2005-08-16 structure of the blue shifted intermediate state of the photoactive yellow protein lacking the N-terminal part
3PHY 1998-05-27 PHOTOACTIVE YELLOW PROTEIN, DARK STATE (UNBLEACHED), SOLUTION STRUCTURE, NMR, 26 STRUCTURES
1NWZ 2003-03-11 0.82 PYP Ultra-high resolution structure of a Bacterial Photoreceptor
3PYP 1999-06-01 0.85 PHOTOACTIVE YELLOW PROTEIN, CRYOTRAPPED EARLY LIGHT CYCLE INTERMEDIATE
1OT6 2004-05-11 0.95 CRYOTRAPPED CRYSTAL STRUCTURE OF THE E46Q MUTANT OF PHOTOACTIVE YELLOW PROTEIN UNDER CONTINUOUS ILLUMINATION AT 110K
1OT9 2004-05-11 1.0 CRYOTRAPPED STATE IN WILD TYPE PHOTOACTIVE YELLOW PROTEIN, INDUCED WITH CONTINUOUS ILLUMINATION AT 110K
2QJ7 2008-05-20 1.05 PYP ultra-high resolution of a bacterial photoreceptor
1OTA 2004-05-11 1.1 E46Q MUTANT OF PHOTOACTIVE YELLOW PROTEIN, P63 AT 295K
1OTB 2004-05-11 1.1 WILD TYPE PHOTOACTIVE YELLOW PROTEIN, P63 AT 295K
1F9I 2000-07-21 1.1 CRYSTAL STRUCTURE OF THE PHOTOACTIVE YELLOW PROTEIN MUTANT Y42F
1ODV 2003-03-18 1.14 Photoactive yellow protein 1-25 deletion mutant
1F98 2000-07-21 1.15 CRYSTAL STRUCTURE OF THE PHOTOACTIVE YELLOW PROTEIN MUTANT T50V
1KOU 2002-04-03 1.16 Crystal Structure of the Photoactive Yellow Protein Reconstituted with Caffeic Acid at 1.16 A Resolution
1UWP 2006-05-10 1.2 Initial Events in the Photocycle of Photoactive Yellow Protein
2QJ5 2008-05-20 1.2 PYP ultra-high resolution of a bacterial photoreceptor
1UWN 2004-03-25 1.2 The Initial Events in the Photocycle of Photoactive Yellow Protein: A Common Mechanism on Light Activation in Photoreceptor Proteins
1UGU 2004-08-10 1.2 Crystal structure of PYP E46Q mutant
6MMD 2019-05-29 1.23 Photoactive Yellow Protein with 3,5-dichlorotyrosine substituted at position 42
1OTD 2004-05-11 1.25 STRONG HYDROGEN BONDS IN PHOTOACTIVE YELLOW PROTEIN AND THEIR ROLE IN ITS PHOTOCYCLE
2ZOH 2009-03-24 1.25 X-ray Crystal Structure of Photoactive Yellow Protein, Wild type, at 295K
2D01 2006-04-04 1.34 Wild Type Photoactive Yellow Protein, P65 Form
6MHI 2019-05-29 1.35 Photoactive Yellow Protein with covalently bound 3,5-dichloro-4-hydroxycinnamic acid chromophore
1D7E 2000-03-31 1.39 CRYSTAL STRUCTURE OF THE P65 CRYSTAL FORM OF PHOTOACTIVE YELLOW PROTEIN
1OTE 2004-05-11 1.4 E46Q MUTANT OF PHOTOACTIVE YELLOW PROTEIN, P65 AT 110K
1OTI 2004-05-11 1.4 E46Q MUTANT OF PHOTOACTIVE YELLOW PROTEIN, P65 AT 295K
2PHY 1995-10-15 1.4 PHOTOACTIVE YELLOW PROTEIN, DARK STATE (UNBLEACHED)
2D02 2006-04-04 1.42 R52Q Mutant of Photoactive Yellow Protein, P65 Form
5GX9 2017-08-30 1.49 PYP mutant - E46Q
2ZOI 2009-03-24 1.5 Neutron Crystal Structure of Photoactive Yellow Protein, Wild type, at 295K
4I3I 2013-03-20 1.6 Structures of IT intermediate of photoactive yellow protein E46Q muntant from time-resolved laue crystallography collected at 14ID APS
5HD3 2016-05-18 1.6 Femtosecond Structural Dynamics Drives the Trans/Cis Isomerization in Photoactive Yellow Protein: Dark structure of photoactive yellow protein
1T19 2005-01-18 1.6 Early intermediate IE2 from time-resolved crystallography of the E46Q mutant of PYP
4BBU 2012-11-14 1.6 The PR2 Photocycle Intermediate of Photoactive Yellow Protein
4I3J 2013-03-20 1.6 Structures of PR1 intermediate of photoactive yellow protein E46Q muntant from time-resolved laue crystallography collected AT 14ID APS
5HD5 2016-05-25 1.6 Femtosecond Structural Dynamics Drives the Trans/Cis Isomerization in Photoactive Yellow Protein: 200 ns time delay photo-activated (light) structure
5HDD 2016-05-25 1.6 Femtosecond Structural Dynamics Drives the Trans/Cis Isomerization in Photoactive Yellow Protein: 800 fs to 1200 fs Structure
4BBV 2012-11-14 1.6 The PB0 Photocycle Intermediate of Photoactive Yellow Protein
1TS0 2005-07-05 1.6 Structure of the pB1 intermediate from time-resolved Laue crystallography
3VE4 2013-03-20 1.6 Structures of ICT and PR1 intermediates from time-resolved laue crystallography
1T1A 2005-01-18 1.6 Late intermediate IL1 from time-resolved crystallography of the E46Q mutant of PYP
4I38 2013-03-20 1.6 Structures of IT intermediates from time-resolved laue crystallography collected at 14ID-B, APS
1T18 2005-01-18 1.6 Early intermediate IE1 from time-resolved crystallography of the E46Q mutant of PYP
1S1Y 2004-06-15 1.6 Photoactivated chromophore conformation in Photoactive Yellow Protein (E46Q mutant) from 10 microseconds to 3 milliseconds
1T1C 2005-01-18 1.6 Late intermediate IL3 from time-resolved crystallography of the E46Q mutant of PYP
4I39 2013-03-20 1.6 Structures of ICT and PR1 intermediates from time-resolved laue crystallography collected at 14ID-B, APS
5HDS 2016-05-25 1.6 Femtosecond Structural Dynamics Drives the Trans/Cis Isomerization in Photoactive Yellow Protein: 3 ps Structure
4WL9 2014-12-17 1.6 Time Resolved Serial Femtosecond Crystallography Captures High Resolution Intermediates of PYP
4B9O 2012-11-14 1.6 The PR0 Photocycle Intermediate of Photoactive Yellow Protein
4I3A 2013-03-20 1.6 Structures of PR1 and PR2 intermediates from time-resolved laue crystallography collected at 14ID-B, APS
4BBT 2012-11-14 1.6 The PR1 Photocycle Intermediate of Photoactive Yellow Protein
4HY8 2013-03-20 1.6 Structures of PR1 and PR2 intermediates from time-resolved laue crystallography
6MKT 2019-05-29 1.6 Photoactive Yellow Protein with 3-chlorotyrosine substituted at position 42
1T1B 2005-01-18 1.6 Late intermediate IL2 from time-resolved crystallography of the E46Q mutant of PYP
5HDC 2016-05-25 1.6 Femtosecond Structural Dynamics Drives the Trans/Cis Isomerization in Photoactive Yellow Protein: 100 fs to 400 fs Structure
4WLA 2014-12-17 1.6 Time Resolved Serial Femtosecond Crystallography Captures High Resolution Intermediates of PYP
1TS8 2005-07-05 1.6 Structure of the pR cis planar intermediate from time-resolved Laue crystallography
3VE3 2013-03-20 1.6 Structure of IT Intermediate from time-resolved laue crystallography
1TS7 2005-07-05 1.6 Structure of the pR cis wobble and pR E46Q intermediates from time-resolved Laue crystallography
1S1Z 2004-06-15 1.6 Photoactivated chromophore conformation in Photoactive Yellow Protein (E46Q mutant) from 10 to 500 nanoseconds
1TS6 2005-07-05 1.6 Structure of the pB2 intermediate from time-resolved Laue crystallography
6MHN 2019-05-29 1.67 Photoactive Yellow Protein with covalently bound 3-chloro-4-hydroxycinnamic acid chromophore
1GSV 2002-02-14 1.75 Crystal structure of the P65 crystal form of photoactive yellow protein G47S mutant
1GSX 2002-02-14 1.79 CRYSTAL STRUCTURE OF THE P65 CRYSTAL FORM OF PHOTOACTIVE YELLOW PROTEIN G47S/G51S MUTANT
3UMD 2012-04-11 1.8 Structure of pB intermediate of Photoactive yellow protein (PYP) at pH 4.
3UME 2012-04-11 1.8 Structure of pB intermediate of Photoactive yellow protein (PYP) at pH 7
1GSW 2002-02-14 1.85 CRYSTAL STRUCTURE OF THE P65 CRYSTAL FORM OF PHOTOACTIVE YELLOW PROTEIN G51S MUTANT
1S4R 2004-04-13 1.9 Structure of a reaction intermediate in the photocycle of PYP extracted by a SVD-driven analysis
1S4S 2004-04-13 1.9 Reaction Intermediate in the Photocycle of PYP, intermediate occupied between 100 micro-seconds to 5 milli-seconds
2PYR 1999-04-06 1.9 PHOTOACTIVE YELLOW PROTEIN, 1 NANOSECOND INTERMEDIATE (287K)
2PYP 1998-04-29 1.9 PHOTOACTIVE YELLOW PROTEIN, PHOTOSTATIONARY STATE, 50% GROUND STATE, 50% BLEACHED
2I9V 2007-02-06 2.2 Structural role of Y98 in PYP: effects on fluorescence, gateway and photocycle recovery
2QWS 2008-03-18 2.5 Neutron and X-ray structural studies of short hydrogen bonds in Photoactive Yellow Protein (PYP)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Photoactive yellow protein P16113 PYP_HALHA