Rapid mapping of protein functional epitopes by combinatorial alanine scanning.


Abstract

A combinatorial alanine-scanning strategy was used to determine simultaneously the functional contributions of 19 side chains buried at the interface between human growth hormone and the extracellular domain of its receptor. A phage-displayed protein library was constructed in which the 19 side chains were preferentially allowed to vary only as the wild type or alanine. The library pool was subjected to binding selections to isolate functional clones, and DNA sequencing was used to determine the alanine/wild-type ratio at each varied position. This ratio was used to calculate the effect of each alanine substitution as a change in free energy relative to that of wild type. Only seven side chains contribute significantly to the binding interaction, and these conserved residues form a compact cluster in the human growth hormone tertiary structure. The results were in excellent agreement with free energy data previously determined by conventional alanine-scanning mutagenesis and suggest that this technology should be useful for analyzing functional epitopes in proteins.

Submission Details

ID: NqNZBJU9

Submitter: Shu-Ching Ou

Submission Date: Oct. 18, 2018, 4:54 p.m.

Version: 1

Publication Details
Weiss GA;Watanabe CK;Zhong A;Goddard A;Sidhu SS,Proc Natl Acad Sci U S A (2000) Rapid mapping of protein functional epitopes by combinatorial alanine scanning. PMID:10908667
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
5OHD 2018-04-11 Putative inactive (dormant) dimeric state of GHR transmembrane domain
5OEK 2018-04-11 Putative active dimeric state of GHR transmembrane domain
1HUW 1994-01-31 2.0 THE CRYSTAL STRUCTURE OF AFFINITY-MATURED HUMAN GROWTH HORMONE AT 2 ANGSTROMS RESOLUTION
1AXI 1998-01-28 2.1 STRUCTURAL PLASTICITY AT THE HGH:HGHBP INTERFACE
1HWG 1997-11-19 2.5 1:2 COMPLEX OF HUMAN GROWTH HORMONE WITH ITS SOLUBLE BINDING PROTEIN
1HGU 1995-12-07 2.5 HUMAN GROWTH HORMONE
1KF9 2002-11-20 2.6 PHAGE DISPLAY DERIVED VARIANT OF HUMAN GROWTH HORMONE COMPLEXED WITH TWO COPIES OF THE EXTRACELLULAR DOMAIN OF ITS RECEPTOR
1A22 1998-04-29 2.6 HUMAN GROWTH HORMONE BOUND TO SINGLE RECEPTOR
2AEW 2005-11-01 2.7 A model for growth hormone receptor activation based on subunit rotation within a receptor dimer
3HHR 1994-04-30 2.8 HUMAN GROWTH HORMONE AND EXTRACELLULAR DOMAIN OF ITS RECEPTOR: CRYSTAL STRUCTURE OF THE COMPLEX
1HWH 1997-11-19 2.9 1:1 COMPLEX OF HUMAN GROWTH HORMONE MUTANT G120R WITH ITS SOLUBLE BINDING PROTEIN
1BP3 1998-08-19 2.9 THE XRAY STRUCTURE OF A GROWTH HORMONE-PROLACTIN RECEPTOR COMPLEX

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
181.4 B,C Somatotropin Q95ML5 GHR_SAIBB
185.4 B,C Somatotropin P79194 GHR_MACMU
190.2 B,C Somatotropin Q9XSZ1 GHR_PAPAN
200.0 B,C Somatotropin P10912 GHR_HUMAN
90.5 A Somatotropin P58343 SOMA_SAIBB
93.2 A Somatotropin P01242 SOM2_HUMAN
90.5 A Somatotropin Q9GMB3 SOMA_CALJA
93.7 A Somatotropin P58757 SOM2_PANTR
96.8 A Somatotropin P33093 SOMA_MACMU
100.0 A Somatotropin P58756 SOMA_PANTR
100.0 A Somatotropin P01241 SOMA_HUMAN