Effects of tryptophan to phenylalanine substitutions on the structure, stability, and enzyme activity of the IIAB(Man) subunit of the mannose transporter of Escherichia coli.


Abstract

The hydrophilic subunit of the mannose transporter (IIAB(Man)) of Escherichia coli is a homodimer that contains four tryptophans per monomer, three in the N-terminal domain (Trp12, Trp33, and Trp69) and one in the C-terminal domain (Trp182). Single and double Trp-Phe mutants of IIABMan and of the IIA domain were produced. Fluorescence emission studies revealed that Trp33 and Trp12 are the major fluorescence emitters, Trp69 is strongly quenched in the native protein and Trp182 strongly blue shifted, indicative of a hydrophobic environment. Stabilities of the Trp mutants of dimeric IIA(Man) and IIAB(Man) were estimated from midpoints of the GdmHCl-induced unfolding transitions and from the amount of dimers that resisted dissociation by SDS (sodium dodecyl sulfate), respectively. W12F exhibited increased stability, but only 6% of the wild-type phosphotransferase activity, whereas W33F was marginally and W69F significantly destabilized, but fully active. Second site mutations W33F and W69F in the background of the W12F mutation reduced protein stability and suppressed the functional defect of W12F. These results suggest that flexibility is required for the adjustments of protein-protein contacts necessary for the phosphoryltransfer between the phosphorylcarrier protein HPr, IIA(Man), IIB(Man), and the incoming mannose bound to the transmembrane IIC(Man)-IID(Man) complex. Study holds ProTherm entries: 5471, 5472, 5473, 5474, 5475, 5476, 5477 Extra Details: bacterial phosphotransferase system; circular dichroism;,fluorescence; intragenic suppression

Submission Details

ID: sWhvQBcF4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:30 p.m.

Version: 1

Publication Details
Markovic-Housley Z;Stolz B;Lanz R;Erni B,Protein Sci. (1999) Effects of tryptophan to phenylalanine substitutions on the structure, stability, and enzyme activity of the IIAB(Man) subunit of the mannose transporter of Escherichia coli. PMID:10422843
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
2JZH 2008-02-19 structure of IIB domain of the mannose transporter of E. coli
2JZO 2008-02-19 Solution NMR structure of the non-productive complex between IIAMannose and IIBMannose of the mannose transporter of the E. coli phosphotransferase system
1VRC 2005-04-19 Complex of enzyme IIAmannose and the histidine-containing phosphocarrier protein HPr from escherichia coli nmr, restrained regularized mean structure
2JZN 2008-02-19 Solution NMR structure of the productive complex between IIAMannose and IIBMannose of the mannose transporter of the E. coli phosphotransferase system
1VSQ 2008-02-19 Solution NMR structure of the productive complex between IIAMannose and IIBMannose of the mannose transporter of the E. coli phosphotransferase system
1PDO 1996-08-01 1.7 PHOSPHOENOLPYRUVATE-DEPENDENT PHOSPHOTRANSFERASE SYSTEM

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 PTS system mannose-specific EIIAB component P69800 PTNAB_SHIFL
100.0 PTS system mannose-specific EIIAB component P69797 PTNAB_ECOLI
100.0 PTS system mannose-specific EIIAB component P69798 PTNAB_ECOL6
100.0 PTS system mannose-specific EIIAB component P69799 PTNAB_ECO57