The folding mechanism of a two-domain protein: folding kinetics and domain docking of the gene-3 protein of phage fd.


Abstract

The gene-3 protein (G3P) of filamentous phages is essential for the infection of Escherichia coli. The carboxy-terminal domain anchors this protein in the phage coat, whereas the two amino-terminal domains N1 and N2 protrude from the phage surface. We analyzed the folding mechanism of the two-domain fragment N1-N2 of G3P (G3P(*)) and the interplay between folding and domain assembly. For this analysis, a variant of G3P(*) was used that contained four stabilizing mutations (IIHY-G3P(*)). The observed refolding kinetics extend from 10 ms to several hours. Domain N1 refolds very rapidly (with a time constant of 9.4 ms at 0.5 M guanidinium chloride, 25 degrees C) both as a part of IIHY-G3P(*) and as an isolated protein fragment. The refolding of domain N2 is slower and involves two reactions with time constants of seven seconds and 42 seconds. These folding reactions of the individual domains are followed by a very slow, spectroscopically silent docking process, which shows a time constant of 6200 seconds. This reaction was detected by a kinetic unfolding assay for native molecules. Before docking, N1 and N2 unfold fast and independently, after docking they unfold slowly in a correlated fashion. A high energy barrier is thus created by domain docking, which protects G3P kinetically against unfolding. The slow domain docking is possibly important for the infection of E.coli by the phage. Upon binding to the F pilus, the N2 domain separates from N1 and the binding site for TolA on domain N1 is exposed. Since domain reassembly is so slow, this binding site remains accessible until pilus retraction has brought N1 close to TolA on the bacterial surface. Study holds ProTherm entries: 16098, 16099, 16100 Extra Details: N2 domain. IIHF-G3P* contains four Trp and 15 Tyr residues, and three Trp residues are in the N1 domain, and only one. W181, is in N2 at a solvent exposed position. Twelve Tyr residues are located within domain N2.,The denaturation process were observed by the fluorescence at 310 nm after excitation at 280 nm (to observe Tyr) to follow N2 domain. protein folding; folding kinetics; gene-3 protein; domain docking; pilus

Submission Details

ID: iAgjydeF

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:47 p.m.

Version: 1

Publication Details
Martin A;Schmid FX,J. Mol. Biol. (2003) The folding mechanism of a two-domain protein: folding kinetics and domain docking of the gene-3 protein of phage fd. PMID:12767837
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
1FGP 1997-06-16 MEMBRANE PENETRATION DOMAIN OF THE MINOR COAT PROTEIN G3P OF PHAGE FD, NMR, 15 STRUCTURES
1G3P 1998-01-28 1.46 CRYSTAL STRUCTURE OF THE N-TERMINAL DOMAINS OF BACTERIOPHAGE MINOR COAT PROTEIN G3P
2G3P 1999-07-19 1.9 STRUCTURE OF THE N-TERMINAL TWO DOMAINS OF THE INFECTIVITY PROTEIN G3P OF FILAMENTOUS PHAGE FD
3DGS 2008-07-22 1.9 Changing the determinants of protein stability from covalent to non-covalent interactions by in-vitro evolution: a structural and energetic analysis
3KNQ 2009-11-24 2.13 Beta Turn Optimization of the Gene-3-Protein of Filamentous Phage Fd

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Attachment protein G3P P69168 G3P_BPM13
100.0 Attachment protein G3P P03661 G3P_BPFD
100.0 Attachment protein G3P P69169 G3P_BPF1