PubMed · 12904651
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Michael A Quail. 2003. High-throughput plasmid extraction using microtiter plates.. https://doi.org/10.1385/1-59259-409-3%3A89
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The amino-terminal domains N1 and N2 of the gene-3-protein of phage fd form a bilobal structural and functional entity that protrudes from the phage tip. Domain N2 initiates the infection of Escherichia coli by binding to the F pilus. This binding results in the dissociation of the two domains and allows N1 to interact with the TolA receptor at the cell surface. The refolding of the N1-N2 fragment begins with the folding of domain N1, which takes a few milliseconds, followed by the folding of domain N2, which is complete within five minutes. The subsequent domain assembly is unusually slow and shows a time-constant of 6200 s at 25 degrees C. We found that the rate of this reaction is controlled by the trans to cis isomerization of the Gln212-Pro213 bond in the hinge subdomain of N2, a region that provides many interactions between N1 and N2 in the gene-3-protein. The substitution of Pro213 by Gly accelerated domain association 30-fold and revealed that the folding of the two individual domains and their assembly are indeed sequential steps in the refolding of the gene-3-protein. In the course of infection, the domains must separate to expose the binding site for TolA on domain N1. The kinetic block of domain reassembly caused by Pro213 isomerization could ensure that after the initial binding of N2 to the F pilus the open state persists until N1 and TolA are close enough for their mutual interaction. Pro213 isomerization might thus serve as a slow conformational switch in the function of the gene-3-protein.
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.
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