The Escherichia coli OmpF (outer-membrane protein F; matrix porin) is a homotrimeric beta-barrel and a member of the bacterial porin superfamily. It is the best characterized porin protein, but has resisted attempts to refold it efficiently in vitro. In the present paper, we report the discovery of detergent-based folding conditions, including dodecylglucoside, which can create pure samples of trimeric OmpF. Whereas outer membrane LPS (lipopolysaccharide) is clearly required for in vivo folding, the artificially refolded and LPS-free trimer has properties identical with those of the outer-membrane-derived form. Thus LPS is not required either for in vitro folding or for structural integrity. Dimeric forms of OmpF have been observed in vivo and are proposed to be folding intermediates. In vitro, dimers occur transiently in refolding of trimeric OmpF and, in the presence of dodecylmaltoside, pure dimer can be prepared. This form has less beta-structure by CD and shows lower thermal stability than the trimer. Study of these proteins at the single-molecule level is possible because each OmpF subunit forms a distinct ion channel. Whereas each trimer contains three channels of equal conductance, each dimer always contains two distinct channel sizes. This provides clear evidence that the two otherwise identical monomers adopt different structures in the dimer and indicates that the asymmetric interaction, characteristic of C3 symmetry, is formed at the dimer stage. This asymmetric dimer may be generally relevant to the folding of oligomeric proteins with odd numbers of subunits such as aspartate transcarbamoylase.

The intrinsically disordered translocation domain (T-domain) of the protein antibiotic colicin N binds to periplasmic receptors of target Escherichia coli cells in order to penetrate their inner membranes. We report here that the specific 27 consecutive residues of the T-domain of colicin N known to bind to the helper protein TolA in target cells also interacts intramolecularly with folded regions of colicin N. We suggest that this specific self-recognition helps intrinsically disordered domains to bury their hydrophobic recognition motifs and protect them against degradation, showing that an impaired self-recognition leads to increased protease susceptibility.

Caf1, a chaperone-usher protein from Yersinia pestis, is a major protective antigen in the development of subunit vaccines against plague. However, recombinant Caf1 forms polymers of indeterminate size. We report the conversion of Caf1 from a polymer to a monomer by circular permutation of the gene. Biophysical evaluation confirmed that the engineered Caf1 was a folded monomer. We compared the immunogenicity of the engineered monomer with polymeric Caf1 in antigen presentation assays to CD4 T-cell hybridomas in vitro, as well as in the induction of antibody responses and protection against subcutaneous challenge with Y. pestis in vivo. In C57BL/6 mice, for which the major H-2 b -restricted immunodominant CD4 T-cell epitopes were intact in the engineered monomer, immunogenicity and protective efficacy were preserved, although antibody titers were decreased 10-fold. Disruption of an H-2 d -restricted immunodominant CD4 T-cell epitope during circular permutation resulted in a compromised T-cell response, a low postvaccination antibody titer, and a lack of protection of BALB/c mice. The use of circular permutation in vaccine design has not been reported previously.

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We present a rapid method for protein tertiary structure analysis which avoids the need for techniques such as circular dichroism and differential scanning calorimetry. Small changes to a protein's noncovalent "soft" structure are detected by exploiting differences in thermal stability and fluorescent reporter binding. It can detect subtle stability differences using micrograms of protein in 2 microL volumes within minutes.