No abstract

The use of PPII helices in protein design is currently hindered by limitations in our understanding of their conformational stability and folding. Recent studies of the snow flea antifreeze protein (sfAFP), a useful model system composed of six PPII helices, suggested that a low denatured state entropy contributes to folding thermodynamics. To get atomic level information on the conformational ensemble and entropy of the reduced denatured state of sfAFP, we have analyzed its chemical shifts and {1H}-15N relaxation parameters by NMR spectroscopy at three experimental conditions. No significant populations of preferred secondary structure were detected. The stiffening of certain N-terminal residues at neutral versus acidic pH leads us to suggest that favorable charge-charge interactions could bias the conformational ensemble to favor the formation of the two disulfide bonds during nascent folding. Despite a high content of flexible glycine residues, the mobility of the sfAFP denatured ensemble is similar for denatured α/β proteins both on fast ps/ns as well as slower μs/ms timescales. These results are in line with a conformational entropy in the denatured ensemble resembling that of typical proteins and suggest that new structures based on PPII helical bundles should be amenable to protein design.