Studies of Early Events of Folding of a Predominately
β‑Sheet Protein Using Fluorescence Correlation Spectroscopy
and Other Biophysical Methods
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Abstract
The
interplay between the early collapse of the unfolded state
and the formation of the secondary structure has been the subject
of extensive research in protein chemistry. In this study, we used
the intestinal fatty acid binding protein (IFABP), a small model protein
with predominately β-sheet structure, to study the early events,
including the early chain collapse and the formation of the secondary
structure. We used a combination of fluorescence correlation spectroscopy
and far-UV circular dichroism (CD) to understand how these early processes
influence the late folding events like the stabilization of the secondary
structure and aggregation. Acid-induced unfolded IFABP was found to
collapse in the presence of low concentrations of added salt and aggregate
at higher concentrations. Both the formation of the collapsed state
and aggregation were conveniently probed by fluorescence correlation
spectroscopy, a sensitive fluorescence technique with single-molecule
resolution. In contrast, the formation of the secondary structure
was monitored by far-UV CD. The results suggested that backbone hydrogen
bond formation, not only the overall hydrophobicity of IFABP, may
play crucial roles in the early collapse. Two mutant proteins positioned
at a crucial nucleating site, namely, G80V and L64G, although being
opposite in their overall hydrophobicity,
collapsed relatively rapidly compared to the wild-type protein. The
interconnection among the early collapse, the formation of the secondary
structure, and aggregation was similar for these two mutants. Another
mutant, G44V, which was identical in its overall hydrophobicity to
G80V
but situated in a region distant from the hydrophobic core, was
found to be very different from G80V and L64G