Nature of the burst-phase signals of protein folding has been the subject of much debate as to whether the signals represent the formation of early intermediates or the non-specific collapse of unfolded polypeptides. To distinguish the two possibilities, the submillisecond folding dynamics of ribonuclease A (RNase A) was examined, and compared with those of the disulfide bond-ruptured analog of RNase A (r-RNase A). The circular dichroism measurements on RNase A showed the burst-phase signal within 320 micros after the initiation of the folding reaction, which was identical to that observed for r-RNase A. In contrast, the burst phase increase in the extrinsic fluorescence from 1-anilino-8-naphthalene sulfonate (ANS) was observed for RNase A but not for r-RNase A. The kinetic titration experiment of the ANS fluorescence intensity showed the presence of a specific binding site for ANS in the fast-refolding component of RNase A. The small-angle X-ray scattering measurements at approximately 22 ms after initiating the folding reaction demonstrated that the burst phase conformations of the medium and slow-refolding components of RNase A were distinctly smaller than that of r-RNase A. These results indicated the difference in the burst phase conformations of RNase A and r-RNase A. Since r-RNase A is denatured in the physiological solution condition, the burst-phase signal of RNase A was interpreted as the formation of the folding intermediate with specific conformations. Study holds ProTherm entries: 19216, 19217, 19218, 19219 Extra Details: protein folding; burst phase intermediate; collapse; ribonuclease A; ANS fluorescence
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:51 p.m.
|Number of data points||8|
|Proteins||Ribonuclease pancreatic ; Ribonuclease pancreatic|
|Assays/Quantities/Protocols||Experimental Assay: Cm prot_conc:4 mg/ml ; Experimental Assay: m prot_conc:4 mg/ml ; Experimental Assay: Cm prot_conc:100 microM ; Experimental Assay: m prot_conc:100 microM ; Experimental Assay: Cm prot_conc:5 microM ; Experimental Assay: m prot_conc:5 microM|
|Libraries||Mutations for sequence KETAAAKFERQHMDSSTSAASSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASV|