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Proteolysis as a probe of thermal unfolding of cytochrome C
Author(s) -
Wang Leyu,
Chen Robert X.,
Kallenbach Neville R.
Publication year - 1998
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/(sici)1097-0134(19980301)30:4<435::aid-prot10>3.0.co;2-j
Subject(s) - chemistry , proteolysis , heme , absorbance , cleavage (geology) , circular dichroism , native state , cytochrome c , population , proteinase k , protein structure , protein secondary structure , crystallography , biophysics , enzyme , biochemistry , chromatography , biology , paleontology , demography , mitochondrion , sociology , fracture (geology)
Recent hydrogen exchange experiments on native cytochrome c implicate a sequential unfolding pathway in contrast to a simple two‐state process. We have studied the heat‐induced unfolding of this protein by using spectroscopic measurements to detect changes in conformation and proteolytic enzyme digestion to identify regions of the protein that are labile. Several spectroscopic profiles were monitored: CD at 222 nm, a measurement of secondary structure change in the protein, the absorbance at 280 nm, involving the local environment of Trp 59, and absorbance at 420 nm, the Soret band of the heme. The apparent T m values for these probes differ, consistent with an unfolding pathway containing intermediates. The limited digestion by proteinase K is consistent with population of an intermediate state in unfolding. We find a single strong region of cleavage at low temperature with retention of structure in each fragment. Proteins 30:435–441, 1998. © 1998 Wiley‐Liss, Inc.