Proteolysis as a probe of thermal unfolding of cytochrome c

Leyu Wang, Robert X. Chen, Neville R. Kallenbach

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)435-441
Number of pages7
JournalProteins: Structure, Function and Genetics
Volume30
Issue number4
DOIs
StatePublished - Mar 1 1998

Fingerprint

Proteolysis
Cytochromes c
Digestion
Hot Temperature
Endopeptidase K
Protein Unfolding
Heme
Hydrogen
Proteins
Peptide Hydrolases
Temperature
Conformations
Population
Experiments
CD 222

Keywords

  • Cytochrome c
  • Proteinase K
  • Proteolysis
  • Thermal unfolding
  • Thermolysin

ASJC Scopus subject areas

  • Genetics
  • Structural Biology
  • Biochemistry

Cite this

Proteolysis as a probe of thermal unfolding of cytochrome c. / Wang, Leyu; Chen, Robert X.; Kallenbach, Neville R.

In: Proteins: Structure, Function and Genetics, Vol. 30, No. 4, 01.03.1998, p. 435-441.

Research output: Contribution to journalArticle

Wang, Leyu ; Chen, Robert X. ; Kallenbach, Neville R. / Proteolysis as a probe of thermal unfolding of cytochrome c. In: Proteins: Structure, Function and Genetics. 1998 ; Vol. 30, No. 4. pp. 435-441.
@article{ffdcc4d828dd4d8db44a588ace885e94,
title = "Proteolysis as a probe of thermal unfolding of cytochrome c",
abstract = "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.",
keywords = "Cytochrome c, Proteinase K, Proteolysis, Thermal unfolding, Thermolysin",
author = "Leyu Wang and Chen, {Robert X.} and Kallenbach, {Neville R.}",
year = "1998",
month = "3",
day = "1",
doi = "10.1002/(SICI)1097-0134(19980301)30:4<435::AID-PROT10>3.0.CO;2-J",
language = "English (US)",
volume = "30",
pages = "435--441",
journal = "Proteins: Structure, Function and Genetics",
issn = "0887-3585",
publisher = "Wiley-Liss Inc.",
number = "4",

}

TY - JOUR

T1 - Proteolysis as a probe of thermal unfolding of cytochrome c

AU - Wang, Leyu

AU - Chen, Robert X.

AU - Kallenbach, Neville R.

PY - 1998/3/1

Y1 - 1998/3/1

N2 - 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.

AB - 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.

KW - Cytochrome c

KW - Proteinase K

KW - Proteolysis

KW - Thermal unfolding

KW - Thermolysin

UR - http://www.scopus.com/inward/record.url?scp=0032031294&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032031294&partnerID=8YFLogxK

U2 - 10.1002/(SICI)1097-0134(19980301)30:4<435::AID-PROT10>3.0.CO;2-J

DO - 10.1002/(SICI)1097-0134(19980301)30:4<435::AID-PROT10>3.0.CO;2-J

M3 - Article

VL - 30

SP - 435

EP - 441

JO - Proteins: Structure, Function and Genetics

JF - Proteins: Structure, Function and Genetics

SN - 0887-3585

IS - 4

ER -