Tags

Type your tag names separated by a space and hit enter

Multiple kinetic intermediates accumulate during the unfolding of horse cytochrome c in the oxidized state.
Biochemistry. 1998 Jun 23; 37(25):9147-55.B

Abstract

The unfolding kinetics of horse cytochrome c in the oxidized state has been studied at 10, 22, and 34 degreesC as a function of guanidine hydrochloride (GdnHCl) concentration. Rapid (millisecond) measurements of far-UV circular dichroism (CD) as well as fluorescence quenching due to tryptophan to heme excitation energy transfer have been used to monitor the unfolding process. At 10 degreesC, the decrease in far-UV CD signal that accompanies unfolding occurs in two phases. The unobservable burst phase is complete within 4 ms, while the slower phase occurs over tens to hundreds of milliseconds. The burst phase unfolding amplitude increases cooperatively with an increase in GdnHCl concentration, exhibiting a transition midpoint of 3.2 M at 10 degreesC. In contrast, no burst phase change in fluorescence occurs during unfolding at 10 degreesC. At 22 and 34 degreesC, both the fluorescence-monitored unfolding kinetics and the far-UV CD-monitored unfolding kinetics are biphasic. At both temperatures, the two probes yield burst phase unfolding transitions that are noncoincident with respect to the transition midpoints as well as the dependency of the burst phase amplitudes on GdnHCl concentration. The results suggest that at least two kinetic unfolding intermediates accumulate during unfolding. One burst phase intermediate, IU1, has lost virtually all the native-state secondary structure, while the other burst phase intermediate, IU2, has lost both secondary structure and native-like compactness. The presence of kinetic unfolding intermediates is also indicated by the nonlinear dependence of the logarithm of the apparent unfolding rate constant on GdnHCl concentration, which is particularly pronounced at 10 and 22 degreesC. Analysis of the burst phase unfolding transitions obtained using the two probes shows that the stabilities of IU1 and IU2 decrease steadily with an increase in temperature from 10 to 34 degreesC, suggesting that the structures present in them are stabilized principally by hydrogen bonding interactions.

Authors+Show Affiliations

National Centre for Biological Sciences, TIFR Centre, Indian Institute of Science, Bangalore.No affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

9636061

Citation

Bhuyan, A K., and J B. Udgaonkar. "Multiple Kinetic Intermediates Accumulate During the Unfolding of Horse Cytochrome C in the Oxidized State." Biochemistry, vol. 37, no. 25, 1998, pp. 9147-55.
Bhuyan AK, Udgaonkar JB. Multiple kinetic intermediates accumulate during the unfolding of horse cytochrome c in the oxidized state. Biochemistry. 1998;37(25):9147-55.
Bhuyan, A. K., & Udgaonkar, J. B. (1998). Multiple kinetic intermediates accumulate during the unfolding of horse cytochrome c in the oxidized state. Biochemistry, 37(25), 9147-55.
Bhuyan AK, Udgaonkar JB. Multiple Kinetic Intermediates Accumulate During the Unfolding of Horse Cytochrome C in the Oxidized State. Biochemistry. 1998 Jun 23;37(25):9147-55. PubMed PMID: 9636061.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Multiple kinetic intermediates accumulate during the unfolding of horse cytochrome c in the oxidized state. AU - Bhuyan,A K, AU - Udgaonkar,J B, PY - 1998/6/24/pubmed PY - 1998/6/24/medline PY - 1998/6/24/entrez SP - 9147 EP - 55 JF - Biochemistry JO - Biochemistry VL - 37 IS - 25 N2 - The unfolding kinetics of horse cytochrome c in the oxidized state has been studied at 10, 22, and 34 degreesC as a function of guanidine hydrochloride (GdnHCl) concentration. Rapid (millisecond) measurements of far-UV circular dichroism (CD) as well as fluorescence quenching due to tryptophan to heme excitation energy transfer have been used to monitor the unfolding process. At 10 degreesC, the decrease in far-UV CD signal that accompanies unfolding occurs in two phases. The unobservable burst phase is complete within 4 ms, while the slower phase occurs over tens to hundreds of milliseconds. The burst phase unfolding amplitude increases cooperatively with an increase in GdnHCl concentration, exhibiting a transition midpoint of 3.2 M at 10 degreesC. In contrast, no burst phase change in fluorescence occurs during unfolding at 10 degreesC. At 22 and 34 degreesC, both the fluorescence-monitored unfolding kinetics and the far-UV CD-monitored unfolding kinetics are biphasic. At both temperatures, the two probes yield burst phase unfolding transitions that are noncoincident with respect to the transition midpoints as well as the dependency of the burst phase amplitudes on GdnHCl concentration. The results suggest that at least two kinetic unfolding intermediates accumulate during unfolding. One burst phase intermediate, IU1, has lost virtually all the native-state secondary structure, while the other burst phase intermediate, IU2, has lost both secondary structure and native-like compactness. The presence of kinetic unfolding intermediates is also indicated by the nonlinear dependence of the logarithm of the apparent unfolding rate constant on GdnHCl concentration, which is particularly pronounced at 10 and 22 degreesC. Analysis of the burst phase unfolding transitions obtained using the two probes shows that the stabilities of IU1 and IU2 decrease steadily with an increase in temperature from 10 to 34 degreesC, suggesting that the structures present in them are stabilized principally by hydrogen bonding interactions. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/9636061/Multiple_kinetic_intermediates_accumulate_during_the_unfolding_of_horse_cytochrome_c_in_the_oxidized_state_ DB - PRIME DP - Unbound Medicine ER -