Ultrafast events in the folding of ferrocytochrome c.
Abstract: Laser flash photolysis and stopped-flow methods have been used to study the dynamic events in the micro- to millisecond time bin in the refolding of horse ferrocytochrome c in the full range of guanidine hydrochloride concentration at pH 12.8 (+/-0.1), 22 degrees C. Under the absolute refolding condition, the earliest relaxation time of the unfolded protein chain is less than 1 micros. The chain then undergoes diffusive dynamics-mediated contraction and expansion, in which intrapolypeptide ligands make transient contacts with the heme iron, giving rise to two distinct kinetic phases of approximately 0.4 and approximately 3 micros. Under moderate to absolute refolding conditions, the rates of these processes show little dependence on the denaturant concentration, indicating the absence of structural element in the incipient or the relaxed state. Chain expansion and contraction events continue until the polypeptide finds a stable and supportive transition state. The crossing of this transition barrier, which rate-limits the folding of alkaline ferrocytochrome c, is characterized by a stopped-flow measured time constant of approximately 3 ms in aqueous solvent. Observed kinetics thus implicate no submillisecond folding structure. The folding kinetics is effectively two state in which the unfolded polypeptide first relaxes to an unstructured chain and then crosses over a late rate-limiting barrier to achieve the native conformation. The experimentally observed rates as a function of guanidine hydrochloride concentration have been simulated by numerically calculated microscopic rates of a simple kinetic model that captures the essential features of folding.
Publication Date: 2005-06-29 PubMed ID: 15982002DOI: 10.1021/bi050384bGoogle Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research studied the micro and millisecond dynamics of the refolding process of horse ferrocytochrome c protein using laser flash photolysis and stopped-flow methods at a specific pH and temperature, revealing two main stages in the folding process.
Methodology
- The study employs laser flash photolysis and stopped-flow methods. These techniques enable the observation of extremely rapid chemical reactions or events, such as the unfolding and refolding of proteins.
- The research was conducted on horse ferrocytochrome c under specific conditions (pH 12.8 +/- 0.1, 22 degrees Celsius) and in different guanidine hydrochloride concentrations.
Findings
- The initial relaxation of the unfolded protein chain takes less than a microsecond. This is followed by a contraction and expansion process driven by diffusive dynamics where certain ligands within the protein make temporary contact with the heme iron. This process results in two additional phases in the kinetics of the protein transformation.
- These processes do not change significantly based on the concentration of the denaturing agent, implicating that they do not depend on the presence of a specific structural element in the initial or relaxed states of the protein.
- The protein continues contracting and expanding until finding a stable transition state. The rate-limiting component of the protein folding process, characterized by a stopped-flow measured time constant of approximately 3 milliseconds in an aqueous solvent, is the crossing of this transition barrier.
- The studies suggest no submillisecond folding structure and describe the folding kinetics as effectively a two-state process: the protein relaxes to an unstructured chain and then crosses a rate limiting barrier to acquire its native conformation.
Conclusion
- The observed folding rates in relation to different concentrations of guanidine hydrochloride were simulated using a basic kinetic model. This model effectively encapsulates the primary characteristics of the folding process.
- This study provides new insights into the process and dynamics of protein folding, with potential applications in better understanding diseases related to protein misfolding.
Cite This Article
APA
Kumar R, Prabhu NP, Bhuyan AK.
(2005).
Ultrafast events in the folding of ferrocytochrome c.
Biochemistry, 44(26), 9359-9367.
https://doi.org/10.1021/bi050384b Publication
Researcher Affiliations
- School of Chemistry, University of Hyderabad, Hyderabad 500046, India.
MeSH Terms
- Animals
- Cytochromes c / chemistry
- Cytochromes c / metabolism
- Horses
- Kinetics
- Protein Folding
- Spectrometry, Fluorescence
Citations
This article has been cited 3 times.- Roterman I, Konieczny L, Banach M, Jurkowski W. Intermediates in the protein folding process: a computational model. Int J Mol Sci 2011;12(8):4850-60.
- Kim S, Chung JK, Kwak K, Bowman SE, Bren KL, Bagchi B, Fayer MD. Native and unfolded cytochrome c--comparison of dynamics using 2D-IR vibrational echo spectroscopy. J Phys Chem B 2008 Aug 14;112(32):10054-63.
- Ye M, Zhang QL, Li H, Weng YX, Wang WC, Qiu XG. Infrared spectroscopic discrimination between the loop and alpha-helices and determination of the loop diffusion kinetics by temperature-jump time-resolved infrared spectroscopy for cytochrome c. Biophys J 2007 Oct 15;93(8):2756-66.
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