Core formation in apomyoglobin

Probing the upper reaches of the folding energy landscape

M. Gulotta, R. Gilmanshin, T. C. Buscher, Robert Callender, R. B. Dyer

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

An acid-destabilized form of apomyoglobin, the so-called E state, consists of a set of heterogeneous structures that are all characterized by a stable hydrophobic core composed of 30-40 residues at the intersection of the A, G, and H helices of the protein, with little other secondary structure and no other tertiary structure. Relaxation kinetics studies were carried out to characterize the dynamics of core melting and formation in this protein. The unfolding and/or refolding response is induced by a laser-induced temperature jump between the folded and unfolded forms of E, and structural changes are monitored using the infrared amide I′ absorbance at 1648-1651 cm-1 that reports on the formation of solvent-protected, native-like helix in the core and by fluorescence emission changes from apomyoglobin's Trp14, a measure of burial of the indole group of this residue. The fluorescence kinetics data are monoexponential with a relaxation time of 14 μs. However, infrared kinetics data are best fit to a biexponential function with relaxation times of 14 and 59 μs. These relaxation times are very fast, close to the limits placed on folding reactions by diffusion. The 14 μs relaxation time is weakly temperature dependent and thus represents a pathway that is energetically downhill. The appearance of this relaxation time in both the fluorescence and infrared measurements indicates that this folding event proceeds by a concomitant formation of compact secondary and tertiary structures. The 59 μs relaxation time is much more strongly temperature dependent and has no fluorescence counterpart, indicating an activated process with a large energy barrier wherein nonspecific hydrophobic interactions between helix A and the G and H helices cause some helix burial but Trp14 remains solvent exposed. These results are best fit by a multiple-pathway kinetic model when U collapses to form the various folded core structures of E. Thus, the results suggest very robust dynamics for core formation involving multiple folding pathways and provide significant insight into the primary processes of protein folding.

Original languageEnglish (US)
Pages (from-to)5137-5143
Number of pages7
JournalBiochemistry
Volume40
Issue number17
StatePublished - May 1 2001

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Relaxation time
Fluorescence
Burial
Kinetics
Infrared radiation
Temperature
Protein folding
Energy barriers
Protein Folding
Hydrophobic and Hydrophilic Interactions
Amides
Freezing
apomyoglobin
Proteins
Melting
Lasers
Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Gulotta, M., Gilmanshin, R., Buscher, T. C., Callender, R., & Dyer, R. B. (2001). Core formation in apomyoglobin: Probing the upper reaches of the folding energy landscape. Biochemistry, 40(17), 5137-5143.

Core formation in apomyoglobin : Probing the upper reaches of the folding energy landscape. / Gulotta, M.; Gilmanshin, R.; Buscher, T. C.; Callender, Robert; Dyer, R. B.

In: Biochemistry, Vol. 40, No. 17, 01.05.2001, p. 5137-5143.

Research output: Contribution to journalArticle

Gulotta, M, Gilmanshin, R, Buscher, TC, Callender, R & Dyer, RB 2001, 'Core formation in apomyoglobin: Probing the upper reaches of the folding energy landscape', Biochemistry, vol. 40, no. 17, pp. 5137-5143.
Gulotta M, Gilmanshin R, Buscher TC, Callender R, Dyer RB. Core formation in apomyoglobin: Probing the upper reaches of the folding energy landscape. Biochemistry. 2001 May 1;40(17):5137-5143.
Gulotta, M. ; Gilmanshin, R. ; Buscher, T. C. ; Callender, Robert ; Dyer, R. B. / Core formation in apomyoglobin : Probing the upper reaches of the folding energy landscape. In: Biochemistry. 2001 ; Vol. 40, No. 17. pp. 5137-5143.
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