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Biophysical Journal 84:1909-1918 (2003)
© 2003 The Biophysical Society

Primary Folding Dynamics of Sperm Whale Apomyoglobin: Core Formation

Miriam Gulotta*, Eduard Rogatsky*, Robert H. Callender* and R. Brian Dyer{dagger}

* Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461; and {dagger} Bioscience Division, Mail Stop J586, Los Alamos National Laboratory, Los Alamos, New Mexico 87545

Correspondence: Address reprint requests to Miriam Gulotta, Tel.: 718-430-2437; Fax: 718-430-8565; E-mail: gulotta{at}aecom.yu.edu.

The structure, thermodynamics, and kinetics of heat-induced unfolding of sperm whale apomyoglobin core formation have been studied. The most rudimentary core is formed at pH* 3.0 and up to 60 mM NaCl. Steady state for ultraviolet circular dichroism and fluorescence melting studies indicate that the core in this acid-destabilized state consists of a heterogeneous composition of structures of ~26 residues, two-thirds of the number involved for horse heart apomyoglobin under these conditions. Fluorescence temperature-jump relaxation studies show that there is only one process involved in Trp burial. This occurs in 20 µs for a 7° jump to 52°C, which is close to the limits placed by diffusion on folding reactions. However, infrared temperature jump studies monitoring native helix burial are biexponential with times of 5 µs and 56 µs for a similar temperature jump. Both fluorescence and infrared fast phases are energetically favorable but the slow infrared absorbance phase is highly temperature-dependent, indicating a substantial enthalpic barrier for this process. The kinetics are best understood by a multiple-pathway kinetics model. The rapid phases likely represent direct burial of one or both of the Trp residues and parts of the G- and H-helices. We attribute the slow phase to burial and subsequent rearrangement of a misformed core or to a collapse having a high energy barrier wherein both Trps are solvent-exposed.




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