Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 403, Issue 4, Pages 495-504Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2010.09.004
Keywords
denatured states; protein folding; loop formation; residual structure; chain stiffness
Categories
Funding
- National Institute of General Medical Sciences [GM074750-04S1]
- University of Montana
- MT NSF EPSCoR [EPS-0701906]
- [GM074750]
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Histidine-heme loop formation in the denatured state of a protein is a sensitive means for probing residual structure under unfolding conditions. In this study, we use a host-guest approach to investigate the relative tendencies of different amino acids to promote residual structure under denaturing conditions. The host for this work is a 6-amino-acid insert of five alanines, followed by a lysine engineered immediately following a unique histidine near the N-terminus of yeast iso-1-cytochrome c. We substitute the fourth alanine in this sequence HAAAXAK (with X = Trp, Phe, Tyr, and Leu). The effects of proline are tested with substitutions at positions 1 and 5 in the insert (HPAAAAK and HAAAAPK, respectively). Thermodynamic studies on His-heme loop formation in 3 M guanidine hydrochloride reveal significant stabilization of residual structure by aromatic amino acids, particularly Trp and Phe, and minimal stabilization of residual structure by Leu. Prolines slightly disfavor His-heme loop formation, presumably due to enhanced chain stiffness. Kinetic studies reveal that much of the change in His-heme loop stability for the aromatic amino acids is caused by a slowdown in the rate of His-heme loop breakage, indicating that residual structure is preferentially stabilized in the closed-loop form of the denatured state. (C) 2010 Elsevier Ltd. All rights reserved.
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