Molecular motions and protein folding: Characterization of the backbone dynamics and folding equilibrium of α2D using 13C NMR spin relaxation Academic Article uri icon

Overview

MeSH Major

  • Hypertension, Pulmonary
  • Infant, Newborn, Diseases
  • Scleroderma, Systemic

abstract

  • De novo protein design enables systematic exploration of the relationship between the amino acid sequences, conformations, and thermodynamics of proteins. The polypeptide α2D is a de novo designed dimeric four-helix bundle with a native-like three-dimensional structure [Hill, R. B.; DeGrado, W. F. J. Am. Chem. Soc. 1998, 120, 1138-1145]. The roles of intramolecular conformational dynamics and folding kinetics in determining the equilibrium properties of α2D have been investigated using novel NMR spin relaxation methods. To facilitate these experiments, the four leucine residues in the α2D monomer were labeled specifically with 13C at the Ca position. Reduced spectral densities [Farrow, N. A.; Zhang, O.; Szabo, A.; Torchia, D. A.; Kay, L. E. J. Biomol. NMR 1995, 6, 153-162] were obtained from spin relaxation data recorded at four static magnetic fields and were interpreted Using the model-free formalism [Lipari, G.; Szabo, A. J. Am. Chem. Soc. 1982, 104, 4546-4559]. Generally, the backbone mobility of α2D is typical of natural proteins. High C(α) order parameters indicate that motions are restricted on the picosecond to nanosecond time scale. Slightly lower order parameters and longer internal correlation times are observed for the most N-terminal and C-terminal sites. Chemical exchange linebroadening is manifest for all leucine 13C(α) spins and results from the folding equilibrium of α2D. The chemical exchange process was characterized using the relaxation-compensated CarrPurcell-Meiboom-Gill experiment [Loria; J. P.; Rance, M.; Palmer, A. G., III. J. Am. Chem. Soc. 1999, 121, 2331-2332]. The folding and unfolding rate constants were measured to be (4.7 ± 0.9) x 106 M-1 S-1 and 15 ± 3 s-1, respectively, and agree with the equilibrium constant for folding of α2D. The 13C(α) chemical shifts for unfolded and folded forms of α2D, obtained from this analysis, indicate that the ensemble of unfolded states includes transiently structured helical conformations. The results both confirm the success of the de novo design strategy and suggest avenues for further improvement of the native-like properties of α2D.

publication date

  • November 29, 2000

Research

keywords

  • Academic Article

Identity

Digital Object Identifier (DOI)

  • 10.1021/ja001129b

Additional Document Info

start page

  • 11610

end page

  • 11619

volume

  • 122

number

  • 47