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Human type-alpha transforming growth factor (hTGF alpha) is a small mitogenic
protein containing 50 amino acids and three disulfide bonds. It has both
sequence and structural homology with epidermal growth factor (EGF). While the
three-dimensional structures of hTGF alpha and other EGF-like proteins have been
studied extensively, relatively little is known about conformational dynamics of
these molecules. In this paper we describe nuclear relaxation measurements which
probe the molecular dynamics of hTGF alpha in aqueous solution at neutral pH. In
order to characterize conformational dynamics of hTGF alpha on both the fast
(i.e., sub-nanosecond) and intermediate nitrogen-15 chemical-exchange (i.e.,
microsecond) time scales, we measured nitrogen-15 relaxation parameters at pH
7.1 +/- 0.1 and a temperature of 30 +/- 0.5 degrees C. Measurements of
nitrogen-15 longitudinal (R1) and transverse (R2) relaxation rates, and 1H-15N
heteronuclear NOE effects, were then interpreted using an extended Lipari-Szabo
analysis [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559;
Clore, G. M., Szabo, A., Bax, A., Kay, L. E., Driscoll, P. C., & Gronenborn,
A. M. (1990) J. Am. Chem. Soc. 112, 4989-4991] to provide estimates of the
locations and amplitudes of fast internal motions and the locations of
nitrogen-15 chemical-exchange line broadening. These results demonstrate that,
under conditions of pH and temperature at which it is tightly bound by the EGF
receptor, hTGF alpha is a highly dynamic molecule. Indeed, some 40% of the
backbone amide groups of hTGF alpha, including many at the interface between the
two subdomains, exhibit significant nitrogen-15 chemical-exchange line
broadening indicative of interconversions between multiple protein conformations
on the microsecond time scale. The distribution of these sites on the
three-dimensional protein structure suggests that these dynamic fluctuations are
due to (i) partial unfolding of the core beta-sheet, (ii) hinge-bending motions
between the N- and C-terminal subdomains, and/or (iii) disulfide bond
isomerization in the solution structure of hTGF alpha at neutral pH.
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