Abstract
The longitudinal relaxation rates ( 1 T 1 app ) of the amide protons of two peptide substrates for protein kinase, Arg-Arg-Ala-Ser-Leu and Leu-Arg-Arg-Ala-Ser-Leu-Gly, were measured as a function of temperature. Exchange rates of these protons with water ( 1 τ exH 2 O ) were also measured, using the saturation-transfer technique. At all temperatures, ( 1 T 1 app ) was found to be the sum of two components: 1 τ exH 2 O and 1 T 1 , the intrinsic relaxation rate. The NH protons of carboxy-terminal residues exchanged >10-fold slower than those of internal residues, and this behavior was duplicated by the NH protons of N-acetyl-serine and N-acetyl-serine amide, respectively. A 50-fold variation of the exchange rates at 25° was detected among the internal residues, with a trend toward progressively slower exchange at positions closer to the carboxy terminus. This trend systematically deviated from rates predicted by studies with model dipeptides ( R. S. Molday, S. W. Englander, and R. G. Kallen, Biochemistry 11, 150 (1972) ). While the ΔH ‡ values ranged from 16 to 19 kcal/mol, differences in rates of exchange were due primarily to unequal entropy barriers. The NH 2 protons of N-acetyl-serine amide exchanged both with water and with each other. For these protons, 1 T 1 app could be adequately described as the sum of three components: 1 T 1 , 1 τ exH 2 O , and 1 τ exNH 2 , the rate of cross-exchange which reflects the rate of intramolecular rotation about the amide CN bond. Despite the various exchange rates of the amide protons, all exhibited similar changes in chemical shift with temperature.
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