Temperature dependence of the longitudinal relaxation rates and exchange rates of the amide protons in peptide substrates of protein kinase

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 CN bond. Despite the various exchange rates of the amide protons, all exhibited similar changes in chemical shift with temperature.