Sequence specific assignment of the proton nuclear magnetic resonance spectrum of barley serine proteinase inhibitor 2

Abstract

The sequence specific assignment of the t H NMR spectrum of barley serine proteinase inhibitor 2 is described. The sequential assignment procedure has been applied to the residues 22 to 83. The 21 residues in the N-terminal part of the structure were shown to be a random coil for which sequential assignment was not immediately possible. For the C-terminal part of the protein starting at Thr-22 and ending with the C-terminal residue Gly-83, sequence specific assignment of all the H s, H ~, Ha', Ha" resonances was achieved and complete sequence specific assignment of the non labile protons of the amino acid residue types of glycine, alanine, threonine, serine, valine, isoleucine, leucine, asparagine, aspartic acid, glutamine, glutamic acid, phenylalanine, tyrosine and tryptophan is reported.

Following co-operative formation of secondary and tertiary structure in a single protein module

“…Anomalous conformational shifts in Thr3 have been observed in smaller fragments (de Prat Gay et al, 1994a;Itzhaki et al, 1995b), which indicate non-random conformations around Trp5. The conformational shifts are different from those of the intact protein (Kjñr et al, 1987; data not shown). There are no residues that are protected against exchange with solvent (see Materials and Methods).…”

“…The H a conformational shifts to random coil are shown in Figure 4(A). On the other hand, the shifts of the majority of residues are very similar to those of the intact protein (Kjñr et al, 1987). Only in two regions do they differ signi®cantly (i.e.…”

“…The main-chain hydrogen bond between Arg46 and Gly64 is absent, because Gly64 is removed. This results in a displacement of the b-sheet around the beginning of b-strand 4, but not a complete disruption of the native-like b-sheet scaffold, as judged by the similarity in confor- (1 ± 63) and the values observed in the intact protein (Kjñr et al, 1987)…”

“…protein is the chemical shift of the same proton but in the intact protein (Kjñr et al, 1987) and d random-coil is the chemical shift for that proton measured in the random-coil models (Wu È thrich, 1986). The È NMR is similar to the general coef®cient of similarity de®ned by Gower (1971) and used in studies of denatured proteins (Shortle & Abeygunawardana, 1993).…”

Hydrogen exchange in chymotrypsin inhibitor 2 probed by mutagenesis

“…Assignment of 1 H-15 N HSQC experiments of wildtype and mutants was carried out by comparison of the observed chemical shifts with the reported assignments (Kjñr et al, 1987) and using our own data (B. D. & A. R. F., unpublished results). The volume integrals of the cross-peaks were calculated using the BRUKER program for each spectrum.…”