Chiral β and random fractional deuteration for the determination of protein sidechain conformation by NMR

Abstract

Stereospecific assignments of the aspartic acid and asparagine fl-protons of the 108 residue protein E. coli thioredoxin have been obtained by the use of chiral deuteration. In addition protein samples have been prepared in which all carbon bound hydrogen positions are substituted to an extent of 75% with deuterium. These random fractionally deuterated samples significantly facilitate the measurement of coupling constants and intraresidue NOE intensities which combined with the stereospecific assignments have provided determination of the first sidechain dihedral angle Z1 for all four asparagine residues and eight of the ten assigned aspartic acid residues.

Deuterium labelling in NMR structural analysis of larger proteins

“…The utility of chiral yff-deuteration in conformational analysis is illustrated in the case of E. coli thioredoxin in which aspartic acid and asparagine were appropriately labelled (LeMaster, 1987). Fig.…”

“…Stimulated by this and other findings in the NMR studies, the E. coli thioredoxin structure was subsequently refined to an R factor of 165 % at 168 A (Katti et al 1989). Table 1 is taken in large part from the earlier NMR analysis (LeMaster, 1987). The last column has been introduced to summarize the corresponding X-ray results.…”

Stereospecific assignments of glycine in proteins by stereospecific deuteration and 15N labeling

“…In some cases, these assignments can be determined from an analysis of homonuclear and heteronuclear vicinal coupling constants or from NOE data (Hyberts et al, 1987;Wagner et al, 1987;Arseniev et al, 1988;Nilges et al, 1990;Xu et al, 1992). Alternatively, isotope labeling has been employed to unambiguously obtain stereospecific assignments (Fischman et al, 1980;LeMaster, 1987;LeMaster and Richards, 1988;Neri et al, 1989). For example, the [3-protons of the aspartic acid and asparagine residues in thioredoxin have been stereospecifically assigned from an analysis of a homonuclear COSY spectrum of the protein in which stereospecifically deuterated aspartic acid was incorporated (LeMaster, 1987).…”

“…Alternatively, isotope labeling has been employed to unambiguously obtain stereospecific assignments (Fischman et al, 1980;LeMaster, 1987;LeMaster and Richards, 1988;Neri et al, 1989). For example, the [3-protons of the aspartic acid and asparagine residues in thioredoxin have been stereospecifically assigned from an analysis of a homonuclear COSY spectrum of the protein in which stereospecifically deuterated aspartic acid was incorporated (LeMaster, 1987).…”

Stereospecific assignments of the leucine methyl resonances in the 1H NMR spectrum of Lactobacillus casei dihydrofolate reductase

“…Selective deuteration has frequently been used to identify the 'H signals from a particular amino acid type [l-6] and has also been used to make stereospecific assignments for methylene protons in Gly [7] and Asp/ Asn residues [8]. The present application is the first example of the use of stereoselective deuterium labelling for the assignment of diastereotopic pairs of methyl resonances in protein NMR spectra.…”