Identification of the C2-1H histidine NMR resonances in chloramphenicol acetyltransferase by a 13C-1H heteronuclear multiple quantum coherence method

AbstractChloramphenicol acetyltransferase (CAT) was used to assess the feasibility of study of specific proton resonances in an enzyme of overall molecular mass 75000. [ring2-13C]Histidine was selectively incorporated into the type III chloramphenicol acetyltransferase (CATIII) using a histidine auxotroph of E. coli. Heteronuclear multiple and single quantum experiments were used to select the C2 protons in the histidyl imidazole ring. One- and two-dimensional spectra revealed six signals out of a total of seven histidine residues in CATIII. pH titration, chemical modification and ligand binding were used to demonstrate that the signal from H195, the histidine at the active site, is not among those observed. Nevertheless, this work demonstrates that selective isotopic enrichment and multiple quantum coherence techniques can be used to distinguish proton resonances in a protein of high molecular mass

Biophysical Analysis of Nucleic Acids

This overview unit provides a thorough overview of biophysical methods used for structure analysis, including X‐ray diffraction, nuclear magnetic resonance, optical spectroscopy, theoretical and computational methods, and single‐molecule methods. Advantages and disadvantages of the methods are compared.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143731/1/cpnc0701.pd

The combined use of selective deuteration and double resonance experiments in assigning the 1H resonances of valine and tyrosine residues of dihydrofolate reductase

AbstractSelective deuteration is a general solution to the resolution problem which limits the application of double resonance experiments to the assignment of the 1H NMR spectra of proteins. Spin-decoupling and NOE experiments have been carried out on Lactobacillus casei dihydrofolate reductase and on selectively deuterated derivatives of the enzyme containing either [γ-2H6]Val or (α,δ2,ϵ1-2H3]His, [α,δ1,δ2,ϵ1,ϵ2,ζ-2H6]Phe, [α,δ1,ϵ3,ζ2,ζ3,η2-2H6]Trp and [α,ϵ1,ϵ2-2H3]Tyr. When combined with ring-current shift calculations based on the crystal structure of the enzyme, these experiments allow us to assign 1H resonances of Val 61, Val 115, Tyr 46 and Tyr 68

Optimising selective deuteration of proteins for 2D 1H NMR detection and assignment studies Application to the Phe residues of Lactobacillus casei dihydrofolate reductase

AbstractA selectively deuterated dihydrofolate reductase from L. casei has been prepared containing partially deuterated aromatic amino acids. This provides simplified 2D NMR spectra and allows signals from all 8 Phe residues to be identified. The pattern of deuteration is such that (i) the only cross-peaks detected in the aromatic region of the 2D COSY spectrum are those between the Phe 2′,6′ and 3′,5′ protons and (ii) chemical shift degeneracy in the aromatic region is removed thus allowing unambiguous assignment of cross-peaks in 2D NOESY spectra required for specific assignment purposes