1H NMR studies of lambda cro repressor. 1. Selective optimization of two-dimensional relayed coherence transfer...

Area of Science:

• Biochemistry
• Structural Biology
• Nuclear Magnetic Resonance Spectroscopy

Background:

• Protein structure determination relies on identifying spin systems within NMR spectra.
• Crowded 1H NMR spectra pose challenges for unambiguous resonance assignment.
• Two-dimensional relayed coherence transfer (RELAY) NMR spectroscopy offers a method for spin system correlation.

Purpose of the Study:

• To optimize RELAY NMR experiments for efficient side chain spin system identification in complex protein spectra.
• To demonstrate the utility of optimized RELAY for analyzing the lambda cro repressor protein.
• To establish a method for rapid and unambiguous resonance assignment in challenging NMR datasets.

Main Methods:

• Utilized two-dimensional relayed coherence transfer (RELAY) NMR spectroscopy.
• Optimized the mixing time in RELAY experiments based on transverse relaxation time (T2) and J couplings.
• Applied recently developed methods for mixing time optimization [Bax, A., & Drobny, G. (1985) J. Magn. Reson, 61, 306-320].

Main Results:

• A 26 ms mixing time yielded strong C alpha H-C gamma H3 RELAY cross peaks for valine, threonine, and isoleucine residues.
• RELAY cross peaks for other spin systems were weak or absent with the optimized mixing time.
• Enabled rapid and unambiguous identification of side chain resonances for valine, isoleucine, threonine, and alanine (by elimination).

Conclusions:

• Optimized RELAY NMR is a powerful tool for analyzing and identifying spin systems in complex protein spectra.
• The optimized 26 ms mixing time facilitates straightforward assignment of specific amino acid side chains.
• This approach significantly aids in structural elucidation of proteins with crowded NMR spectra.