Chain-selective isotopic labeling for NMR studies of large multimeric proteins: application to hemoglobin

Area of Science:

• Biophysics
• Structural Biology
• Nuclear Magnetic Resonance (NMR) Spectroscopy

Background:

• Multidimensional, multinuclear NMR offers potential for studying protein allostery and cooperativity.
• Severe resonance overlap in NMR spectra of proteins with non-equivalent subunits hinders resonance assignment.
• Chain-selective labeling is a technique to address spectral overlap in complex protein systems.

Purpose of the Study:

• To apply chain-selective labeling to carbonmonoxy-hemoglobin A (HbCO A) to overcome resonance overlap.
• To extend resonance assignments of key amino acid residues, including surface histidines involved in the Bohr effect.
• To demonstrate the utility of heteronuclear NMR with chain-selective labeling for large, multimeric proteins.

Main Methods:

• Application of chain-selective labeling to recombinant (15)N-labeled HbCO A.
• Two-dimensional heteronuclear multiple quantum coherence (HMQC) experiments.
• Analysis of C2-proton (H epsilon(1)) and C4-proton (H delta(2)) chemical shifts for histidines.
• Assignment of H delta(1), H epsilon(1), and N epsilon(1) resonances for tryptophan residues.

Main Results:

• Successful application of chain-selective labeling to HbCO A.
• Assignment of chemical shifts for all 13 surface histidines per alpha beta dimer.
• Assignment of resonances for all three tryptophan residues per alpha beta dimer.
• Demonstration of heteronuclear NMR's capability with chain-selective labeling for large proteins.

Conclusions:

• Chain-selective labeling effectively reduces resonance overlap in complex protein NMR.
• This technique facilitates resonance assignment in key regions of multimeric proteins like hemoglobin.
• Provides a viable approach for elucidating the solution structure of large proteins.