Protein conformation change of myoglobin upon ligand binding probed by ultraviolet resonance Raman spectroscopy.

The research article explores the conformational changes in myoglobin, a type of protein, upon binding with a ligand, using ultraviolet resonance Raman Spectroscopy. Significant changes were observed at specific tryptophan and tyrosine residues following carbon monoxide binding, implying induced changes in the regions containing these residues due to iron movement.

Methodology

• The researchers used ultraviolet resonance Raman Spectroscopy (UVRR) with 244 nm excited light to investigate the conformational changes in myoglobin (Mb) upon ligand binding.
• The study carried out spectral analyses of two types of myoglobins—sperm whale (sw) normal (wild-type) and W7F and W14F mutant forms, as well as horse (h) myoglobin.
• The experiments were conducted in the absence of a ligand (deoxy state) and in the presence of carbon monoxide (CO), a gaseous ligand.

Findings

• By analyzing the UVRR spectra, changes in the structural or environmental conditions of specific protein residues, including Trp7, Trp14, and Tyr151, were observed when comparing the deoxy and CO-bound states.
• Among these, the Trp7 residue (near the N-terminus) and the Tyr151 residue (in the C-terminus) showed definite changes upon CO binding, while Trp14, Tyr103, and Tyr146 did not demonstrate any significant change.
• The experiment also used a model compound for kinetically examining the effect of solvent on the spectral changes of tyrosine residues.
• Comparing spectral differences between CO and n-butyl isocyanide-bound forms revealed smaller differences than those displayed between deoxy and CO-bound forms, suggesting that the structural transition in the N- and C-terminal regions of the protein is driven by the motion of iron on the proximal side of the heme (a component of the protein).
• Despite notable movement of the His64 (histidine) residue stimulated by binding with large ligands—proven by previous X-ray crystallographic studies—the UVRR spectra did not detect solvent exposure of His64 for the n-butyl isocyanide-bound form. This factor calls for further investigation.

Conclusion

• The research gives valuable insights into the conformational changes that occur in myoglobin upon ligand binding, which are crucial for understanding the molecular mechanisms involving protein function and behavior.
• The study also underscores the potential of UVRR spectroscopy in probing protein residue level structural transitions, which can be essential for various biomedical and biochemical applications.