Methyl rotation barriers in proteins from 2H relaxation data. Implications for protein structure

Area of Science:

• Biophysics
• Structural Biology
• Computational Chemistry

Background:

• Methyl group rotation is crucial for protein dynamics and function.
• Understanding methyl rotation barriers provides insights into the protein's hydrophobic core.
• Nuclear Magnetic Resonance (NMR) relaxation data and molecular dynamics (MD) simulations are key tools for studying protein dynamics.

Purpose of the Study:

• To determine the temperature-dependent correlation times characterizing fast methyl motion in the SH3 domain of alpha-spectrin.
• To experimentally measure methyl rotation barriers and compare them with computational predictions.
• To investigate the accuracy of NMR structures by analyzing methyl rotation barriers.

Main Methods:

• Collected side-chain 2H and backbone 15N relaxation data at multiple temperatures for the SH3 domain.
• Analyzed relaxation data to determine temperature-dependent correlation times (tau_f) for methyl motion.
• Performed molecular dynamics simulations to model methyl rotation and calculate activation energies (barriers).

Main Results:

• Experimental data yielded an average methyl rotation barrier of 2.8 ± 0.9 kcal/mol for the SH3 domain, consistent with a fluid hydrophobic core.
• MD simulations predicted average barriers of 3.1-3.5 kcal/mol, closely matching experimental findings.
• Analysis of NMR structures revealed higher barriers (4-6 kcal/mol), often due to tight packing and structure calculation artifacts, with some exceeding 10 kcal/mol.

Conclusions:

• Experimental methyl rotation barriers are representative of the protein's internal dynamics and hydrophobic core characteristics.
• NMR structures may inaccurately represent methyl group environments, potentially leading to overestimated rotation barriers.
• Methyl rotation barriers can serve as a valuable metric for assessing the quality and accuracy of protein structural models.