Measuring Protein Structural Heterogeneity with Two-Dimensional Infrared Spectroscopy

Abstract

Protein structure and heterogeneity is particularly difficult to measure due to lack of experimental techniques that combine structural sensitivity and sub-microsecond time resolution. Two dimensional spectroscopy is a new optical technique that measures protein structure and dynamics with ultrafast time resolution. The delocalized backbone C=O (Amide-I) vibrations reflect the global secondary structure of the protein. A 13C=18O isotope label on a residue red-shifts its frequency by ∼60 cm-1, isolating the site from the main amide band. The label provides a unique spectroscopic handle on the structure (distances), heterogeneity, and hydrogen-bonding environment (solvent exposure) of the labeled residues, and the ultrafast time resolution is able to distinguish between different fast-exchanging conformational states. We apply this new method to NTL9, a 39-residue α/β mini protein, by isotope labeling five different sites, including a dual-label across a type-I beta-turn. The structural interpretation is enabled by spectral simulations based on a recent Markov state model (MSM) built from millisecond-long molecular dynamics trajectories. Structures are assigned by matching the measured frequencies and lineshapes to simulated spectra for each Markov state. The excellent qualitative agreement between theory and experiment provides a solid set of structural constraints. We find a number of sub-states with different configurations, particularly in flexible regions of the protein, such as the type-I beta turn. Specifically, we find a significant population of bulged turn configurations. The results show that residues in the first and last turns of the helix exhibit multiple hydrogen-bonding environments reflecting the greater solvent-exposure within these regions of the backbone. Finally, the lineshapes serve to characterize the flexibility and stability of the backbone at the different sites. We find that β-strands remain relatively rigid whereas the turn and helix regions show increased flexibility, qualitatively matching b-factors extracted from crystallography.