Potassium channels are responsible for the selective permeation of K+ ions across cell membranes. K+ ions permeate in single file through the selectivity filter, a narrow pore lined by backbone carbonyls that compose 4 K+ binding sites. Here, we report 2D IR spectra of a semisynthetic KcsA channel with site-specific 13C18O isotope labels in the selectivity filter. The ultrafast time-resolution of 2D IR spectroscopy provides an instantaneous snapshot of the multi-ion configurations and structural distributions that occur spontaneously in the filter. Two elongated features are resolved, revealing the statistical weighting of two structural conformations. The spectra are reproduced by MD simulations of structures with water separating two K+ ions in the binding sites, ruling out configurations with ions occupying adjacent sites.

Polyglutamine (polyQ) sequences are found in a variety of proteins, and mutational expansion of the polyQ tract is associated with many neurodegenerative diseases. We study the amyloid fibril structure and aggregation kinetics of K 2 Q 24 K 2 W, a model polyQ sequence. Two structures have been proposed for amyloid fibrils formed by polyQ peptides. By forming fibrils composed of both 12 C and 13 C monomers, made possible by protein expression in Escherichia coli, we can restrict vibrational delocalization to measure 2D IR spectra of individual monomers within the fibrils. The spectra are consistent with a β-turn structure in which each monomer forms an antiparallel hairpin and donates two strands to a single β-sheet. Calculated spectra from atomistic molecular-dynamics simulations of the two proposed structures confirm the assignment. No spectroscopically distinct intermediates are observed in rapid-scan 2D IR kinetics measurements, suggesting that aggregation is highly cooperative. Although 2D IR spectroscopy has advantages over linear techniques, the isotope-mixing strategy will also be useful with standard Fourier transform IR spectroscopy.Huntington disease | isotope dilution | antiparallel β-sheets | two-dimensional infrared spectroscopy N umerous neurodegenerative diseases, including Huntington disease, are associated with the mutational expansion of CAG repeats in specific genes. This expansion causes an increase in the length of normally benign polyglutamine (polyQ) tracts in expressed proteins (1, 2). In vivo, polyQ tracts occur embedded within larger proteins, such as the huntingtin protein. Full length proteins are impractical for detailed structural studies, and so isolated polyQ sequences are often used instead in experiments and simulations (3-5). Although isolated polyQ sequences may not aggregate in precisely the same manner as they would in fulllength proteins (3, 6, 7), it has been established that such models yield data that are relevant to some, if not all, polyQ pathologies (8). Additionally, proteolytic polyQ-containing fragments, rather than full-length proteins, may be the primary toxic species in Huntington disease (9) and some types of spinocerebellar ataxia (10). As a consequence, simple polyQ peptides remain an attractive alternative to more complicated proteins for understanding polyQmediated aggregation.A plethora of structures have been suggested for polyQ aggregates, ranging from α-helical coiled coils (11) or β-helices (12) to amyloid fibrils comprising stacked, linear β-sheets (13). Current consensus favors fibrillar structures, but the precise arrangement of the peptides within the fibril remains unclear. Most amyloid proteins form parallel β-sheets within fibrils, although antiparallel sheets do occur (14). Parallel sheets have been observed for glutamine-rich hexapeptides (15), but NMR studies (16), as well as the results presented here, suggest that sequences approaching the pathological length of 40 glutamines adopt an antiparallel structure. Fig. 1 shows two models that hav...

Urea/water is an archetypical “biological” mixture, and is especially well known for its relevance to protein thermodynamics, as urea acts as a protein denaturant at high concentration. This behavior has given rise to an extended debate concerning urea’s influence on water structure. Based on a variety of methods and of definitions of water structure, urea has been variously described as a structure-breaker, a structure-maker, or as remarkably neutral towards water. Because of its sensitivity to microscopic structure and dynamics, vibrational spectroscopy can help resolve these debates. We report experimental and theoretical spectroscopic results for the OD stretch of HOD/H2O/urea mixtures (linear IR, 2DIR, and pump-probe anisotropy decay) and for the CO stretch of urea-D4/D2O mixtures (linear IR only). Theoretical results are obtained using existing approaches for water, and a modification of a frequency map developed for acetamide. All absorption spectra are remarkably insensitive to urea concentration, consistent with the idea that urea only very weakly perturbs water structure. Both this work and experiments by Rezus and Bakker, however, show that water’s rotational dynamics are slowed down by urea. Analysis of the simulations casts doubt on the suggestion that urea immobilizes particular doubly hydrogen bonded water molecules.

Using classical molecular dynamics simulations, the 2D infrared (IR) spectroscopy of CN(-) solvated in D2O is investigated. Depending on the force field parametrizations, most of which are based on multipolar interactions for the CN(-) molecule, the frequency-frequency correlation function and observables computed from it differ. Most notably, models based on multipoles for CN(-) and TIP3P for water yield quantitatively correct results when compared with experiments. Furthermore, the recent finding that T1 times are sensitive to the van der Waals ranges on the CN(-) is confirmed in the present study. For the linear IR spectrum, the best model reproduces the full widths at half maximum almost quantitatively (13.0 cm(-1) vs. 14.9 cm(-1)) if the rotational contribution to the linewidth is included. Without the rotational contribution, the lines are too narrow by about a factor of two, which agrees with Raman and IR experiments. The computed and experimental tilt angles (or nodal slopes) α as a function of the 2D IR waiting time compare favorably with the measured ones and the frequency fluctuation correlation function is invariably found to contain three time scales: a sub-ps, 1 ps, and one on the 10-ps time scale. These time scales are discussed in terms of the structural dynamics of the surrounding solvent and it is found that the longest time scale (≈10 ps) most likely corresponds to solvent exchange between the first and second solvation shell, in agreement with interpretations from nuclear magnetic resonance measurements.

Vibrational sum frequency generation (SFG) has become a very promising technique for the study of proteins at interfaces, and it has been applied to important systems such as anti-microbial peptides, ion channel proteins, and human islet amyloid polypeptide. Moreover, so-called “chiral” SFG techniques, which rely on polarization combinations that generate strong signals primarily for chiral molecules, have proven to be particularly discriminatory of protein secondary structure. In this work, we present a theoretical strategy for calculating protein amide I SFG spectra by combining line-shape theory with molecular dynamics simulations. We then apply this method to three model peptides, demonstrating the existence of a significant chiral SFG signal for peptides with chiral centers, and providing a framework for interpreting the results on the basis of the dependence of the SFG signal on the peptide orientation. We also examine the importance of dynamical and coupling effects. Finally, we suggest a simple method for determining a chromophore’s orientation relative to the surface using ratios of experimental heterodyne-detected signals with different polarizations, and test this method using theoretical spectra.