Amide vibrations are delocalized across the hydrophobic interface of a transmembrane helix dimer

Abstract

The tertiary interactions between amide-I vibrators on the separate helices of transmembrane helix dimers were probed by ultrafast 2D vibrational photon echo spectroscopy. The 2D IR approach proves to be a useful structural method for the study of membrane-bound structures. The 27-residue human erythrocyte protein Glycophorin A transmembrane peptide sequence: KKITLIIFG79VMAGVIGTILLISWG94IKK was labeled at G79 and G94 with 13 tertiary interaction ͉ vibrational spectra ͉ multidimensional spectroscopy T wo-dimensional IR spectroscopy (1-6) is a promising new method with which to probe structures and their motions in complex systems. Recent applications of this method to biologically related molecules have yielded novel structural and dynamical results not readily obtainable by other methods for small peptides (7,8), soluble helices (9, 10), membrane-bound helices (11), lipids (12), ␤-sheets (13, 14), and model secondary structures (15, 16). The 2D IR approach is one that can be immediately applicable to a wide variety of sample types ranging from solutions to solids, including aqueous and lipid environments. The method exposes interactions between spatially nearby vibrational modes by converting three pulse photon echo signals into 2D spectral maps in the IR, analogous to 2D NMR spectra.The amide-I vibrational bands of polypeptides are highly degenerate because, for each residue, their frequencies are approximately the same and the interactions among them are not strong enough to separately display them as individual, assignable transitions. The combination of multiple isotope selection and 2D IR spectroscopy that spreads the transitions into two dimensions proves to be a useful strategy that can simplify and expose the underlying transitions of the diffuse amide bands and allow their features to be characterized at a residue level. For example the 2D IR spectrum of a water-soluble helix selectively substituted with 13 CA 16 O and 13 CA 18 O provided characteristics of pairs of coupled residues and the visualization of the sequence dependence of the structural dynamics (17). This paper introduces a comparable approach to investigate tertiary interactions between vibrational modes in Glycophorin A (GpA), a transmembrane (TM) dimer of interacting helices (Fig. 1). The GpA TM helix provides an excellent prototype for the study of TM helix association, a key event in the folding of membrane proteins (18). The dimer also is an appropriate model for these investigations, because it is stable in a variety of micelles, including SDS (19), and a solution NMR structure is available (20). There are two Gly residues at the dimer interface that allow an intimate contact of the main chain, a feature frequently observed in TM helical interfaces (21,22). Because vibrational coupling is sensitive to distance, the proximity of the backbones is advantageous. GpA also was selected because verification of the applicability of the 2D IR method to membrane protein aggregates is of great interest and because there is a chronic paucity o...

Tidal surge in the M2 proton channel, sensed by 2D IR spectroscopy

“…The presence of more than one such conformation will broaden the IR bandwidth or cause a splitting into multiple transitions. That each amide-I mode for an isolated oscillator has a characteristic bandwidth and shape is based on experiments on transitions for many different proteins, peptides, and their isotopologues (3,4,7). Therefore, the observation of one amide-I band with the characteristic width and shape of an isolated amide group is strongly suggestive of there being one peak in the conformational distribution wherein all the glycines have the same frequency (backbone, side chain, and water distribution) within a small range.…”

“…The 2D IR spectra create residue level images of both instantaneous conformational distributions as well as fast dynamics in proteins (3-6) by means of 13 C ¼ 18 O labeling of helix backbone amide units (7,8), which act as probes of local solvent spectral density through measures of the frequencyfrequency correlation function of the amide-I vibration. The motion of water molecules around the amide groups in proteins and peptides causes fluctuations in the amide-I vibrational frequencies.…”

Vibrational Coupling between Helices Influences the Amide I Infrared Absorption of Proteins: Application to Bacteriorhodopsin and Rhodopsin

“…[13][14][15][16][17][18][19] Several recent studies have instead focused on the IR spectral features of transition dipole coupling (TDC) between secondary structure elements, such as those between β -sheets [20][21][22] or α-helices in a helix-dimer. 23,24 This coupling is important to consider as it influences the band positions associated with the secondary structure absorption and therefore the interpretation of the IR spectra. Recently, the 1D and 2D-IR spectra of the helix dimer pair have been investigated both experimentally and theoretically and it was found that there is an exciton coupling interaction between the amide I transitions of the respective helices.…”

“…Recently, the 1D and 2D-IR spectra of the helix dimer pair have been investigated both experimentally and theoretically and it was found that there is an exciton coupling interaction between the amide I transitions of the respective helices. 23,24 The ability to measure such coupling interactions could enable 3D structure determination by providing important constraints. Studies of the predominantly helical globular protein myoglobin have also concluded that it is not possible to accurately replicate the spectra of a many-helix protein without including long-range interactions between helices.…”

Discovery of competing anaerobic and aerobic pathways in umpolung amide synthesis allows for site-selective amide 18 O-labeling

“…Aside from the need to greatly generalize any one of these developments, it remains a challenge to routinely incorporate chiral nonnatural amino acids. Siteselective labeling of amide oxygen, despite its power as a technique for the spectroscopic study of protein structure (22), also remains quite difficult beyond the use of methods that label the precursor carboxylic acids through a combination of acid and H 2 18 O (23-26).…”