Search

We used two-dimensional IR bioimaging to study the structural heterogeneity of formalin-fixed mouse pancreas. Images were generated from the hyperspectral data sets by plotting quantities associated with the amide I vibrational mode, which is created by the backbone carbonyl stretch. Images that measure the fundamental vibrational frequencies, cross peaks, and anharmonic shifts are presented. Histograms are generated for each quantity, providing averaged values and distributions around the mean that serve as metrics for protein structures. Images were generated from tissue that had been stored in a formalin fixation for 3, 8, and 48 weeks. Over this period, all three metrics show that that the β-sheet content of the samples increased, consistent with protein aggregation. Our results indicate that formalin fixation does not entirely arrest the degradation of a protein structure in pancreas tissue.

Vibrational spectroscopy is an essential tool in chemical analyses, biological assays, and studies of functional materials. Over the past decade, various coherent nonlinear vibrational spectroscopic techniques have been developed and enabled researchers to study time-correlations of the fluctuating frequencies that are directly related to solute-solvent dynamics, dynamical changes in molecular conformations and local electrostatic environments, chemical and biochemical reactions, protein structural dynamics and functions, characteristic processes of functional materials, and so on. In order to gain incisive and quantitative information on the local electrostatic environment, molecular conformation, protein structure and interprotein contacts, ligand binding kinetics, and electric and optical properties of functional materials, a variety of vibrational probes have been developed and site-specifically incorporated into molecular, biological, and material systems for time-resolved vibrational spectroscopic investigation. However, still, an all-encompassing theory that describes the vibrational solvatochromism, electrochromism, and dynamic fluctuation of vibrational frequencies has not been completely established mainly due to the intrinsic complexity of intermolecular interactions in condensed phases. In particular, the amount of data obtained from the linear and nonlinear vibrational spectroscopic experiments has been rapidly increasing, but the lack of a quantitative method to interpret these measurements has been one major obstacle in broadening the applications of these methods. Among various theoretical models, one of the most successful approaches is a semiempirical model generally referred to as the vibrational spectroscopic map that is based on a rigorous theory of intermolecular interactions. Recently, genetic algorithm, neural network, and machine learning approaches have been applied to the development of vibrational solvatochromism theory. In this review, we provide comprehensive descriptions of the theoretical foundation and various examples showing its extraordinary successes in the interpretations of experimental observations. In addition, a brief introduction to a newly created repository Web site (http://frequencymap.org) for vibrational spectroscopic maps is presented. We anticipate that a combination of the vibrational frequency map approach and state-of-the-art multidimensional vibrational spectroscopy will be one of the most fruitful ways to study the structure and dynamics of chemical, biological, and functional molecular systems in the future.

Proteins exhibit structural fluctuations over decades of time scales. From the picosecond side chain motions to aggregates that form over the course of minutes, characterizing protein structure over these vast lengths of time is important to understanding their function. In the past 15 years, two-dimensional infrared spectroscopy (2D IR) has been established as a versatile tool that can uniquely probe proteins structures on many time scales. In this review, we present some of the basic principles behind 2D IR and show how they have, and can, impact the field of protein biophysics. We highlight experiments in which 2D IR spectroscopy has provided structural and dynamical data that would be difficult to obtain with more standard structural biology techniques. We also highlight technological developments in 2D IR that continue to expand the scope of scientific problems that can be accessed in the biomedical sciences.

A high-sensitivity metal-carbonyl-based IR probe is described that can be incorporated into proteins or other biomolecules in very high yield via Click chemistry. A two-step strategy is demonstrated. First, a methionine auxotroph is used to incorporate the unnatural amino acid azidohomoalanine at high levels. Second, a tricarbonyl (η(5)-cyclopentadienyl) rhenium(I) probe modified with an alkynyl linkage is coupled via the Click reaction. We demonstrate these steps using the C-terminal domain of the ribosomal protein L9 as a model system. An overall incorporation level of 92% was obtained at residue 109, which is a surface-exposed residue. Incorporation of the probe into a surface site is shown not to perturb the stability or structure of the target protein. Metal carbonyls are known to be sensitive to solvation and protein electrostatics through vibrational lifetimes and frequency shifts. We report that the frequencies and lifetimes of this probe also depend on the isotopic composition of the solvent. Comparison of the lifetimes measured in H2O versus D2O provides a probe of solvent accessibility. The metal carbonyl probe reported here provides an easy and robust method to label very large proteins with an amino-acid-specific tag that is both environmentally sensitive and a very strong absorber.

Infrared spectroscopy is playing an important role in the elucidation of amyloid fiber formation, but the coupling models that link spectra to structure are not well tested for parallel β-sheets. Using a synthetic macrocycle that enforces a two stranded parallel β-sheet conformation, we measured the lifetimes and frequency for six combinations of doubly (13)C═(18)O labeled amide I modes using 2D IR spectroscopy. The average vibrational lifetime of the isotope labeled residues was 550 fs. The frequencies of the labels ranged from 1585 to 1595 cm(-1), with the largest frequency shift occurring for in-register amino acids. The 2D IR spectra of the coupled isotope labels were calculated from molecular dynamics simulations of a series of macrocycle structures generated from replica exchange dynamics to fully sample the conformational distribution. The models used to simulate the spectra include through-space coupling, through-bond coupling, and local frequency shifts caused by environment electrostatics and hydrogen bonding. The calculated spectra predict the line widths and frequencies nearly quantitatively. Historically, the characteristic features of β-sheet infrared spectra have been attributed to through-space couplings such as transition dipole coupling. We find that frequency shifts of the local carbonyl groups due to nearest neighbor couplings and environmental factors are more important, while the through-space couplings dictate the spectral intensities. As a result, the characteristic absorption spectra empirically used for decades to assign parallel β-sheet secondary structure arises because of a redistribution of oscillator strength, but the through-space couplings do not themselves dramatically alter the frequency distribution of eigenstates much more than already exists in random coil structures. Moreover, solvent exposed residues have amide I bands with >20 cm(-1) line width. Narrower line widths indicate that the amide I backbone is solvent protected inside the macrocycle. This work provides calculated and experimentally verified couplings for parallel β-sheets that can be used in structure-based models to simulate and interpret the infrared spectra of β-sheet containing proteins and protein assemblies, such as amyloid fibers.

Vibrational and electronic transition dipole strengths are often good probes of molecular structures, especially in excitonically coupled systems of chromophores. One cannot determine transition dipole strengths using linear spectroscopy unless the concentration is known, which in many cases it is not. In this paper, we report a simple method for measuring transition dipole moments from linear absorption and 2D IR spectra that does not require knowledge of concentrations. Our method is tested on several model compounds and applied to the amide I(') band of a polypeptide in its random coil and α-helical conformation as modulated by the solution temperature. It is often difficult to confidently assign polypeptide and protein secondary structures to random coil or α-helix by linear spectroscopy alone, because they absorb in the same frequency range. We find that the transition dipole strength of the random coil state is 0.12 ± 0.013 D(2), which is similar to a single peptide unit, indicating that the vibrational mode of random coil is localized on a single peptide unit. In an α-helix, the lower bound of transition dipole strength is 0.26 ± 0.03 D(2). When taking into account the angle of the amide I(') transition dipole vector with respect to the helix axis, our measurements indicate that the amide I(') vibrational mode is delocalized across a minimum of 3.5 residues in an α-helix. Thus, one can confidently assign secondary structure based on exciton delocalization through its effect on the transition dipole strength. Our method will be especially useful for kinetically evolving systems, systems with overlapping molecular conformations, and other situations in which concentrations are difficult to determine.

We describe a methodology for studying protein kinetics using a rapid-scan technology for collecting 2D IR spectra. In conjunction with isotope labeling, 2D IR spectroscopy is able to probe the secondary structure and environment of individual residues in polypeptides and proteins. It is particularly useful for membrane and aggregate proteins. Our rapid-scan technology relies on a mid-IR pulse shaper that computer generates the pulse shapes, much like in an NMR spectrometer. With this device, data collection is faster, easier, and more accurate. We describe our 2D IR spectrometer, as well as protocols for (13)C(18)O isotope labeling, and then illustrate the technique with an application to the aggregation of the human islet amyloid polypeptide implicated in type 2 diabetes., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Reports that two-dimensional (2D) IR spectroscopy can be designed to eliminate the diagonal peaks and expose only the crosspeaks needed for structure determination. Polarization conditions for 2D-IR spectroscopy; Use of 1,3-cyclohexanedione as a model system for the development of the method; Applications of 2D IR to proteins, peptides and other complex molecules.

Summary: The pH-controlled M2 protein from influenza A is a critical component of the virus and serves as a target for the aminoadamantane antiflu agents that block its H+ channel activity. To better understand its H+ gating mechanism, we investigated M2 in lipid bilayers with a new combination of IR spectroscopies and theory. Linear Fourier transform infrared (FTIR) spectroscopy was used to measure the precise orientation of the backbone carbonyl groups, and 2D infrared (IR) spectroscopy was used to identify channel-lining residues. At low pH (open state), our results match previously published solid-state NMR and X-ray structures remarkably well. However, at neutral pH when the channel is closed, our measurements indicate that a large conformational change occurs that is consistent with the transmembrane α-helices rotating by one amino acid register—a structural rearrangement not previously observed. The combination of simulations and isotope-labeled FTIR and 2D IR spectroscopies provides a noninvasive means of interrogating the structures of membrane proteins in general and ion channels in particular. [Copyright &y& Elsevier]