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2D infrared (IR) spectroscopy is a cutting-edge technique, with applications in subjects as diverse as the energy sciences, biophysics and physical chemistry. This book introduces the essential concepts of 2D IR spectroscopy step-by-step to build an intuitive and in-depth understanding of the method. This unique book introduces the mathematical formalism in a simple manner, examines the design considerations for implementing the methods in the laboratory, and contains working computer code to simulate 2D IR spectra and exercises to illustrate involved concepts. Readers will learn how to accurately interpret 2D IR spectra, design their own spectrometer and invent their own pulse sequences. It is an excellent starting point for graduate students and researchers new to this exciting field. Computer codes and answers to the exercises can be downloaded from the authors' website, available at www.cambridge.org/9781107000056.
Femtosecond two-dimensional infrared spectroscopy in combination with isotope labeling and molecular dynamics simulation has been used to investigate the structures and dynamics of nonfolding peptides and collagen peptides. Homopolymeric peptides are simple yet important, serve as model systems for investigating the intrinsic propensity of protein folding, especially for those disordered or unfolded peptides in aqueous solution. Here the full structure of (Ala)5 has been studied. Two different isotope-labeled peptides, Ala-(13C)Ala-(13C,18O)Ala-Ala-Ala, and Ala-Ala-(13C,18O)Ala-(13C)Ala-Ala were strategically designed to simplify the four-oscillator system into three two-oscillator systems. By utilizing the different polarization dependence of diagonal and cross peaks, coupling constant β and angle θ between transition dipoles has been extracted through spectral fitting. The coupling constant is around 4 cm-1 and angle around 100. These parameters were related to the dihedral angles characterizing the peptide backbone structure through DFT calculated maps. The derived dihedrals are all located in the polyproline-II region. These results were compared to the conformations sampled by hamiltonian replica-exchange MD simulation with 3 different CHARMM force fields: C22, C36 and Drude. The C22 force field predicted too high α-helix population, whereas C36 predicted that polyproline-II is the dominate conformation, consistent with experimental findings. The Drude model predicted dominating beta-sheet. Since the 2D-IR derived results were obtained from fitting to a single set of structural parameters, the effect of structural fluctuation within one conformation and structural transition between different conformations was also discussed. The C36 MD trajectories were used to simulate 2D IR spectra using the sum-over-state method and the time-averaging approximation (TAA) method. Reasonable agreement with the experimental data was achieved. Collagen is the most abundant protein in mammal. Its structural properties are important for biological functions. Here, the structure and thermal melting of a model collagen peptide, (PPG)10, has been investigated. The temperature dependent linear IR spectra of the unlabeled peptide and two isotopomers (either 13C-16O or 13C-18O labeled on the 4th glycine residue) showed that the triple helix unravels with increasing temperature and leads to greater solvent exposure. With some adjustments of calculation parameters according to linear IR spectra, 2D IR spectral simulation based on MD trajectories and TAA reasonably reproduced experimental spectra taken at the parallel and perpendicular polarization conditions. Further refinement of models and parameters are needed to improve the simulation. Our results for model nonfolding peptides and collagen peptides contribute to the fundamental understanding of peptide structure and dynamics, and to the further development of theoretical models for simulating 2D IR spectra.
The amide II' diagonal provides a measure of the degree of exchange and the cross peaks between the structurally sensitive amide I/I' vibration and the solvent exposure sensitive amide II and II' modes reveal the location of exchange. Partial exchange of the secondary structure of ubiquitin is revealed by correlation of the different amide signatures through analysis of cross peak line shapes, positions and amplitudes. Results provide direct evidence for a highly stable helix and labile "--Sheet structure.
Two-Dimensional Optical Spectroscopy discusses the principles and applications of newly emerging two-dimensional vibrational and optical spectroscopy techniques. It provides a detailed account of basic theory required for an understanding of two-dimensional vibrational and electronic spectroscopy. It also bridges the gap between the formal developm
The advent of laser-based sources of ultrafast infrared pulses has extended the study of very fast molecular dynamics to the observation of processes manifested through their effects on the vibrations of molecules. In addition, non-linear infrared spectroscopic techniques make it possible to examine intra- and intermolecular interactions and how such interactions evolve on very fast time scales, but also in some instances on very slow time scales. Ultrafast Infrared Vibrational Spectroscopy is an advanced overview of the field of ultrafast infrared vibrational spectroscopy based on the scientific research of the leading figures in the field. The book discusses experimental and theoretical topics reflecting the latest accomplishments and understanding of ultrafast infrared vibrational spectroscopy. Each chapter provides background, details of methods, and explication of a topic of current research interest. Experimental and theoretical studies cover topics as diverse as the dynamics of water and the dynamics and structure of biological molecules. Methods covered include vibrational echo chemical exchange spectroscopy, IR-Raman spectroscopy, time resolved sum frequency generation, and 2D IR spectroscopy. Edited by a recognized leader in the field and with contributions from top researchers, including experimentalists and theoreticians, this book presents the latest research methods and results. It will serve as an excellent resource for those new to the field, experts in the field, and individuals who want to gain an understanding of particular methods and research topics.
Proteins and peptides associated with cell membranes play a vital role in cell signaling and cell health. From ion channels that allow cells to transduce electrical signals to antibiotic peptides that break open cell membranes, membrane-associated and membrane-bound proteins are of interest due to their importance. However, due to the nature of lipid membranes, the ion channels and other membrane proteins have historically been more challenging to study since they cannot be simply in aqueous solution like soluble proteins. Increased knowledge of protein expression and purification in the last few decades has made membrane-associated and membrane-bound proteins and peptides more readily studied, however, there is still much to learn about the basic function of these macromolecules. For example, one of the most fundamental processes is ion channel conduction. In potassium channels, potassium ions flow at nearly the diffusion limit with exquisite selectivity when the channel is open. The previously established mechanism for ion transduction has been recently called into question. Two-dimensional infrared spectroscopy is an excellent method to study membrane-bound proteins and peptides because of its structural sensitivity, inherent time resolution, and ability to be modeled from structural and computational results. In this dissertation, methods with which to study potassium channels, as well as other membrane-bound peptides are developed, along with strategies to study surfaces, including working with proteins bound in a single lipid bilayer. First, a voltage and pH-sensitive antimicrobial peptide is studied using surface-enhanced 2D IR spectroscopy and an applied voltage. Since the peptide is in a bilayer tethered to a surface, the sample is no longer isotropic and relative intensities of spectral peaks allowed for the extraction of insertion angles upon a change in pH and a change in voltage. Insertion angles were determined through modeling the spectra based on the peptide structure and helical coupling values. Next, progress to studying the selectivity filter of two potassium channels is outlined, including experiment looking at an ester label in the selectivity filter. The ester label causes a water pocket in the ion channel to collapse and changes the binding sites for ions in the selectivity filter. Using waiting time analysis, the dynamics of the labeled residue can be measured. Finally advances to polarization controls to distinguish bulk and surface signals is theoretically developed to create a surface specific spectroscopy. Finally, a chapter is included to disseminate the work presented in this dissertation to the public.
Plasma membranes are the main liaisons between the intercellular and extracellular environment, playing a critical role in numerous biological processes. Recent research has challenged the long-standing “fluid mosaic model,” representing membranes as densely packed, heterogeneous environments. Within these complex membranes are transmembrane proteins which comprise up to 50% of the membrane mass, and are themselves diverse in sequence, structure, and function. Combining two-dimensional infrared spectroscopy (2D IR) and molecular dynamics simulation (MD), this thesis explores membrane complexity from two perspectives: first, it addresses the sequence heterogeneity in transmembrane peptides; and second, it explores the effect this crowded environment has on the lipids themselves and the implications this has on future membrane studies. Site-specific hydration of transmembrane peptides was probed using singly isotope-labeled pH (Low) Insertion Peptides, or pHLIPs. These peptides are a class of small transmembrane helices containing ~30% polar residues. By including a single-residue 13C=18O isotope label on the pHLIP backbone, IR experiments effectively produce a single-residue spectrum separate from the main peptide peak. With computational models to connect atomistic structure from MD to infrared frequency shifts, these site-specific spectra reveal local hydration as far as 1 nm into the hydrophobic membrane core. Crowding experiments probed dynamics at the lipid-water interface of model membranes as a function of transmembrane peptide concentration. These dynamics, drawn from time-dependent 2D IR, trend non-monotonically with peptide concentration, revealing three dynamical regimes: a pure lipid-like, a bulk-like, and a crowded regime. Through similar computational methods, these dynamics were linked to water structure at the lipid-water interface, which is perturbed by peptide insertion. Finally, preliminary work has been carried out in developing transient 2D IR methods for applications to protein folding. The first pH-jump 2D IR experiment has been performed by implementing an ultraviolet pump laser to a photoacid-containing sample. Pumping the sample with UV dissociates the photoacid, causing an instantaneous, local pH drop, and the effect on the sample is probed by 2D IR. This new method extends the picosecond-scale 2D IR experiment to a micro-to-millisecond timescale, and has potential for studying pH-initiated conformational transitions such as protein folding and polymerization
An estimated 35% of the human proteome is intrinsically disordered. Disordered proteins play a key role in physiologic and pathologic regulation, recognition, and signaling making protein disorder the subject of increasing investigation. Since disordered samples do not generate x-ray quality crystals and since they have conformations that interconvert faster than the time resolution of NMR or ESR, little is known about their structure or function. By combining isotope-edited two-dimensional infrared spectroscopy (2D IR) with spectral modeling based on molecular dynamics simulations, this work will show that one can measure the residual structure and conformational heterogeneity of a putatively disordered sequence. This methodology was first used to study the structure and dynamics of the tryptophan zipper 2 (TZ2) peptide. The TZ2 peptide is a 12 residue engineered sequence that employs the "tryptophan zipper" motif to stabilize a p-hairpin fold. For this work five isotopologues of TZ2 were synthesized, the p-turn label K8, the midstrand labels T10 and T3T1O, the N-terminal label S1, and the unlabeled peptide UL. Temperature dependent FTIR and 2D IR studies in conjunction with modeling revealed that the TZ2 peptide at low temperatures exists as a folded peptide with a type I' turn and a small previously unobserved subpopulation of a bulged loop structure. Upon heating a fraying of the peptides termini is observed, in addition the population of the type I' turn increases relative to the population of the bulged turn. The TZ2 peptide provided a model hairpin system to test the utility of different forms of analysis and isotope labeling patterns for the subsequent study of disordered hairpin peptides. This methodology was next employed to gain insight into the structure of the elastin-like peptide GVGVPGVG, a prototypical disordered system. For this work nine isotopologues were studied in addition to size dependent variants based on the VPGVG sequence and point mutants variants of the form GVGXPGVG. The conformational ensemble was found to contain a high population of irregular P-turns possessing two peptide hydrogen bonds to the proline C=O (see figure below). Further, it was found that this population grows as a function of 1) side-chain volume for the peptide series GVGXPGVG, where X = Gly, Ala, and Val, and 2) polymer size for the peptide series GVG(VPGVG)a, where n = 1-6 and 251. These findings provide new insight into the molecular origin of the mechanical properties of biopolymers containing XPG turns including collagen (X = Pro), elastin (X = Val), mussel byssus (X = Gly), dragline spider silk (X = Gly), and wheat glutenin (X = Gln).`