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Understanding the structure and dynamics of proteins is essential to understanding their roles and functions in these physiological processes. In this thesis, I describe the implementation of an ultrafast nonlinear spectroscopic technique, two-dimensional infrared (2D IR) spectroscopy to probe the structure and dynamics of ion channels and amyloid fibers. Regarding ion channels, I describe the combination of semisynthesis, 2D IR spectroscopy and molecular dynamic (MD) simulations in addressing the longstanding question of ion permeation through the selectivity filter of a potassium ion channel. I show that ions and water alternate through the filter and that these ions cannot occupy adjacent binding sites. Furthermore, 2D IR experiments revealed a flipped state that is predicted by MD simulations but not observed in x-ray crystallography. In another aspect of this work, we show that the collapsed state of the filter is structurally different in low K+ and low pH. Moreover, our work also reveals how the large conformational motions of the protein are coupled to structural changes in the selectivity filter, as evidenced by a change in the ion occupancy. In a second research direction, I developed an optical technique to quantify photoactivatable fluorophores with fluorescence microscopy. This technique allows for the quantification of a limitless number of fluorophores, and corrects for stochastic events such as fluorescence intermittency. This work can be extended to the study of amyloids, where determining the number of proteins in a prefibrillar aggregates is necessary for understanding their roles in amyloid related diseases. Finally, using 2D IR spectroscopy we describe the effect of common solvents on the anharmonicity of small molecule chromophores. The data indicates that the carbonyl anharmonicity, and, subsequently, the Stark tuning rate, is an intrinsic property of the carbonyl vibrational probes, which have important implications on the interpretation of carbonyl vibrational frequency shifts in the condensed phase.
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.
This book discusses how biological molecules exert their function and regulate biological processes, with a clear focus on how conformational dynamics of proteins are critical in this respect. In the last decade, the advancements in computational biology, nuclear magnetic resonance including paramagnetic relaxation enhancement, and fluorescence-based ensemble/single-molecule techniques have shown that biological molecules (proteins, DNAs and RNAs) fluctuate under equilibrium conditions. The conformational and energetic spaces that these fluctuations explore likely contain active conformations that are critical for their function. More interestingly, these fluctuations can respond actively to external cues, which introduces layers of tight regulation on the biological processes that they dictate. A growing number of studies have suggested that conformational dynamics of proteins govern their role in regulating biological functions, examples of this regulation can be found in signal transduction, molecular recognition, apoptosis, protein / ion / other molecules translocation and gene expression. On the experimental side, the technical advances have offered deep insights into the conformational motions of a number of proteins. These studies greatly enrich our knowledge of the interplay between structure and function. On the theoretical side, novel approaches and detailed computational simulations have provided powerful tools in the study of enzyme catalysis, protein / drug design, protein / ion / other molecule translocation and protein folding/aggregation, to name but a few. This work contains detailed information, not only on the conformational motions of biological systems, but also on the potential governing forces of conformational dynamics (transient interactions, chemical and physical origins, thermodynamic properties). New developments in computational simulations will greatly enhance our understanding of how these molecules function in various biological events.
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.
Theoretical Vibrational Spectroscopy of Proteins Lu Wang Under the supervision of Professor James L. Skinner At the University of Wisconsin-Madison Vibrational spectroscopy, such as linear and two-dimensional infrared (IR) spectroscopy, is widely utilized to study the structure and dynamics of peptides and proteins. Interpretation of the experiment, or a direct assignment of the complex experimental spectra to the underlying protein structure, can be difficult. Molecular dynamics (MD) simulations offer a complementary approach to provide high-resolution structural and temporal information of proteins, although they are limited by factors such as force field accuracy and are not directly comparable to spectroscopic experiments. We have developed vibrational frequency maps for proteins that generate instantaneous site frequencies directly from MD simulations. We combine the frequency maps with established nearest-neighbor frequency shift and coupling schemes and a mixed quantum/classical framework to form a theoretical strategy for calculating protein linear and 2D IR spectra in the amide I region. This theoretical method provides a means to bridge spectroscopic experiments and molecular simulations, which allows a critical assessment of MD simulations by comparison to experiment, and enables the interpretation of experimental spectra at the molecular level. In this dissertation, we present the development of the vibrational frequency maps and provide the theoretical protocol that allows the calculation of protein vibrational spectra directly from MD simulations. We validate the theoretical method by applying it to peptides with various secondary structures in aqueous solution, and apply it to a few biologically relevant problems. For instance, we have studied the thermal unfolding transition of the villin headpiece subdomain (HP36) using IR spectra calculations. We follow the unfolding process of HP36 by monitoring its spectral changes as a function of temperature. With the help of isotope labeling, we are able to capture the feature that helix 2 of HP36 loses its secondary structure before global unfolding occurs, in agreement with experiment. In collaboration with the Zanni group and the de Pablo group at University of Wisconsin, we have also carried out studies on IAPP, a peptide closely related to type 2 diabetes. By combining theoretical modeling with extensive computer simulations and spectroscopic experiments, we have investigated the structure and dynamics of IAPP in aqueous solution, in the fibril form and in the vicinity of lipid membranes.
Complete understanding of protein function requires knowledge of protein conformational dynamics, or the structural fluctuations of a protein. However, characterization of protein dynamics is challenged by protein complexity, as they are large, heterogeneous molecules with potentially important motions on very fast timescales. This complexity demands the use of a technique with high spatial and temporal resolution. Two-dimensional infrared (2D IR) spectroscopy has emerged as a powerful tool for the characterization and direct measurement of molecular heterogeneity and dynamics due to its excellent spatial and temporal resolution. However, application to proteins is hindered by their severely congested spectra due to the large number of similar bonds. To overcome this issue, proteins can be site-specifically labeled with spectrally resolved IR probes that are active in the transparent frequency region (~1800 - 2500 cm-1) and are sensitive to their environment. The studies presented here take advantage of the combination of site-specific labeling and IR spectroscopy to study the environments and dynamics at specific locations in three distinct protein systems. Herein, I describe our investigations of dynamic complexes of proteins that have challenged experimental characterization with conventional methods: plastocyanin (Pc) and its binding partner cytochrome f (cyt f); cytochrome P450cam (P450cam) and substrates or its redox partner, putidaredoxin; and the SH3Sho1 domain and the proline-rich (PR) recognition motif of its binding partner Pbs2. In addition, we describe my attempts at improving the experimental technique of site-specific IR spectroscopy as a general biophysical approach for protein characterization. Overall, I present evidence for the importance of fast dynamics in protein function and illustrate the rich information provided by 2D IR spectroscopy to complement existing biophysical methods.
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).`
Simulations of two-dimensional infrared (2DIR) spectroscopy of several proteins are presented. Applications of 2DIR spectroscopy to protein folding, protein aggregation, and photosensing are reported. We demonstrate that 2DIR spectroscopy is an excellent probe of protein structure and dynamics. Our simulations predict future experiments as well as provide detailed explanations of previous experiments. We also present simulations of the related two-dimensional ultraviolet and two-dimensional stimulated resonance Raman spectroscopies, which are shown to provide complementary information to 2DIR spectroscopy. This thesis can be viewed as a guide for the design and analysis of future two-dimensional optical measurements on proteins.