Vibrational Spectroscopic Map, Vibrational Spectroscopy, and Intermolecular Interaction.

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.

Structural Ensemble of the Insulin Monomer.

Experimental evidence suggests that monomeric insulin exhibits significant conformational heterogeneity, and modifications of apparently disordered regions affect both biological activity and the longevity of pharmaceutical formulations, presumably through receptor binding and fibrillation/degradation, respectively. However, a microscopic understanding of conformational heterogeneity has been lacking. Here, we integrate all-atom molecular dynamics simulations with an analysis pipeline to investigate the structural ensemble of human insulin monomers. We find that 60% of the structures present at least one of the following elements of disorder: melting of the A-chain N-terminal helix, detachment of the B-chain N-terminus, and detachment of the B-chain C-terminus. We also observe partial melting and extension of the B-chain helix and significant conformational heterogeneity in the region containing the B-chain ß-turn. We then estimate hydrogen-exchange protection factors for the sampled ensemble and find them in line with experimental results for KP-insulin, although the simulations underestimate the importance of unfolded states. Our results help explain the ready exchange of specific amide sites that appear to be protected in crystal structures. Finally, we discuss the implications for insulin function and stability.

Refinement of Peptide Conformational Ensembles by 2D IR Spectroscopy: Application to Ala‒Ala‒Ala.

Characterizing ensembles of intrinsically disordered proteins is experimentally challenging because of the ill-conditioned nature of ensemble determination with limited data and the intrinsic fast dynamics of the conformational ensemble. Amide I two-dimensional infrared (2D IR) spectroscopy has picosecond time resolution to freeze structural ensembles as needed for probing disordered-protein ensembles and conformational dynamics. Also, developments in amide I computational spectroscopy now allow a quantitative and direct prediction of amide I spectra based on conformational distributions drawn from molecular dynamics simulations, providing a route to ensemble refinement against experimental spectra. We performed a Bayesian ensemble refinement method on Ala-Ala-Ala against isotope-edited Fourier-transform infrared spectroscopy and 2D IR spectroscopy and tested potential factors affecting the quality of ensemble refinements. We found that isotope-edited 2D IR spectroscopy provides a stringent constraint on Ala-Ala-Ala conformations and returns consistent conformational ensembles with the dominant ppII conformer across varying prior distributions from many molecular dynamics force fields and water models. The dominant factor influencing ensemble refinements is the systematic frequency uncertainty from spectroscopic maps. However, the uncertainty of conformer populations can be significantly reduced by incorporating 2D IR spectra in addition to traditional Fourier-transform infrared spectra. Bayesian ensemble refinement against isotope-edited 2D IR spectroscopy thus provides a route to probe equilibrium-complex protein ensembles and potentially nonequilibrium conformational dynamics.

The dynamics of peptide-water interactions in dialanine: An ultrafast amide I 2D IR and computational spectroscopy study.

We present a joint experimental and computational study of the dynamic interactions of dialanine (Ala-Ala) with water, comparing the results of ultrafast 2D IR and infrared transient absorption spectroscopy of its amide I vibration with spectra modeled from molecular dynamics (MD) simulations. The experimental data are analyzed to describe vibrational frequency fluctuations, vibrational energy relaxation, and chemical exchange processes. The origin of these processes in the same underlying fluctuating forces allows a common description in terms of the fluctuations and conformational dynamics of the peptide and associated solvent. By comparing computational spectroscopy from MD simulations with multiple force fields and water models, we describe how the dynamics of water hydrogen bond fluctuations and switching processes act as a source of friction that governs the dephasing and vibrational relaxation, and provide a description of coupled water and peptide motions that give rise to spectroscopic exchange processes.

Computational IR Spectroscopy of Insulin Dimer Structure and Conformational Heterogeneity.

We have investigated the structure and conformational dynamics of insulin dimer using a Markov state model (MSM) built from extensive unbiased atomistic molecular dynamics simulations and performed infrared spectral simulations of the insulin MSM to describe how structural variation within the dimer can be experimentally resolved. Our model reveals two significant conformations to the dimer: a dominant native state consistent with other experimental structures of the dimer and a twisted state with a structure that appears to reflect a ∼55° clockwise rotation of the native dimer interface. The twisted state primarily influences the contacts involving the C-terminus of insulin's B chain, shifting the registry of its intermolecular hydrogen bonds and reorganizing its side-chain packing. The MSM kinetics predict that these configurations exchange on a 14 µs time scale, largely passing through two Markov states with a solvated dimer interface. Computational amide I spectroscopy of site-specifically 13C18O labeled amides indicates that the native and twisted conformation can be distinguished through a series of single and dual labels involving the B24F, B25F, and B26Y residues. Additional structural heterogeneity and disorder is observed within the native and twisted states, and amide I spectroscopy can also be used to gain insight into this variation. This study will provide important interpretive tools for IR spectroscopic investigations of insulin structure and transient IR kinetics experiments studying the conformational dynamics of insulin dimer.

Insulin Dissociates by Diverse Mechanisms of Coupled Unfolding and Unbinding.

The protein hormone insulin exists in various oligomeric forms, and a key step in binding its cellular receptor is dissociation of the dimer. This dissociation process and its corresponding association process have come to serve as paradigms of coupled (un)folding and (un)binding more generally. Despite its fundamental and practical importance, the mechanism of insulin dimer dissociation remains poorly understood. Here, we use molecular dynamics simulations, leveraging recent developments in umbrella sampling, to characterize the energetic and structural features of dissociation in unprecedented detail. We find that the dissociation is inherently multipathway with limiting behaviors corresponding to conformational selection and induced fit, the two prototypical mechanisms of coupled folding and binding. Along one limiting path, the dissociation leads to detachment of the C-terminal segment of the insulin B chain from the protein core, a feature believed to be essential for receptor binding. We simulate IR spectroscopy experiments to aid in interpreting current experiments and identify sites where isotopic labeling can be most effective for distinguishing the contributions of the limiting mechanisms.

Studying Protein-Protein Binding through T-Jump Induced Dissociation: Transient 2D IR Spectroscopy of Insulin Dimer.

Insulin homodimer associates through the coupled folding and binding of two partially disordered monomers. We aim to understand this dynamics by observing insulin dimer dissociation initiated with a nanosecond temperature jump using transient two-dimensional infrared spectroscopy (2D IR) of amide I vibrations. With the help of equilibrium FTIR and 2D IR spectra, and through a systematic study of the dependence of dissociation kinetics on temperature and insulin concentration, we are able to decompose and analyze the spectral evolution associated with different secondary structures. We find that the dissociation under all conditions is characterized by two processes whose influence on the kinetics varies with temperature: the unfolding of the ß sheet at the dimer interface observed as exponential kinetics between 250 and 1000 µs and nonexponential kinetics between 5 and 150 µs that we attribute to monomer disordering. Microscopic reversibility arguments lead us to conclude that dimer association requires significant conformational changes within the monomer in concert with the folding of the interfacial ß sheet. While our data indicates a more complex kinetics, we apply a two-state model to the ß-sheet unfolding kinetics to extract thermodynamic parameters and kinetic rate constants. The association rate constant, ka (23 °C) = 8.8 × 10(5) M(-1) s(-1) (pH 0, 20% EtOD), is approximately 3 orders of magnitude slower than the calculated diffusion limited association rate, which is explained by the significant destabilizing effect of ethanol on the dimer state and the highly positive charge of the monomers at this pH.