Session K50: Physics of Proteins III: Experimental and Computational Studies on the Structure and Conformational Dynamics of Proteins

Dynamics and Mechanism of UVR8 Photoreceptor

UVR8 (UV RESISTANCE LOCUS 8) proteins are a class of UV-B photoreceptors in high plants. UVR8 is a homodimer that dissociates into monomers upon UV-B irradiation (280 to 315 nm), which triggers various protective mechanisms against UV damages. Uniquely, UVR8 does not contain any external chromophores and utilizes the natural amino acid tryptophan (Trp) to perceive UV-B light. Each UVR8 monomer has 14 tryptophan residues. However, only the epicenter two Trp (W285 W233) residues are critical to the light-induced dimer-to-monomer transformation. Here, combining time-resolved spectroscopy and extensive site-directed mutations, we have revealed the entire dynamics of UV perception to lead to monomerization, including a series of critical dynamical processes of a striking energy-flow network, exciton charge separation and recombination, charge neutralization, salt bridge zipping and protein solvation, providing a complete molecular picture of the initial biological function.   

From photon to biological signaling: emerging principles in the biophysical mechanism of photoreceptor activation

Photosensory proteins convert photon absorbance into protein conformational changes that initiate biological signaling. Six distinct classes of photoreceptors with diverse folds, chromophores, and initial photochemical events have been studied in detail. We analyzed results for well-studied members of each of these photoreceptors to identify emerging trends in the molecular mechanism of their function. First, the primary photochemical event usually occurs on an ultrafast time scale and involves only very small atomic motions limited to the light-absorbing chromophore. A key question is how these ultrafast, localized conformational changes trigger large-amplitude, long-lived conformational changes for signaling. Time-resolved studies show that in all six receptor types the initial photochemical event is followed by much slower intramolecular proton transfer, and subsequently by large protein conformational changes. A dual role for these proton transfer events is emerging, causing changes in hydrogen bonding in some photoreceptors and changes in electrostatics in others, which then drive conformational changes for receptor activation. These results provide a broad mechanistic and energetic framework for dissecting the mechanism of photoreceptor function.   

Dynamics of Critical Charge Separation in UVR8 Light-induced Monomerization

Photoreceptors exist in many living organisms to fulfill critical biological functions including photoprotection, vision and regulation of life cycles. UVR8 (UV RESISTANCE LOCUS 8) is a UV-B (ultraviolet-B, 280-315 nm) photoreceptor in plants. In the absence of UV light, UVR8 is a protein homodimer, and UV absorption leads to UVR8 monomerization, triggering various UV-protective and photomorphogenesis signaling events. UVR8 does not contain any external chromophore and uses natural amino acid tryptophan (Trp) for light perception. Each UVR8 monomer has 14 tryptophan residues. At the dimer interface, flanked by critical inter-subunit salt-bridges, two symmetrical “Trp pyramid” clusters are formed by close packing of Trp residues, serving as the reaction center. Here, with extensive mutagenesis studies, femtosecond resolved spectroscopy and computational methods, we revealed the reaction dynamics of charge separation during UVR8 light-induced monomerization.   

A Study of Boson Peak and Fracton in Protein Lysozyme by Terahertz Time-domain Spectroscopy

Terahertz time-domain spectroscopy and low-frequency Raman scattering have been performed on protein hen egg white lysozyme to investigate the boson peak (BP) and fracton dynamics. The BP dynamics is a universal feature in the glassy states and the fracton dynamics is a universal feature of fractal objects. In the α(v)/ν² plot of the infrared spectrum, where α(v) is attenuation, the boson peak of lysozyme was detected at about 0.58 THz at room temperature. In Raman spectrum, the BP was observed at about 0.82 THz at room temperature from Raman susceptibility divided frequency, which is χ''(ν)/ν spectrum. Neither the imaginary part of complex dielectric constant nor Raman susceptibility shows absorption peak around their respective BP frequency. In the frequency region above the BP, the fracton behavior has been observed both in the infrared and Raman spectrum of lysozyme. The fracton region has a frequency range from 0.58 THz to 3.3 THz in the infrared spectrum, and 0.82 THz to 2.6 THz in the case of Raman spectrum.   

Cellular-Scale Proton Transfer in Weak Magnetic Fields

The problem of proton transfer within the presence of a weak magnetic field is examined on the cellular scale by way of examples such as the asymmetric double potential well and quantum tunneling. The effect(s) resulting from perturbing a system with a weak magnetic field are then hypothesized.   

Electrostatic effects on facilitated dissociation of molecular ligands

Electrostatic interactions between molecular ligands and their binding sites control many aspects in biomachinery from gene regulation to molecular recognition. In this study, unbinding of multivalent cationic ligands from charged polymeric binding sites is considered. Our molecular dynamics (MD) study is inspried by the good accord between the experiments and MD simulations of single-molecule studies of protein-DNA interactions (Kamar et al, PNAS, 2017). We consider univalent salt concentrations spanning a thousandfold range, together with various concentrations of excess ligands to reveal the ionic effects on spontaneous and facilitated dissociation (FD) unbinding mechanisms. We treat electrostatic interactions both at a Debye-H\”{u}ckel level, as well as by the more precise approach of considering all ionic species explicitly. Both treatments show that salt dependence of FD process becomes weaker with increasing free ligand concentration. Our simulations also predict a variety of FD regimes as a function of free ligand concentration (Erbas et al, arXiv, 2017).   

Model for Concentration-Dependent Effects of Charge Regulation on Protein Solution Thermodynamics and Liquid-liquid Phase Separation

We study, theoretically, the effects of charge regulation of an eye lens protein, bovine gammaB-crystallin, on its thermodynamic properties. In a recent analysis we found that near neutral pH, because of continual exchange of protons with the solvent, approximately 400 charge patterns of this protein occur often enough to affect protein-protein interactions. We build a free energy model that incorporates orientation-dependent short-range protein-protein interactions and accommodates specific pairs of protonation patterns. The effective potential includes simplified, screened electrostatic interactions between pattern pairs, hard-core interactions, and dispersion forces. We apply conditions for multiple chemical equilibria and for multicomponent phase separation of proteins that are taken to have fixed patterns. The first-order concentration-dependent effect is that pattern probabilities increase with increasing concentration for members of those pairs of patterns that have more net protein-protein attraction. The model gives a way to study relationships between phase boundaries and chemical equilibria, because the intersection of the chemical equilibrium curve with the multicomponent phase boundaries yields the binary liquid-liquid phase boundary.   

Understanding the structural and mechanical properties of transmembrane proteins

Transmembrane proteins (TM) are embedded in cell membranes and play a crucial role in transport, cellular interactions, and signaling. TM proteins account for nearly a third of all natural proteins and serve as targets for a wide array of drug therapies. However, due to a lack of available high-resolution data, many features of the structure of TM proteins are not well known. We analyzed the composition, side chain conformations, and packing fraction of buried residues in TM proteins using available protein crystal structures. We find that buried residues in TM and soluble proteins have similar packing fraction distributions. In addition, we show that the hard-sphere plus stereochemical constraint model is able to recapitulate the side chain dihedral angle conformations of buried residues observed in crystal structures. This result emphasizes that steric interactions are dominant in determining the structure of buried residues, even in the TM proteins. Thus, we find that the structure of buried residues in TM is similar to that for core residues in soluble proteins.   

Underlying subwavelength pinhole affects the optical properties of microcavity surface plasmon resonance biosensors

Our previous work showed that submicron dielectric cores covered in gold proved to be a very sensitive surface plasmon biosensors capable of real-time monitoring of selected biomolecular interactions. The microcavity surface plasmon resonance sensors (MSPRS) were used to analyze conformational changes of bound biomolecules as the oxidation state changes. However, the biosensor gold shell generates a subwavelength nanoaperture at the point of contact between the dielectric core and the glass substrate. The surrounding structure is believed to excite stationary plasmon resonances at the biosensor’s surface evidentiated through spectral minima and maxima in the visible range. In this work we present data showing that the size of the underlying pinhole drastically affects the optical properties of the biosensor and therefore its sensitivity. Visible range optical spectroscopy is used to study the wavelength shift and spectral light intensity as the refractive index is changing at the outer shell surface. Scanning Electron Microscopy (SEM) and Focused Ion Beam (FIB) were used to study the characteristics of the underlying subwavelength aperture and microfluidics was used as a flexible platform for housing fabricated biosensors.   

Protein Evolution and Emergence of Novel Catalytic Functions

Enzyme evolution underlies major expansions of metabolic complexity with profound biological implications. In this presentation, I will discuss emergence of cyclization reactions catalyzed by terpene synthases. Cyclic terpenes mediate numerous biological functions in modern plants (such as attracting pollinators and repelling microbial pathogens), and provide bioactive compounds for human use, including artemisinin, the most effective treatment for malaria currently available. Guided by the available structural, kinetic, and sequence data, we have constructed mutant libraries which include combinations of amino acids responsible for inducing cyclization reactions in an enzyme that produces beta-farnesene, a linear hydrocarbon chain. We have used measurements of kinetic rates and mass spectrometry in order to assess catalytic efficiency and specificity of the mutant enzymes. Using spin glass models adapted from statistical physics, we have inferred evolutionary patterns of enzyme energetics, as well as sequence signatures of active vs. inactive terpene synthases. Our studies provide quantitative insights into evolutionary dynamics of a major enzyme family, and highlight the importance of epistasis in molecular evolution.   

Critical Phenomena in the Temperature-pressure-crowding Phase Diagram of a Large Protein

In the cell, large proteins fold and perform complex functions through global structural rearrangements. Such large-scale dynamics are influenced by solvent fluctuations and the excluded volume from surrounding macromolecules. This requires a protein to be susceptible to small environmental agitations, yet stable to maintain structural integrity. These apparently competing behaviors are commonly exhibited by physical systems near a critical point, where distinct phases merge; a concept that is contrary to previous studies indicating proteins fold/unfold into well-defined phases on the pressure-temperature plane. Here, by characterizing the behavior of a large (~45 kDa) two-domain protein, phosphoglycerate kinase (PGK), on the temperature (T), pressure (P), and crowding volume-fraction (φ_c) phase diagram, we demonstrate a critical transition where several phases coexist. Above a critical point at a certain T and P, intermediate conformations between folded and unfolded phases disappear. When φ_c increases, this point moves along the T - P plane. Remarkably, crowding places PGK near a critical surface in its natural parameter space, where large conformational changes can occur without thermodynamic penalties.   

Structural-elastic Determination of the Lifetime of Biomolecules under Force

The lifetime of protein domains and ligand-receptor complexes under force is crucial for both mechanochemistry and mechanobiology. However, how force affects the lifetime remains poorly understood. Currently, most of the models are derived based on a presumed shape of the energy surface, which restricts the scope of their applications to explain experimental data. Here we report a novel analytical expression of the force-dependent rate (the reciprocal of lifetime) of protein unfolding or ligand-receptor complex rupturing based on the structural-elastic properties of the molecules. This new model is able to fit a wide scope of experimentally measured force-dependent rates, including many experimental data showing complex derivation from Bell’s model, such as the "catch-to-slip" behaviour. Most importantly, the best-fitting parameters of our model directly inform us of the differential structural-elastic properties of the molecules between the transition and native states. Further, combined with the structural-elastic properties of the native state that can be obtained from the molecular structure and all-atom molecular dynamics simulations, the structural-elastic properties of the transition state of the molecule can be uniquely determined from the values of fitting parameters.   

Inter-head Tension of Cytoplasmic Dynein Regulates the Coordination between Two Heads

Studying the coordination between two heads of a motor protein is crucial to understand the walking mechanism of the motor protein on cytoskeletal tracks. Previous experiments found that inter-head tension of a cytoplasmic dynein was able to regulate the coordination between its two heads. However, the molecular origin is largely unknown. Here we utilized a structure-based coarse-grained model to investigate the structural changes of a cytoplasmic dynein monomer responding to opposite forces. We identified the molecular origin of the coupling between the AAA ring of a cytoplasmic dynein and its binding affinity to microtubules. Importantly, our simulation explained why this coupling is regulated by the direction of forces. Based on this work, we established an analytical expression of the detach rate of cytoplasmic dynein with respect to forces, which matches well with experimental findings. Our results provide a molecular basis to understand the walking pattern of cytoplasmic dynein.