Session D06: Physics of Proteins I: Structure & Dynamics of Proteins I

Revealing the origin of multiphasic dynamic behaviors in cyanobacteriochrome

Cyanobacteriochromes are photoreceptors in cyanobacteria that exhibit a wide spectral coverage and unique photophysical properties from the photoinduced isomerization of a linear tetrapyrrole chromophore. Here, we integrate femtosecond-resolved fluorescence and transient-absorption methods and unambiguously showed the significant solvation dynamics occurring at the active site from a few to hundreds of picoseconds. These motions of local water molecules and polar sidechains are continuously convoluted with the isomerization reaction, leading to a nonequilibrium processes with continuous active-site motions. By mutations of critical residues at the active site, the modified local structures become looser, resulting in faster solvation relaxations and isomerization reaction. The observation of solvation dynamics is significant and critical to the correct interpretation of often observed multiphasic dynamic behaviors and thus the previously invoked ground-state heterogeneity may not be relevant to the excited-state isomerization reaction.   

Photoswitching of Terahertz Structural Dynamics in the Photosynthesis Photo Protective Orange Carotenoid Protein

Protein long-range structural vibrations enable efficient conformational transitions during biological function.  Conformational changes play a critical role in the regulation of activity of the photo protective protein, orange carotenoid protein (OCP).  Exposure to bright blue-green light results in conversion from the compact orange resting state (OCP^o) to the open active red state (OCP^R) which can then interact with the light harvesting antenna, the phycobilisome (PBS), to induce fluorescent quenching.  We examine how structural vibrations may actuate the transition using anisotropic terahertz microspectroscopy (ATM).   We find that the intramolecular vibrations switch with photo excitation, with reversible shifting of the anisotropic bands in the ca. 50 cm^-1 range.  Comparison with normal mode ensemble analysis calculations indicates that the measured changes coincide with the loss of the H-bonds between residues Y201 and W288 and the keto group of the carotenoid in the C terminal domain.  This change in the internal dynamics with the initial photoexcitation may provide the motional bias towards the subsequent reorganization of the N-terminal domain (NTD) and the eventual opening between the NTD and the C- terminal domain necessary for the interaction with the phycobilisome.   

Probing Protein Motions for Sequence Fidelity Control or Information Detection along DNA

In template-based polymerization or transcription elongation, fidelity is mainly controlled by RNA polymerase (RNAP) selectivity. Although it is understood that RNAP operates in non-equilibrium nucleotide addition cycles to improve accuracy, it is not clear how it communicates with DNA template to assist cognate nucleotide incorporation. We have combined kinetic modeling with all-atom molecular dynamics (MD) simulation to show most critical residue motions supporting the nucleotide 'recognition' and selectivity, from phage T7 to SARS-CoV-2 viral replication. The atomistic MD simulations are also useful in demonstrating hydrogen bonding (HB) interaction at the protein-DNA interface. The rate-limiting collective HB dynamics is revealed in our recent work showing spontaneous stepping of a transcription factor (TF) protein along DNA. Modeled to coarse-grained level, protein-DNA electrostatics maintain significant impacts, for example, with the TF protein preferentially associating with one DNA strand in facilitated diffusion. Additionally, we have found that the protein-DNA associations can be notably impacted by protein rotation or re-orientation, which lead to a hierarchical free energy landscape of protein diffusion and dissociation along DNA for target search.     

Directional information flow as a tool for analyzing protein allostery

Dynamical network analysis is an invaluable tool for quantifying protein allostery. Existing workflows that apply dynamical network models to molecular dynamics simulations use time-symmetric metrics for identifying correlated motions between residue pairs. However, these methods do not capture the directionality of information flow from which causal relationships can be determined. In recent years, the transfer entropy measure of information flow has been applied to study causal relationships in protein dynamics. We used transfer entropy to develop a workflow for generating causal protein networks based on residue fluctuations and applied it to study allosteric communication in the SARS-CoV-2 main protease, a protein that binds allosteric ligands at various sites. We identified directional information flow between residue contacts emerging at various time lags and determined whether residues generate or receive information from their neighbors. Lastly, we used our transfer entropy network to quantify the directionality of optimal paths, allowing us to determine causal relationships between important binding sites of substrate ligands or drug molecules.   

Photo-response of Photoreceptor Proteins Based on Graphene Field Effect Transistors

Based on the unique properties of graphene, efforts have been made to realize bioelectronic devices, which combine the biological functionalities of a biomolecule to an inorganic component. A detection method can be developed based on optical properties of biomolecules that generate electrical readout. Photoreceptor yellow protein is one kind of light-sensitive protein, which is activated by light and absorbs photons. We have fabricated graphene field-effect transistor (GFETs) devices for biomolecular detection of photoreceptor yellow proteins in the visible range. Experimentally, current-voltage measurement by tuning the gate voltage has been carried out to elucidate the electronic properties of GFETs based biosensors. The wavelength dependent photo-response due to the absorption spectrum of photoreceptor proteins has also been studied under varied illuminations. The device shows a strong photo-response for a low concentration solution of photoreceptor yellow protein.   

From quantum connectivity to biological networks: A window into proteins

While the backbone topologies of protein structures are well understood, a general overarching framework built from the crucial side-chain interactions between the constituent amino acids is not as well-developed. I will present a multidisciplinary approach for characterizing side-chain based protein structure networks. Using a historical context, I will describe the manner in which it synthesizes concepts from quantum physics and chemistry, the biology of polymer conformations, matrix mathematics, and percolation theory. I then discuss a method for constructing the protein structure network and mining the wealth of static, dynamic and evolutionary information contained in the graph spectra of key proteins involved in human health and disease. Finally, as an illustrative example, I will demonstrate the insights that the network approach combined with dynamics can provide in the case of the SARS-CoV-2 spike protein in terms of traits implicated in the virulence exhibited by the current global pandemic. 

In collaboration with:Smitha Vishveshwara, Moitrayee Bhattacharyya, Vasundhara Gadiyaram, Sambit Ghosh, Arinnia Anto, Anushka Halder, Anasuya Dighe, Varsha Subramanyan.    

Crystal Symmetry Effects on Protein Anisotropic Absorption

Long-range protein structural vibrations provide dynamical access to intermediate conformations during biological function.  Anisotropic Terahertz Microspectroscopy provides a method of measuring these vibrations.  To identify the measured resonances with structural motions we compare the spectra to normal mode ensemble analysis calculations. The anisotropic absorbance depends on the orientation of the molecules relative to the light polarization. This orientation is set by the crystal symmetry group and crystal facet perpendicular to the light propagation.  We determine this polarization plane using X-ray crystal face indexing for monoclinic and triclinic hen egg white lysozyme crystals. For monoclinic crystals, unit cell parameters are in agreement with fully hydrated monoclinic symmetry seen in 2D4K.pdb, and with a polarization plane given by the [101] face. Using this, the calculated monoclinic spectra show agreement with the measured spectra. For triclinic crystals, face indexing of the polarization plane did not correspond to a simple index plane. The polarization plane was determined by combining indexing of the other facets with the measured dimensions. The resulting calculated triclinic spectra agreed with observed asymmetry in the spectra and spectral resonances.   

Quantifying viscous coupling between global diffusion and conformational dynamics from simulated protein trajectories

In the simulation of protein folding and binding, it is convenient to analyze the results in a body-fixed reference system.The transition from a lab frame to the protein’s body-fixed frame kinematically isolates the protein’s internal fluctuations. However, it also reveals dynamical couplings between those fluctuations and global diffusion. We present a Langevin equation of motion in the body-fixed frame which explicitly quantifies the instantaneous coupling, via inertial and viscous forces, between the molecule’s global diffusion and its internal conformational fluctuations. Couplings due to inertial effects are expected to have little relevance at low Reynolds numbers. Couplings due to viscous forces, however, cannot be dismissed in principle and require careful examination. We hypothesize that these coupling terms could facilitate slow fluctuations necessary for the protein’s function. Our formalism helps to quantify the extent of the global-internal couplings directly from a Molecular Dynamics simulation trajectory, thus identifying the conditions in which each coupling term actively participates in the dynamics. Additionally, in the quasi-rigid limit of tightly folded proteins, we show how the equations can be linearized and subsequently parameterized from fluctuation statistics obtained via Molecular Dynamics simulation. This leads to coarse-grained equations of motion for the molecule and a description of ballistic and diffusive modes of motion and their timescales. We demonstrate the approach by studying the coupling contributions and linearizations of some model systems.   

Unified free energy landscapes of μ-conotoxins reveal prefolding predictors of folding pathway classification

Conotoxins are short, disulfide-rich peptide toxins produced by cone snails. Their strong affinity for receptors involved in neuromuscular transmission makes them promising therapeutic leads. While at least one conotoxin-based analgesic has been brought to market to date, a greater understanding of the overall subclasses of short disulfide-rich peptides is needed so that the entire class may be developed as a search space rather than a pool of single therapeutic candidates.

The μ conotoxin class exists in a folding spectrum from ordered to disordered--hirudin-like to BPTI-like. In hirudin-like folding, formation of disulfides occurs indiscriminately followed by reshuffling, leading to an ensemble of kinetically trapped isomers. In BPTI-like folding, formation of disulfides occurs through step-by-step formation of the native disulfide bonds. Here, we employ the composite diffusion map approach to study the unified free energy surface of pre-folded equilibrium. We show that in the absence of the disulfides the conotoxins can be thought of as disordered polymers. We identify the most important collective modes of the unified folding landscape and demonstrate that diffusion distance from the projected native state is a predictor of where the conotoxins fall on the folding continuum.  Overall, this work sheds important light on the folding processes and free energy landscapes of different μ-conotoxins and represents an important step in design space exploration.   

A many-body effect renders universal sub-diffusion of water on the surface of different proteins

Dynamics of interfacial water is critical for the function of the enclosed protein. By combining neutron scattering and molecular dynamics simulation on proteins with different surface structures and chemistries including intrinsically disordered proteins, demonstrated that, although the average mobility of the interfacial water molecules depends on the structure of the underlying proteins, they present a universal sub-diffusive motion with a common power law. Further analysis of the simulation trajectories, and analytical modeling reveal that it is the inter-water volume-exclusion effect that overrides the surface differences among proteins and renders the surface water a universal sub-diffusive power law. The present findings significantly recast existing ideas in molecular biophysics that assumes the water dynamics is strongly regulated by the surface properties of the enclosed protein. Instead, we showed that, the power law of diffusive motion of hydration water is independent of the underlying protein and this independence results from the inter-water interaction, which has been rarely considered in the past when describing the diffusion of interfacial water.