Conformational Fluctuations Research Articles

Conformational dynamics of androgen receptors bound to agonists and antagonists

The androgen receptor (AR) is critical in the progression of prostate cancer (PCa). Small molecule antagonists that bind to the ligand binding domain (LBD) of the AR have been successful in treating PCa. However, the structural basis by which the AR antagonists manifest their therapeutic efficacy remains unclear, due to the lack of detailed structural information of the AR bound to the antagonists. We have performed accelerated molecular dynamics (aMD) simulations of LBDs bound to a set of ligands including a natural substrate (dihydrotestosterone), an agonist (RU59063) and three antagonists (bicalutamide, enzalutamide and apalutamide) as well as in the absence of ligand (apo). We show that the binding of AR antagonists at the substrate binding pocket alter the dynamic fluctuations of H12, thereby disrupting the structural integrity of the agonistic conformation of AR. Two antagonists, enzalutamide and apalutamide, induce considerable structural changes to the agonist conformation of LBD, when bound close to H12 of AR LBD. When the antagonists bind to the pocket with different orientations having close contact with H11, no significant conformational changes were observed, suggesting the AR remains in the functionally activated (agonistic) state. The simulations on a drug resistance mutant F876L bound to enzalutamide demonstrated that the mutation stabilizes the agonistic conformation of AR LBD, which compromises the efficacy of the antagonists. Principal component analysis (PCA) of the structural fluctuations shows that the binding of enzalutamide and apalutamide induce conformational fluctuations in the AR, which are markedly different from those caused by the agonist as well as another antagonist, bicalutamide. These fluctuations could only be observed with the use of aMD.

Identification of the structural features of quinazoline derivatives as EGFR inhibitors using 3D-QSAR modeling, molecular docking, molecular dynamics simulations and free energy calculations

Epidermal growth factor receptor (EGFR) is a promising target for the treatment of different types of malignant tumors. Therefore, a combined molecular modeling study was performed on a series of quinazoline derivatives as EGFR inhibitors. The optimum ligand-based CoMFA and CoMSIA models showed reliable and satisfactory predictability (with R2 cv=0.681, R2 ncv=0.844, R2 pred=0.8702 and R2 cv=0.643, R2 ncv=0.874, R2 pred=0.6423). The derived contour maps provide structural features to improve inhibitory activity. Furthermore, the contour maps, molecular docking, and molecular dynamics (MD) simulations have good consistency, illustrating that the derived models are reliable. In addition, MD simulations and binding free energy calculations were also carried out to understand the conformational fluctuations at the binding pocket of the receptor. The results indicate that hydrogen bond, hydrophobic and electrostatic interactions play significant roles on activity and selectivity. Furthermore, amino acids Val31, Lys50, Thr95, Leu149 and Asp160 are considered as essential residues to participate in the ligand-receptor interactions. Overall, this work would offer reliable theoretical basis for future structural modification, design and synthesis of novel EGFR inhibitors with good potency. Communicated by Ramaswamy H. Sarma

Impacts of mutations on dynamic allostery of adenylate kinase.

Escherichia coli adenylate kinase (AK) is composed of CORE domain and two branch domains: LID and AMP-binding domain (AMPbd). AK exhibits considerable allostery in a reversible phosphoryl transfer reaction, which is largely attributed to the relative motion of LID and AMPbd with respect to CORE. Such an allosteric conformational change is also evident in the absence of ligands. Recent studies showed that the mutations in branch domains can adjust dynamic allostery and alter the substrate affinity and enzyme activity. In this work, we use all-atom molecular dynamics simulation to study the impacts of mutations in branch domains on AK's dynamic allostery by comparing two double mutants, i.e., LID mutant (Val135Gly, Val142Gly) and AMPbd mutant (Ala37Gly, Ala55Gly), with wild-type. Two mutants undergo considerable conformational fluctuation and exhibit deviation from the initially extended apo state to more compact structures. The LID domain in the LID mutant adjusts its relative position and firmly adheres to CORE. More strikingly, AMPbd mutations affect the relative positions of both the AMPbd domain and remote LID domain. Both domains undergo considerable movement, especially the inherent hinge swing motion of the flexible LID domain. In both mutants, the mutations can enhance the inter-domain interaction. The results about the conformation change of AK in both mutants are in line with the experiment of AK's affinity and activity. As revealed by our findings, the flexibility of branch domains and their inherent motions, especially LID domain, is highly relevant to dynamic allostery in the AK system.

Unfolding of Dynamical Events in the Early Stage of Insulin Dimer Dissociation.

The dissociation of an insulin dimer is an important biochemical event that could also serve as a prototype of dissociations in similar biomolecular assemblies. We use a recently developed multidimensional free energy landscape for insulin dimer dissociation to unearth the microscopic and mechanistic aspects of the initial stages of the process that could hold the key to understanding the stability and the rate. The following sequence of events occurs in the initial stages: (i) The backbone hydrogen bonds break partially at the antiparallel β-sheet junction, (ii) the two α-helices (chain B) move away from each other while several residues (chain A) move closer, and (iii) a flow of adjacent water molecules occurs into the junction region. Interestingly, the intermonomeric center-to-center distance does not increase, but the number of native contacts exhibits a sharp decrease. Subsequent steps involve further disengagement of hydrophobic groups. This process is slow because of an entropic bottleneck created by the existence of the large configuration space available in the native state (NS), which is inhabited by low-frequency conformational fluctuations. We carry out a density-of-states analyses in the dimer NS to unearth distinctive features not present in the monomers. These low-frequency modes are also responsible for a large entropic stabilization of the NS. Hydrophobic disengagement in the early stage leads to the formation of a twisted intermediate state which itself is a metastable minimum (IS-1). The subsequent progress leads to another dimeric complex (IS-2), which is on the dissociative pathway and characterized by a further decrease in the native contacts. The dissociation process provides insights into the workings of a biomolecular assembly.

Ultra-sensitive lock-in amplifier coupled oscillatory magnetic tweezers for piconewton force manipulation applications

We have developed lock-in amplifier coupled oscillatory magnetic tweezers, aiming to synchronize the oscillatory magnetic force application and single-molecule response detection at the applied oscillatory frequency by incorporating an optical lock-in amplifier detection. The designed home-built lock-in amplifier detection instrument enables an exact reference signal input and hence reveals an accurate and sensitive magnetic response synchronization. We have further demonstrated the approach with the rhodamine 6G stained super-paramagnetic beads by monitoring the below-the-noise-background weak fluorescence signal changes due to the magnetic response of the super-paramagnetic beads under the oscillatory force manipulation. The integration of the lock-in amplifier and the oscillating magnetic tweezers can significantly expand the application of the magnetic tweezers for signal detection below the noise background, such as adapting to the important applications in the detailed exploration of mechanical properties of biomolecules and studies of the protein conformational fluctuation dynamics.

In silico analysis and identification of antiviral coumarin derivatives against 3-chymotrypsin-like main protease of the novel coronavirus SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) is a pandemic viral disease caused by SARS-CoV-2 that generated serious damages for both the human population and the global economy. Therefore, it is currently considered as one of the most important global health problems of human societies and there is an urgent need for potent drugs or vaccines which can effectively combat this virus. The chymotrypsin-like protease (3CLpro) of SARS-CoV-2 plays a key role in the viral replication inside the host and thus is a promising drug target to design and develop effective antiviral drugs against SARS and other coronaviruses. This study evaluated some antiviral coumarin phytochemicals as potential inhibitors of coronaviruses 3CLpro by in silico approaches such as molecular docking, ADMET prediction, molecular dynamics simulation, and MM-PBSA binding energy calculation. Natural coumarin derivatives were docked to the 3CLpro of SARS-CoV-2 and for further investigation, docked to the 3CLpro of SARS-CoV and MERS-CoV. The docking scores of these natural compounds were compared with 3CLpro referenced inhibitors (ritonavir and lopinavir) and co-crystal inhibitor N3. Molecular docking studies suggested more than half of the coumarin phytochemicals had favorable interaction at the binding pocket of the coronaviruses 3CLpro and exhibited better binding affinities toward 3CLpro than ritonavir and lopinavir. Most antiviral phytochemicals interact strongly with one or both the catalytic dyad residues (His41 and Cys145) and the other key residues of SARS-CoV-2 main protease. Further, MD simulation and binding free energy calculations using MM-PBSA were carried out for three 3CLpro-coumarin complexes and 3CLpro-N3/lopinavir. The results confirmed that the 3CLpro-glycycoumarin, 3CLpro-oxypeucedanin hydrate, and 3CLpro-inophyllum P complexes were highly stable, experience fewer conformation fluctuations and share a similar degree of compactness. Also, the pharmacokinetics and drug-likeness studies showed good results for the selected coumarin phytochemicals.Therefore, the coumarin phytochemicals could be used as antiviral agents in the treatment of COVID-19 after further studies.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s11030-021-10230-6.

Dynamics and rheology of ring-linear blend semidilute solutions in extensional flow: Single molecule experiments

Ring polymers exhibit unique flow properties due to their closed chain topology. Despite recent progress, we have not yet achieved a full understanding of the nonequilibrium flow behavior of rings in nondilute solutions where intermolecular interactions greatly influence chain dynamics. In this work, we directly observe the dynamics of DNA rings in semidilute ring-linear polymer blends using single molecule techniques. We systematically investigate ring polymer relaxation dynamics from high extension and transient and steady-state stretching dynamics in a planar extensional flow for a series of ring-linear blends with varying ring fraction. Our results show multiple molecular subpopulations for ring relaxation in ring-linear blends, as well as large conformational fluctuations for rings in a steady extensional flow, even long after the initial transient stretching process has subsided. We further quantify the magnitude and characteristic time scales of ring conformational fluctuations as a function of blend composition. Interestingly, we find that the magnitude of ring conformational fluctuations follows a nonmonotonic response with increasing ring fraction, first increasing at low ring fraction and then substantially decreasing at large ring fraction in ring-linear blends. A unique set of ring polymer conformations are observed during the transient stretching process, which highlights the prevalence of molecular individualism and supports the notion of complex intermolecular interactions in ring-linear polymer blends. In particular, our results suggest that transient intermolecular structures form in ring-linear blends due to a combination of direct forces due to linear chains threading through open rings and indirect forces due to hydrodynamic interactions; these combined effects lead to large conformational fluctuations of rings over distributed time scales. Taken together, our results provide a new molecular understanding of ring polymer dynamics in ring-linear blends in the nonequilibrium flow.

Dynamics and rheology of ring-linear blend semidilute solutions in extensional flow. Part I: Modeling and molecular simulations

We use Brownian dynamics (BD) simulations and single molecule experiments to investigate the influence of topological constraints and hydrodynamic interactions on the dynamics and rheology of solutions of ring-linear polymer blends at the overlap concentration. We find agreement between simulation and experiment in which rings in solution blends exhibit large conformational fluctuations. A subpopulation of rings shows extension overshoots in the startup of the flow, and other populations display tumbling and tank-treading at the steady state. Ring polymer fluctuations increase with the blend fraction of linear polymers and are peaked at a ring Weissenberg number WiR≈1.5. On the contrary, linear and ring polymers in pure solutions show a peak in fluctuations at the critical coil-stretch Weissenberg number Wi=0.5. BD simulations show that extension overshoots on the startup of the flow are due to flow-induced intermolecular ring-linear polymer hooks, whereas fluctuations at the steady state are dominated by intermolecular hydrodynamic interactions (HIs). This is supported by simulations of bidisperse linear polymer solution blends, which show similar trends in conformational dynamics between rings and linear polymers with a matched contour length. Compared to BD simulations, single molecule experiments show quantitatively larger fluctuations, which could arise because experiments are performed on higher molecular weight polymers with stronger topological constraints. To this end, we have advanced the understanding of the effects of topological interactions and intermolecular HIs on the dynamics of semidilute ring-linear polymer blend solutions.

Transition-State Compressibility and Activation Volume of Transient Protein Conformational Fluctuations.

Proteins are dynamic entities that intermittently depart from their ground-state structures and undergo conformational transitions as a critical part of their functions. Central to understanding such transitions are the structural rearrangements along the connecting pathway, where the transition state plays a special role. Using NMR relaxation at variable temperature and pressure to measure aromatic ring flips inside a protein core, we obtain information on the structure and thermodynamics of the transition state. We show that the isothermal compressibility coefficient of the transition state is similar to that of short-chain hydrocarbon liquids, implying extensive local unfolding of the protein. Our results further indicate that the required local volume expansions of the protein can occur not only with a net positive activation volume of the protein, as expected from previous studies, but also with zero activation volume by compaction of remote void volume, when averaged over the ensemble of states.

It Is Good to Be Flexible: Energy Transport Facilitated by Conformational Fluctuations in Light-Harvesting Polymers.

We investigate the mechanism of energy transfer between ruthenium(II) (Ru) and osmium(II) (Os) polypyridyl complexes affixed to a polyfluorene backbone (PF-RuOs) using a combination of time-resolved emission spectroscopy and coarse-grained molecular dynamics (CG MD). Photoexcitation of a Ru chromophore initiates Dexter-style energy hopping along isoenergetic complexes followed by sensitization of a lower-energy Os trap. While we can determine the total energy transfer rate within an ensemble of solvated PF-RuOs from time-dependent Os* emission spectra, heterogeneity of the system and inherent polymer flexibility give rise to highly multiexponential kinetics. We developed a three-part computational kinetic model to supplement our spectroscopic results: (1) CG MD model of PF-RuOs that simulates molecular motions out to 700 ns, (2) energy transfer kinetic simulations in CG MD PF-RuOs that produce time-resolved Ru and Os excited-state populations, and (3) computational experiments that interrogate the mechanisms by which motion aids energy transfer. Good agreement between simulated and experimental emission transients reveals that our kinetic model accurately simulates the molecular motion of PF-RuOs during energy transfer. Simulated results indicate that pendant flexibility allows 81% of the excited state to sensitize an Os trap compared to a 48% occupation when we treat pendants statically. Our computational experiments show how static pendants are only able to engage in local energy transfer. The excited state equilibrates across a domain of complexes proximal to the initial excitation and becomes trapped within that unique, frozen locality. Side-chain flexibility enables pendants to swing in and out of the original domain spreading the excited state out to ±30 pendant complexes away from the initial excitation.

Base pair fluctuations in helical models for nucleic acids.

A statistical method is developed to estimate the maximum amplitude of the base pair fluctuations in a three dimensional mesoscopic model for nucleic acids. The base pair thermal vibrations around the helix diameter are viewed as a Brownian motion for a particle embedded in a stable helical structure. The probability to return to the initial position is computed, as a function of time, by integrating over the particle paths consistent with the physical properties of the model potential. The zero time condition for the first-passage probability defines the constraint to select the integral cutoff for various macroscopic helical conformations, obtained by tuning the twist, bending, and slide motion between adjacent base pairs along the molecule stack. Applying the method to a short homogeneous chain at room temperature, we obtain meaningful estimates for the maximum fluctuations in the twist conformation with ∼10.5 base pairs per helix turn, typical of double stranded DNA helices. Untwisting the double helix, the base pair fluctuations broaden and the integral cutoff increases. The cutoff is found to increase also in the presence of a sliding motion, which shortens the helix contour length, a situation peculiar of dsRNA molecules.

Autism-associated SHANK3 missense point mutations impact conformational fluctuations and protein turnover at synapses.

Members of the SH3- and ankyrin repeat (SHANK) protein family are considered as master scaffolds of the postsynaptic density of glutamatergic synapses. Several missense mutations within the canonical SHANK3 isoform have been proposed as causative for the development of autism spectrum disorders (ASDs). However, there is a surprising paucity of data linking missense mutation-induced changes in protein structure and dynamics to the occurrence of ASD-related synaptic phenotypes. In this proof-of-principle study, we focus on two ASD-associated point mutations, both located within the same domain of SHANK3 and demonstrate that both mutant proteins indeed show distinct changes in secondary and tertiary structure as well as higher conformational fluctuations. Local and distal structural disturbances result in altered synaptic targeting and changes of protein turnover at synaptic sites in rat primary hippocampal neurons.

Distinguishing different excitation pathways in two-dimensional terahertz-infrared-visible spectroscopy.

In condensed molecular matter, low-frequency modes (LFMs) associated with specific molecular motions are excited at room temperature and determine essential physical and chemical properties of materials. LFMs, with typical mode energies of up to ∼500 cm-1 (62 meV), contribute significantly to thermodynamic parameters and functions (e.g., heat capacity and entropy) and constitute the basis for room temperature molecular dynamics (e.g., conformational fluctuations and change). LFMs are often analyzed indirectly by the measurement of their effect on specific high-frequency modes (HFMs); the LFM-HFM coupling is reflected in the lineshape, as well as in the spectral and angular diffusion of the HFM. Two-dimensional terahertz-infrared-visible (2D TIRV) spectroscopy allows measuring the LFM-HFM coupling directly and can thereby provide new insights into the strength and nature of the coupling and the character of LFMs. However, the interference between the different signals generated by different excitation pathways can complicate 2D TIRV spectra, preventing a straightforward analysis. Here, we develop an experimental method to distinguish different excitation pathways in 2D TIRV spectroscopy and plot them separately in different quadrants of a 2D spectrum. We validate this method by measuring the spectra of CaF2 and nitrogen gas. For CaF2, only sum-frequency mixing between infrared and terahertz fields generates the signal. In contrast, for N2, only difference-frequency mixing is observed. We then use this method to separate sum- and difference-frequency pathways in the 2D TIRV spectrum of liquid water, verifying the previous interpretation of the lineshape of the 2D TIRV spectrum of water.

Monoubiquitination of KRAS at Lysine104 and Lysine147 Modulates Its Dynamics and Interaction with Partner Proteins.

KRAS, a 21 kDa guanine nucleotide-binding protein that functions as a molecular switch, plays a key role in regulating cellular growth. Dysregulation of this key signaling node leads to uncontrolled cell growth, a hallmark of cancer cells. KRAS undergoes post-translational modification by monoubiquitination at various locations, including at lysine104 (K104) and lysine147 (K147). Previous studies have suggested that K104 stabilizes helix-2/helix-3 interactions and K147 is involved in nucleotide binding. However, the impact of monoubiquitination at these residues on the overall structure, dynamics, or function of KRAS is not fully understood. In this study, we examined KRAS monoubiquitination at these sites using data from extensive (12 μs aggregate time) molecular dynamics simulations complemented by nuclear magnetic resonance spectroscopy data. We found that ubiquitin forms dynamic nonspecific interactions with various regions of KRAS and that ubiquitination at both sites modulates conformational fluctuations. In both cases, ubiquitin samples a broad range of conformational space and does not form long-lasting noncovalent contacts with KRAS but it adopts several preferred orientations relative to KRAS. To examine the functional impact of these preferred orientations, we performed a systematic comparison of the dominant configurations of the ubiquitin/KRAS simulated complex with experimental structures of KRAS bound to regulatory and effector proteins as well as a model membrane. Results from these analyses suggest that conformational selection and population shift may minimize the deleterious effects of KRAS ubiquitination at K104 and K147 on binding to some but not all interaction partners. Our findings thus provide new insights into the steric effects of ubiquitin and suggest a potential avenue for therapeutic targeting.

Transient kinetic studies of the antiviral Drosophila Dicer-2 reveal roles of ATP in self-nonself discrimination.

Some RIG-I-like receptors (RLRs) discriminate viral and cellular dsRNA by their termini, and Drosophila melanogaster Dicer-2 (dmDcr-2) differentially processes dsRNA with blunt or 2 nucleotide 3'-overhanging termini. We investigated the transient kinetic mechanism of the dmDcr-2 reaction using a rapid reaction stopped-flow technique and time-resolved fluorescence spectroscopy. Indeed, we found that ATP binding to dmDcr-2's helicase domain impacts association and dissociation kinetics of dsRNA in a termini-dependent manner, revealing termini-dependent discrimination of dsRNA on a biologically relevant time scale (seconds). ATP hydrolysis promotes transient unwinding of dsRNA termini followed by slow rewinding, and directional translocation of the enzyme to the cleavage site. Time-resolved fluorescence anisotropy reveals a nucleotide-dependent modulation in conformational fluctuations (nanoseconds) of the helicase and Platform-PAZ domains that is correlated with termini-dependent dsRNA cleavage. Our study offers a kinetic framework for comparison to other Dicers, as well as all members of the RLRs involved in innate immunity.

Universal Relation between Instantaneous Diffusivity and Radius of Gyration of Proteins in Aqueous Solution.

Protein conformational fluctuations are highly complex and exhibit long-term correlations. Here, molecular dynamics simulations of small proteins demonstrate that these conformational fluctuations directly affect the protein's instantaneous diffusivity D_{I}. We find that the radius of gyration R_{g} of the proteins exhibits 1/f fluctuations that are synchronous with the fluctuations of D_{I}. Our analysis demonstrates the validity of the local Stokes-Einstein-type relation D_{I}∝1/(R_{g}+R_{0}), where R_{0}∼0.3 nm is assumed to be a hydration layer around the protein. From the analysis of different protein types with both strong and weak conformational fluctuations, the validity of the Stokes-Einstein-type relation appears to be a general property.

Understanding the Optimal Cooperativity of Human Glucokinase: Kinetic Resonance in Nonequilibrium Conformational Fluctuations.

The cooperativity of a monomeric enzyme arises from dynamic correlation instead of spatial correlation and is a consequence of nonequilibrium conformation fluctuations. We investigate the conformation-modulated kinetics of human glucokinase, a monomeric enzyme with important physiological functions, using a five-state kinetic model. We derive the non-Michealis-Menten (MM) correction term of the activity (i.e., turnover rate), predict its relationship to cooperativity, and reveal the violation of conformational detailed balance. Most importantly, we reproduce and explain the observed resonance effect in human glucokinase (i.e., maximal cooperativity when the conformational fluctuation rate is comparable to the catalytic rate). With the realistic parameters, our theoretical results are in quantitative agreement with the reported measurement by Miller and co-workers. The analysis can be extended to a general chemical network beyond the five-state model, suggesting the generality of kinetic cooperativity and resonance.

Compressive and Tensile Deformations Alter ATP Hydrolysis and Phosphate Release Rates in Actin Filaments.

Actin filament networks in eukaryotic cells are constantly remodeled through nucleotide state controlled interactions with actin binding proteins, leading to macroscopic structures such as bundled filaments, branched filaments, and so on. The nucleotide (ATP) hydrolysis, phosphate release, and polymerization/depolymerization reactions that lead to the formation of these structures are correlated with the conformational fluctuations of the actin subunits at the molecular scale. The resulting structures generate and experience varying levels of force and impart cells with several functionalities such as their ability to move, divide, transport cargo, etc. Models that explicitly connect the structure to reactions are essential to elucidate a fundamental level of understanding of these processes. In this regard, a bottom-up Ultra-Coarse-Grained (UCG) model of actin filaments that can simulate ATP hydrolysis, inorganic phosphate release (Pi), and depolymerization reactions is presented in this work. In this model, actin subunits are represented using coarse-grained particles that evolve in time and undergo reactions depending on the conformations sampled. The reactions are represented through state transitions, with each state represented by a unique effective coarse-grained potential. Effects of compressive and tensile strains on the rates of reactions are then analyzed. Compressive strains tend to unflatten the actin subunits, reduce the rate of ATP hydrolysis, and increase the Pi release rate. On the other hand, tensile strain flattens subunits, increases the rate of ATP hydrolysis, and decrease the Pi release rate. Incorporating these predictions into a Markov State Model highlighted that strains alter the steady-state distribution of subunits with ADPPi and ADP nucleotide, thus identifying possible additional factors underlying the cooperative binding of regulatory proteins to actin filaments.

DNA Looping and DNA Conformational Fluctuations Can Accelerate Protein Target Search.

Protein searching and binding to specific sites on DNA is a fundamentally important process that marks the beginning of all major cellular transformations. While the dynamics of protein-DNA interactions in in vitro settings is well investigated, the situation is much more complex for in vivo conditions because the DNA molecules in live cells are packed into chromosomal structures where they are undergoing strong dynamic and conformational fluctuations. In this work, we present a theoretical investigation on the role of DNA looping and DNA conformational fluctuations in the protein target search. It is based on a discrete-state stochastic analysis that allows for explicit calculations of dynamic properties, which is also supplemented by Monte Carlo computer simulations. It is found that for stronger nonspecific interactions between DNA and proteins the search occurs faster on the DNA looped conformation in comparison with the unlooped conformation, and the fastest search is observed when the loop is formed near the target site. It is also shown that DNA fluctuations between the looped and unlooped conformations influence the search dynamics, and this depends on the magnitude of conformational transition rates and on which conformation is more energetically stable. Physical-chemical arguments explaining these observations are presented. Our theoretical study suggests that the geometry and conformational changes in DNA are additional factors that might efficiently control the gene regulation processes.

Combining Single-Molecule Fluorescence and MD-Simulations to Delineate the Kinetics and Regulation of Proteins

Protein conformational kinetics and its regulation occur on many time- and length-scales. While for example post-translational modifications act very locally, specific protein-protein interaction typically affect larger domains, and global changes (like crowding) affect the whole protein (complex) non-specifically. In a first part we use a combination of single-molecule FRET, FRET-FCS, ns-FCS and MD simulations to delineate the timescales for information transfer from the hydrolysis event in the nucleotide binding site of the heat shock protein 90 (Hsp90) to large conformational changes in the Hsp90 dimer[1]. This allosteric process occurs via a hierarchical mechanism that involves timescales from pico- to microseconds. In a second part we show protein regulation under different degrees of localization, and present the effects on the Hsp90 chaperone system. Interestingly using single-molecule FRET, we find that similar functional and conformational steady states are caused by completely different underlying kinetics. We will introduce a new mechanistic concept, namely functional stimulation through conformational confinement[2]. Finally, we use a network of distance distributions obtained with single molecule FRET to investigate a Hsp90 - model client interaction[3]. We find negligible effects of the client on the open-close equilibrium of Hsp90, but strong effects on Hsp90's conformational fluctuations. This hints towards an additional (orthogonal) regulation mechanism. Altogether, these applications demonstrate that a combination of (single-molecule) methods is ideal to investigate the biophysics of multi-component (protein) systems. [1] S. Wolf et al., Allosteric action of nucleotides on Hsp90 across several time- and length scales. BioRxiv, 2020.02.15.950725 (2020); https://doi.org/10.1101/2020.02.15.950725 [2] S. Schmid and T. Hugel, Controlling protein function by fine-tuning conformational flexibility. eLife, 9, 1013 (2020); https://doi.org/10.7554/eLife.57180 [3] Hellenkamp et al., Multidomain structure and correlated dynamics determined by self-consistent FRET networks. Nat Methods, 14 (2), 174 (2017). https://doi.org/10.1038/nmeth.4081