A Mode Evolution Metric to Extract Reaction Coordinates for Biomolecular Conformational Transitions.

The complex, multidimensional energy landscape of biomolecules makes the extraction of suitable, nonintuitive collective variables (CVs) that describe their conformational transitions challenging. At present, dimensionality reduction approaches and machine learning (ML) schemes are employed to obtain CVs from molecular dynamics (MD)/Monte Carlo (MC) trajectories or structural databanks for biomolecules. However, minimum sampling conditions to generate reliable CVs that accurately describe the underlying energy landscape remain unclear. Here, we address this issue by developing a Mode evolution Metric (MeM) to extract CVs that can pinpoint new states and describe local transitions in the vicinity of a reference minimum from nonequilibrated MD/MC trajectories. We present a general mathematical formulation of MeM for both statistical dimensionality reduction and machine learning approaches. Application of MeM to MC trajectories of model potential energy landscapes and MD trajectories of solvated alanine dipeptide reveals that the principal components which locate new states in the vicinity of a reference minimum emerge well before the trajectories locally equilibrate between the associated states. Finally, we demonstrate a possible application of MeM in designing efficient biased sampling schemes to construct accurate energy landscape slices that link transitions between states. MeM can help speed up the search for new minima around a biomolecular conformational state and enable the accurate estimation of thermodynamics for states lying on the energy landscape and the description of associated transitions.

Multiple Functional Protein-Protein Interaction Interfaces Allosterically Regulate ATP-Binding in Cyclin-Dependent Kinase-1.

The ATP-dependent phosphorylation activity of cyclin-dependent kinase 1 (CDK1), an essential enzyme for cell cycle progression, is regulated by interactions with Cyclin-B, substrate, and Cks proteins. We have recently shown that active site acetylation in CDK1 abrogated binding to Cyclin-B which posits an intriguing long-range communication between the catalytic site and the protein-protein interaction (PPI) interface. Now, we demonstrate a general allosteric link between the CDK1 active site and all three of its PPI interfaces through atomistic molecular dynamics (MD) simulations. Specifically, we examined ATP binding free energies to CDK1 in native nonacetylated (K33wt) and acetylated (K33Ac) forms as well as the acetyl-mimic K33Q and the acetyl-null K33R mutant forms, which are accessible in vitro. In agreement with experiments, ATP binding is stronger in K33wt relative to the other three perturbed states. Free energy decomposition reveals, in addition to expected local changes, significant and selective nonlocal entropic responses to ATP binding/perturbation of K33 from the αC $$ \alpha C $$ -helix, activation loop (A-loop), and αG $$ \alpha G $$ - α $$ \alpha $$ H segments in CDK1 which interface with Cyclin-B, substrate, and Cks proteins, respectively. Statistical analysis reveals that while entropic responses of protein segments to active site perturbations are on average correlated with their dynamical changes, such correlations are lost in about 9%-48% of the dataset depending on the segment. Besides proving the bi-directional communication between the active site and the CDK1:Cyclin-B interface, our study uncovers a hitherto unknown mode of ATP binding regulation by multiple PPI interfaces in CDK1.

A water-soluble, cell-permeable Mn(ii) sensor enables visualization of manganese dynamics in live mammalian cells.

Central roles of Mn2+ ions in immunity, brain function, and photosynthesis necessitate probes for tracking this essential metal ion in living systems. However, developing a cell-permeable, fluorescent sensor for selective imaging of Mn2+ ions in the aqueous cellular milieu has remained a challenge. This is because Mn2+ is a weak binder to ligand-scaffolds and Mn2+ ions quench fluorescent dyes leading to turn-off sensors that are not applicable for in vivo imaging. Sensors with a unique combination of Mn2+ selectivity, µM sensitivity, and response in aqueous media are necessary for not only visualizing labile cellular Mn2+ ions live, but also for measuring Mn2+ concentrations in living cells. No sensor has achieved this combination thus far. Here we report a novel, completely water-soluble, reversible, fluorescent turn-on, Mn2+ selective sensor, M4, with a K d of 1.4 µM for Mn2+ ions. M4 entered cells within 15 min of direct incubation and was applied to image Mn2+ ions in living mammalian cells in both confocal fluorescence intensity and lifetime-based set-ups. The probe was able to visualize Mn2+ dynamics in live cells revealing differential Mn2+ localization and uptake dynamics under pathophysiological versus physiological conditions. In a key experiment, we generated an in-cell Mn2+ response curve for the sensor which allowed the measurement of the endogenous labile Mn2+ concentration in HeLa cells as 1.14 ± 0.15 µM. Thus, our computationally designed, selective, sensitive, and cell-permeable sensor with a 620 nM limit of detection for Mn2+ in water provides the first estimate of endogenous labile Mn2+ levels in mammalian cells.

Do quantum interference effects manifest in acyclic aliphatic molecules with anchoring groups?

The ability to control single molecule electronic conductance is imperative for achieving functional molecular electronics applications such as insulation, switching, and energy conversion. Quantum interference (QI) effects are generally used to control electronic transmission through single molecular junctions by tuning the molecular structure or the position of the anchoring group(s) in the molecule. While previous studies focussed on the QI between σ and/or π channels of the molecular backbone, here, we show that single molecule electronic devices can be designed based on QI effects originating from the interactions of anchoring groups. Furthermore, while previous studies have concentrated on the QI mostly in conjugated/cyclic systems, our study showcases that QI effects can be harnessed even in the simplest acyclic aliphatic systems-alkanedithiols, alkanediamines, and alkanediselenols. We identify band gap state resonances in the transmission spectrum of these molecules whose positions and intensities depend on the chain length, and anchoring group sensitive QI between the nearly degenerate molecular orbitals localized on the anchoring groups. We predict that these QI features can be harnessed through an external mechanical stimulus to tune the charge transport properties of single molecules in the break-junction experiments.

Capturing the Polarization Response of Solvated Proteins under Constant Electric Fields in Molecular Dynamics Simulations.

We capture and compare the polarization response of a solvated globular protein ubiquitin to static electric (E-fields) using atomistic molecular dynamics simulations. We collectively follow E-field induced changes, electrical and structural, occurring across multiple trajectories using the magnitude of the protein dipole vector (Pp ). E-fields antiparallel to Pp induce faster structural changes and more facile protein unfolding relative to parallel fields of the same strength. While weak E-fields (0.1-0.5 V/nm) do not unfold ubiquitin and produce a reversible polarization, strong E-fields (1-2 V/nm) unfold the protein through a pathway wherein the helix:ß-strand interactions rupture before those for the ß1-ß5 clamp. Independent of E-field direction, high E-field induced structural changes are also reversible if the field is switched off before Pp exceeds 2 times its equilibrium value. We critically examine the dependence of water properties, protein rotational diffusion and E-field induced protein unfolding pathways on the thermostat/barostat parameters used in our simulations.

Biophysical properties of the isolated spike protein binding helix of human ACE2.

The entry of the severe acute respiratory syndrome coronavirus 2 virus in human cells is mediated by the binding of its surface spike protein to the human angiotensin-converting enzyme 2 (ACE2) receptor. A 23-residue long helical segment (SBP1) at the binding interface of human ACE2 interacts with viral spike protein and therefore has generated considerable interest as a recognition element for virus detection. Unfortunately, emerging reports indicate that the affinity of SBP1 to the receptor-binding domain of the spike protein is much lower than that of the ACE2 receptor itself. Here, we examine the biophysical properties of SBP1 to reveal factors leading to its low affinity for the spike protein. Whereas SBP1 shows good solubility (solubility > 0.8 mM), circular dichroism spectroscopy shows that it is mostly disordered with some antiparallel ß-sheet content and no helicity. The helicity is substantial (>20%) only upon adding high concentrations (≥20% v/v) of 2,2,2-trifluoroethanol, a helix promoter. Fluorescence correlation spectroscopy and single-molecule photobleaching studies show that the peptide oligomerizes at concentrations >50 nM. We hypothesized that mutating the hydrophobic residues (F28, F32, and F40) of SBP1, which do not directly interact with the spike protein, to alanine would reduce peptide oligomerization without affecting its spike binding affinity. Whereas the mutant peptide (SBP1mod) shows substantially reduced oligomerization propensity, it does not show improved helicity. Our study shows that the failure of efforts, so far, to produce a short SBP1 mimic with a high affinity for the spike protein is not only due to the lack of helicity but is also due to the heretofore unrecognized problem of oligomerization.

Identification of a copper ion recognition peptide sequence in the subunit II of cytochrome c oxidase: a combined theoretical and experimental study.

The role of the pentapeptide, NHSFM, derived from the surface exposed part of the metal ion binding loop of the subunit II of cytochrome c oxidase on the maturation of the binuclear purple CuA center of the enzyme has been investigated using several experimental and computational methods. The copper ion was found to form 1:1 complex of the pentapeptide with a binding constant ~ 104 M-1 to 105 M-1, where a 4 ligand coordination from the peptide in a type 2 copper center was revealed. The pH dependence of the metal-peptide was associated with a [Formula: see text] of ~ 10 suggesting deprotonation of the N-terminal amine. EXAFS studies as well as DFT calculations of the metal-peptide complexes revealed pH dependent changes in the metal-ligand bond distances. Spectroscopic properties of the metal peptides calculated from TDDFT studies agreed with the experimental results. Restrained molecular dynamics (RMD) simulations indicated coordination of a carbonyl oxygen from the asparagine (N) side chain and of water molecules apart from histidine (H), methionine (M) and terminal amine of asparagine (N) in a distorted square planar geometry of Cu-NHSFM. Analyses of the backbone distances as well as B-factors for the metal peptide suggested that the peptide backbone becomes more compact and rigid on binding of the metal ion. This indicated that binding of copper ion to this pentapeptide in the protein possibly cause movement of the protein backbone bringing other coordinating residues closer to the copper ion, and thus helping in sequential uptake of copper ions to the protein.

Estimating the Directional Flexibility of Proteins from Equilibrium Thermal Fluctuations.

The directional flexibility of proteins is an equilibrium molecular property which is accessible to both experiment and computation. Single molecule force spectroscopy (SMFS) experiments report effective directional spring constants to describe the collective anisotropic response of a protein structure to mechanical pulling forces applied along selected axes. On the other hand, computational methods have thus far employed either indirect force based nonequilibrium simulations or coarse-grained elastic network models (ENM) to predict protein directional spring constants. Here, we examine the ability of equilibrium atomistic Molecular Dynamics (MD) simulations to estimate the directional flexibility and mechanical anisotropy of proteins. MD-derived effective directional spring constants are found to correlate well with SMFS spring constants (ρ2 = 0.97-0.99; Adj R2 = 0.92-0.99) and unfolding forces (ρ2 = 0.85-0.97; Adj R2 = 0.63-0.91) for five different globular proteins. Specifically, the computed spring constants reproduce the mechanical anisotropy reported by SMFS along five different directions of green fluorescence protein (GFP) and six directions of the immunoglobulin-binding B1 domain of streptococcal protein G (GB1). Further, protein dynamics as captured in MD can be translated into spring constants which can distinguish the N-C directional flexibility of ubiquitin (Ub) from two structurally homologous small ubiquitin-like modifier (SUMO1 and SUMO2) isoforms. We apply our computational framework to study the mechanical anisotropy of Ub along the seven lysine-C-term directions which are functionally relevant. We show that Ub possesses two distinct flexibility scales along these directions which roughly differ by an order of magnitude. Further, our studies reveal that the mechanical anisotropy of Ub is modified in contrasting ways by the binding of two partner proteins (UBCH5A and UEV) which attach and recognize these biomolecular tag proteins. On the basis of equilibrium MD benchmarks for flexibility along 2485 bond vectors in Ub, we propose and validate a new covariance-propagation scheme to extract spring constants from ENM normal modes. We also critically examine the ability of ENM to predict directional flexibility of proteins and suggest modifications to improve these intuitive and scalable descriptions.

Role of Ligand Binding Site in Modulating the Mechanical Stability of Proteins with ß-Grasp Fold.

Despite many studies on ligand-modulated protein mechanics, a comparative analysis of the role of ligand binding site on any specific protein fold is yet to be made. In this study, we explore the role of ligand binding site on the mechanical properties of ß-grasp fold proteins, namely, ubiquitin and small ubiquitin related modifier 1 (SUMO1). The terminal segments directly connected through hydrogen bonds constitute the ß-clamp geometry (or mechanical clamp), which confers high mechanical resilience to the ß-grasp fold. Here, we study ubiquitin complexed with CUE2-1, a ubiquitin-binding domain (UBD) from yeast endonuclease protein Cue2, using a combination of single-molecule force spectroscopy (SMFS) and steered molecular dynamics (SMD) simulations. Our study reveals that CUE2-1 does not alter the mechanical properties of ubiquitin, despite directly interacting with its ß-clamp. To explore the role of ligand binding site, we compare the mechanical properties of the ubiquitin/CUE2-1 complex with that of previously studied SUMO1/S12, another ß-grasp protein complex, using SMD simulations. Simulations on the SUMO1/S12 complex corroborate previous experimentally observed enhancement in the mechanical stability of SUMO1, even though S12 binds away from the ß-clamp. Differences in ligand binding-induced structural impact at the transition state of the two complexes explain the differences in ligand modulated protein mechanics. Contrary to previous reports, our study demonstrates that direct binding of ligands to the mechanical clamp does not necessarily alter the mechanical stability of ß-grasp fold proteins. Rather, binding interactions away from the clamp can reinforce protein stability provided by the ß-grasp fold. Our study highlights the importance of binding site and binding modes of ligands in modulating the mechanical stability of ß-grasp fold proteins.

Multiscale modelling reveals higher charge transport efficiencies of DNA relative to RNA independent of mechanism.

In this study, we compare the charge transport properties of multiple double-stranded (ds)RNA sequences with corresponding dsDNA sequences. Recent studies have presented a contradictory picture of relative charge transport efficiencies in A-form DNA : RNA hybrids and dsDNA. Using a multiscale modelling framework, we compute conductance of dsDNA and dsRNA using Landauer formalism in the coherent limit and Marcus-Hush theory in the incoherent limit. We find that dsDNA conducts better than dsRNA in both the charge transport regimes. Our analysis shows that the structural differences in the twist angle and slide of dsDNA and dsRNA are the main reasons behind the higher conductance of dsDNA in the incoherent hopping regime. In the coherent limit however, for the same base pair length, the conductance of dsRNA is higher than that of dsDNA for the morphologies where dsRNA has a smaller end-to-end length relative to that of dsDNA.

Transient Raman Snapshots of the Twisted Intramolecular Charge Transfer State in a Stilbazolium Dye.

Optically triggered twisted intramolecular charge transfer (TICT) states in donor-acceptor chromophores form the molecular basis for designing bioimaging probes that sense polarity, microviscosity, and pH in vivo. However, a lack of predictive understanding of the "twist" localization precludes a rational design of TICT-based dyes. Here, using femtosecond stimulated Raman spectroscopy, we reveal a distinct Raman signature of the TICT state for a stilbazolium-class mitochondrial staining dye. Resonance-selective probing of 4-N,N-diethylamino-4″-N'-methyl-stilbazolium tosylate (DEST) tracks the excited-state structure of the dye as it relaxes to a TICT state on a picosecond time scale. The appearance of a remarkably blue-shifted C=C stretching mode at 1650 cm-1 in the TICT state is attributed to the "twist" of a single bond adjacent to the ethylenic π-bridge in the DEST backbone based on detailed electronic structure calculations and vibrational mode analysis. Our work demonstrates that the π-bridge, connecting the donor and acceptor moieties, influences the spatial location of the "twist" and offers a new perspective for designing organelle-specific probes through cogent tuning of backbone dynamics.

Variance of Atomic Coordinates as a Dynamical Metric to Distinguish Proteins and Protein-Protein Interactions in Molecular Dynamics Simulations.

Protein dynamics is a manifestation of the complex trajectories of these biomolecules on a multidimensional rugged potential energy surface (PES) driven by thermal energy. At present, computational methods such as atomistic molecular dynamics (MD) simulations can describe thermal protein conformational changes in fully solvated environments over millisecond timescales. Despite these advances, a quantitative assessment of protein dynamics remains a complicated topic, intricately linked to issues such as sampling convergence and the identification of appropriate reaction coordinates/structural features to describe protein conformational states and motions. Here, we present the cumulative variance of atomic coordinate fluctuations (CVCF) along trajectories as an intuitive PES sensitive metric to assess both the extent of sampling and protein dynamics captured in MD simulations. We first examine the sampling problem in model one- (1D) and two-dimensional (2D) PES to demonstrate that the CVCF when traced as a function of the sampling variable (time in MD simulations) can identify local and global equilibria. Further, even far from global equilibrium, a situation representative of standard MD trajectories of proteins, the CVCF can distinguish different PES and therefore resolve the resultant protein dynamics. We demonstrate the utility of our CVCF analysis by applying it to distinguish the dynamics of structurally homologous proteins from the ubiquitin family (ubiquitin, SUMO1, SUMO2) and ubiquitin protein-protein interactions. Our CVCF analysis reveals that differential side-chain dynamics from the structured part of the protein (the conserved ß-grasp fold) present distinct protein PES to distinguish ubiquitin from SUMO isoforms. Upon binding to two functionally distinct protein partners (UBCH5A and UEV), intrinsic ubiquitin dynamics changes to reflect the binding context even though the two proteins have similar binding modes, which lead to negligible (sub-angstrom scale) structural changes.

UV-Visible Lysine-Glutamate Dimer Excitations in Protein Charge Transfer Spectra: TDDFT Descriptions Using an Optimally Tuned CAM-B3LYP Functional.

Recent reports of distinctive UV-vis absorption profiles for monomeric proteins rich in charged amino acids that span 250-800 nm have opened up a new label-free optical spectral window for probing biomolecular structure and interactions. Combined experimental-computational studies have revealed that such broad absorption profiles of these proteins arise from photoexcited charge transfer (CT) transitions in spatially proximal charged amino acids such as lysine (Lys) and glutamate (Glu). Here, using time-dependent density functional theory (TDDFT) with an optimally tuned CAM-B3LYP functional, we refine the computed UV-vis spectra for Lys-Glu dimers within protein folds and quantify the percentage CT character of the constituent transitions. The optimally tuned functionals are derived through a careful analysis of the CAM-B3LYP parameter space for Lys-Glu dimers as a function of amino-acid conformation and side chain separation. Our studies reveal that the tuned Lys-Glu dimer spectrum spans 150-650 nm and possesses 5 specific types of CT excitations with diverse and large spatial charge separation length scales of 2-10 Å. These include inter-/intra-residue peptide backbone to peptide backbone (BB-CT) excitations spanning 160-210 nm, inter-/intra-residue peptide backbone to side chain (BS-CT) excitations spanning 160-260 nm, and side chain to side chain (SS-CT) excitations, which show the broadest absorption range spanning 260-650 nm.

Allosteric Regulation of Cyclin-B Binding by the Charge State of Catalytic Lysine in CDK1 Is Essential for Cell-Cycle Progression.

Cyclin-dependent kinase 1 (CDK1) is essential for cell-cycle progression. While dependence of CDK activity on cyclin levels is well established, molecular mechanisms that regulate their binding are less understood. Here, we report for the first time that CDK1:cyclin-B binding is not default but rather determined by the evolutionarily conserved catalytic residue, lysine-33 in CDK1. We demonstrate that the charge state of this lysine allosterically remodels the CDK1:cyclin-B interface. Cell cycle-dependent acetylation of lysine-33 or its mutation to glutamine, which mimics acetylation, abrogates cyclin-B binding. Using biochemical approaches and atomistic molecular dynamics simulations, we have uncovered both short-range and long-range effects of perturbing the charged state of the catalytic lysine, which lead to inhibition of kinase activity. Specifically, although loss of the charge state of catalytic lysine did not impact ATP binding significantly, it altered its orientation in the active site. In addition, the catalytic lysine also acts as an intra-molecular electrostatic tether at the active site to orient structural elements interfacing with cyclin-B. Physiologically, opposing activities of SIRT1 and P300 regulate acetylation and thus control the charge state of lysine-33. Importantly, cells expressing acetylation mimic mutant of Cdc2/CDK1 in yeast are arrested in G2 and fail to divide, indicating the requirement of the deacetylated state of the catalytic lysine for cell division. Thus, by illustrating the molecular role of the catalytic lysine and cell cycle-dependent deacetylation as a determinant of CDK1:cyclin-B interaction, our results redefine the current model of CDK1 activation and cell-cycle progression.

Ultrafast photoactivation of C─H bonds inside water-soluble nanocages.

Light energy absorbed by molecules can be harnessed to activate chemical bonds with extraordinary speed. However, excitation energy redistribution within various molecular degrees of freedom prohibits bond-selective chemistry. Inspired by enzymes, we devised a new photocatalytic scheme that preorganizes and polarizes target chemical bonds inside water-soluble cationic nanocavities to engineer selective functionalization. Specifically, we present a route to photoactivate weakly polarized sp3 C─H bonds in water via host-guest charge transfer and control its reactivity with aerial O2. Electron-rich aromatic hydrocarbons self-organize inside redox complementary supramolecular cavities to form photoactivatable host-guest charge transfer complexes in water. An ultrafast C─H bond cleavage within ~10 to 400 ps is triggered by visible-light excitation, through a cage-assisted and solvent water-assisted proton-coupled electron transfer reaction. The confinement prolongs the lifetime of the carbon-centered radical to enable a facile yet selective reaction with molecular O2 leading to photocatalytic turnover of oxidized products in water.

Differences in the mechanical unfolding pathways of apo- and copper-bound azurins.

Metalloproteins carry out diverse biological functions including metal transport, electron transfer, and catalysis. At present, the influence of metal cofactors on metalloprotein stability is not well understood. Here, we report the mechanical stability and unfolding pathway of azurin, a cupredoxin family protein with ß-barrel topology and type I copper-binding centre. Single-molecule force spectroscopy (SMFS) experiments reveal 2-state and 3-state unfolding pathways for apo-azurin. The intermediate in the 3-state pathway occurs at an unfolding contour length of 7.5 nm from the native state. Steered molecular dynamics (SMD) simulations show that apo-azurin unfolds via a first transition state (TS) where ß2Β-ß8 and ß7-ß8 strand pairs rupture to form the intermediate, which subsequently unfolds by the collective rupture of remaining strands. SMFS experiments on holo-azurin exhibit an additional 4-state pathway besides the 2-state and 3-state pathways. The unfolding contour length leading to the first intermediate is 6.7 nm suggesting a sequestration of ~1 nm polypeptide chain length by the copper. SMD simulations reveal atomistic details of the copper sequestration and predict a combined ß4-ß7 pair and copper coordination sphere rupture to create the third TS in the 4-state pathway. Our systematic studies provide detailed mechanistic insights on modulation of protein mechanical properties by metal-cofactors.

Optically sensing phospholipid induced coil-helix transitions in the phosphoinositide-binding motif of gelsolin.

We present a systematic experimental and computational study of phospholipid induced peptide coil-helix transitions which are relevant in the context of proteins mediating cytoskeletal rearrangement via membrane binding. We developed a sensitive Förster resonance energy transfer (FRET) based assay to address whether coil-helix transitions in phospholipid binding motifs of actin-binding proteins can be induced by physiologically-relevant concentrations (1-20 µM) of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) phospholipids. Based on inter-residue distance constraints obtained from Molecular Dynamics (MD) simulations of a 20 residue peptide (Gel 150-169) from the actin-severing protein gelsolin, we synthetized and labeled the peptide with a tryptophan donor and IAEDANS acceptor pair. Upon addition of PI(4,5)P2 micelles and mixed vesicles containing PI(4,5)P2 and phosphatidylcholine to the peptide, we observed a decrease in the tryptophan emission intensity with increasing concentrations of PI(4,5)P2. The IAEDANS emission spectra showed a more complex profile exhibiting a blue shift of the emission peak and non-monotonic changes in the intensity profile with increasing concentrations of PI(4,5)P2. We showed that the IAEDANS acceptor emission response is a result of both intrinsic polarity sensitivity of the acceptor in the vicinity of the membrane surface and fluorescence energy transfer from the donor. Importantly, the fluorescence lifetime of the donor (tryptophan) showed a monotonous decrease with increasing mol% of PI(4,5)P2 from 1.13 ± 0.10 ns in the absence of phospholipids to 0.25 ± 0.03 ns in the presence of 100% PI(4,5)P2 micelles. We also showed a concomitant increase in FRET efficiency with increasing PI(4,5)P2 levels indicating a PI(4,5)P2 concentration dependent coil-helix transition. Our studies demonstrate that membrane PI(4,5)P2 concentrations as low as 2.5-5 µM can trigger helix-coil conformational changes in gelsolin relevant for triggering regulatory processes in the cell.