A class of analog computers built from large numbers of microscopic probabilistic machines is discussed. It is postulated that such computers are implemented in biological systems as ensembles of protein molecules. The formalism is based on an abstract computational model referred to as Protein Molecule Machine (PMM). A PMM is a continuous-time first-order Markov system with real input and output vectors, a finite set of discrete states, and the input-dependent conditional probability densities of state transitions. The output of a PMM is a function of its input and state. The components of input vector, called generalized potentials, can be interpreted as membrane potential, and concentrations of neurotransmitters. The components of output vector, called generalized currents, can represent ion currents, and the flows of second messengers. An Ensemble of PMMs (EPMM) is a set of independent identical PMMs with the same input vector, and the output vector equal to the sum of output vectors of individual PMMs. The paper suggests that biological neurons have much more sophisticated computational resources than the presently popular models of artificial neurons.
Motivated by single-molecule experiments on synaptic membrane protein domains, we use a stochastic lattice model to study protein reaction and diffusion processes in crowded membranes. We find that the stochastic reaction-diffusion dynamics of synaptic proteins provide a simple physical mechanism for collective fluctuations in synaptic domains, the molecular turnover observed at synaptic domains, key features of the single-molecule trajectories observed for synaptic proteins, and spatially inhomogeneous protein lifetimes at the cell membrane. Our results suggest that central aspects of the sin…
The interaction of proteins with small molecules is fundamental to their function in living organisms and it is widely studied in drug development. Here we compare optical trapping dynamics of streptavidin and biotinylated streptavidin using a double nanohole optical trap in a metal film. Consistent and clearly distinct behavior is seen between the protein with and without the small molecule binding. The real-time dynamics at the single protein level are accessible with this technique, which also has advantages of not requiring tethering to a surface or the need for exogeneous markers.
Protein-surface interactions cause the desirable effect of controlled protein adsorption onto biodevices as well as the undesirable effect of protein fouling. The key to controlling protein-surface adsorptions is to identify and quantify the main adsorption mechanisms: adsorptions that occur (1) while depositing a protein solution onto dry surfaces and (2) after the deposition onto wet surfaces. Bulk measurements cannot reveal the dynamic protein adsorption pathways and thus cannot differentiate between the two adsorption mechanisms. We imaged the interactions of single streptavidin molecules …
Single-molecule narrowfield microscopy is a versatile tool to investigate a diverse range of protein dynamics in live cells and has been extensively used in bacteria. Here, we describe how these methods can be extended to larger eukaryotic, yeast cells, which contain sub-cellular compartments. We describe how to obtain single-molecule microscopy data but also how to analyse these data to track and obtain the stoichiometry of molecular complexes diffusing in the cell. We chose glucose mediated signal transduction of live yeast cells as the system to demonstrate these single-molecule techniques …
We present an effective method for estimating the motion of proteins from the motion of attached probe particles in single-molecule experiments. The framework naturally incorporates Langevin dynamics to compute the most probable trajectory of the protein. By using a perturbation expansion technique, we achieve computational costs more than four orders of magnitude smaller than the conventional gradient descent method without loss of simplicity in the computation algorithm. We present illustrative applications of the method using simple models of single-molecule experiments and confirm that the…
We examine the limits on viral composition that are set by the electrostatic interactions effected by the charge on the viral proteins, the single-stranded viral RNA molecule and monovalent salt ions in the solution. Within the mean-field model of viral energetics we demonstrate the prime importance of the salt concentration for the assembly of a virus. We find that the encapsidation of the viral RNA molecule is thermodynamically suppressed in solutions with high concentrations of monovalent salt. This effect is significantly less important in viruses with proteins whose charge distribution pr…
We have earlier reported the iMOLSDOCK technique to perform induced-fit peptide-protein docking. iMOLSDOCK uses the mutually orthogonal Latin squares (MOLS) technique to sample the conformation and the docking pose of the small molecule ligand and also the flexible residues of the receptor protein, and arrive at the optimum pose and conformation. In this paper we report the extension carried out in iMOLSDOCK to dock nonpeptide small molecule ligands to receptor proteins. We have benchmarked and validated iMOLSDOCK with a dataset of 34 protein-ligand complexes with nonpeptide small molecules as…
We investigate the influences of the excluded volume of molecules on biochemical reaction processes on 2-dimensional surfaces using a model of signal transduction processes on biomembranes. We perform simulations of the 2-dimensional cell-based model, which describes the reactions and diffusion of the receptors, signaling proteins, target proteins, and crowders on the cell membrane. The signaling proteins are activated by receptors, and these activated signaling proteins activate target proteins that bind autonomously from the cytoplasm to the membrane, and unbind from the membrane if activate…
A new method for finding the minimum free energy pathway (MFEP) of ions and small molecule transportation through a protein based on the three-dimensional reference interaction site model (3D-RISM) theory combined with the string method has been proposed. The 3D-RISM theory produces the distribution function, or the potential of mean force (PMF), for transporting substances around and inside proteins. By applying the string method to the PMF surface, one can readily determine the MFEP of the transporting substances inside a protein. The method has been applied to consider the Na+ conduction pa…
Imaging single proteins has been a long-standing ambition for advancing various fields in natural science, as for instance structural biology, biophysics and molecular nanotechnology. In particular, revealing the distinct conformations of an individual protein is of utmost importance. Here, we show the imaging of individual proteins and protein complexes by low-energy electron holography. Samples of individual proteins and protein complexes on ultraclean freestanding graphene were prepared by soft-landing electrospray ion beam deposition, which allows chemical- and conformational-specific sele…
Many of the existing stochastic models of gene expression contain the first-order decay reaction term that may describe active protein degradation or dilution. If the model variable is interpreted as the molecule number, and not concentration, the decay term may also approximate the loss of protein molecules due to cell division as a continuous degradation process. The seminal model of that kind leads to gamma distributions of protein levels, whose parameters are defined by the mean frequency of protein bursts and mean burst size. However, such models (whether interpreted in terms of molecule …
We propose a tight-binding model to investigate electronic transport properties of single helical protein molecules incorporating both the helical symmetry and the possibility of multiple charge transfer pathways. Our study reveals that due to existence of both the multiple charge transfer pathways and helical symmetry, the transport properties are quite rigid under influence of envi- ronmental fluctuations which indicates that these biomolecules can serve as better alternatives in nanoelectronic devices than its other biological counterparts e.g., single-stranded DNA.
In this paper we review the applicability of autofluorescent proteins for single-molecule imaging in biology. The photophysical characteristics of several mutants of the Green Fluorescent Protein (GFP) and those of DsRed are compared and critically discussed for their use in cellular biology. The alternative use of two-photon excitation at the single-molecule level or Fluorescence Correlation Spectroscopy is envisaged for the study of individual autofluorescent proteins. Single-molecule experiments performed in live cells using eGFP and preferably eYFP fusion proteins are reviewed. Finally, th…
DNA-interacting proteins have roles multiple processes, many operating as molecular machines which undergo dynamic metastable transitions to bring about their biological function. To fully understand this molecular heterogeneity, DNA and the proteins that bind to it must ideally be interrogated at a single molecule level in their native in vivo environments, in a time-resolved manner fast to sample the molecular transitions across the free energy landscape. Progress has been made over the past decade in utilising cutting-edge tools of the physical sciences to address challenging biological que…
Following the ideas of Davydov's soliton theory, we study the bio-energy transport in protein molecules. By using a quantum Brownian motion model for a phonon dressed vibrational exciton, we calculate the time-dependence on the mean square distance, diffusion coefficient, and energy of the vibrational exciton. We find the time-dependence by solving the quantum Langevin equation and find oscillatory behaviors due to the super-diffusive non-ohmic dissipation. We find that the vibrational exciton gains an overall energy due to the coupling to the phonon bath; it also dissipates its energy to the …
Many proteins interact with and deform double-stranded DNA in cells. Single-molecule experiments have studied the elasticity of DNA with helix-deforming proteins, including proteins that bend DNA. These experiments increase the need for theories of DNA elasticity which include helix-deforming proteins. Previous theoretical work on bent DNA has examined a long DNA molecule with many nonspecifically binding proteins. However, recent experiments used relatively short DNA molecules with a single, well-defined bend site. Here we develop a simple theoretical description of the effect of a single ben…
Complex DNA topological structures, including polymer loops, are frequently observed in biological processes when protein molecules simultaneously bind to several distant sites on DNA. However, the molecular mechanisms of formation of these systems remain not well understood. Existing theoretical studies focus only on specific interactions between protein and DNA molecules at target sequences. However, the electrostatic origin of primary protein-DNA interactions suggests that interactions of proteins with all DNA segments should be considered. Here we theoretically investigate the role of non-…
In this article, we discuss metal-protein interactions in the Ag-lysozyme complex by spectroscopic measurements. The analysis of the variation in relative intensities of SERS bands reveal the orientation and the change in conformation of the protein molecules on the Ag surface with time. The interaction kinetics of metal-protein complexes has been analyzed over a period of three hours via both Raman and absorption measurements. Our analysis indicates that the Ag nanoparticles most likely interact with Trp-123 which is in close proximity to Phe-34 of the lysozyme molecule.
We present a single-molecule tool called the CoPro (Concentration of Proteins) method that uses millisecond imaging with convolution analysis, automated image segmentation and super-resolution localization microscopy to generate robust estimates for protein concentration in different compartments of single living cells, validated using realistic simulations of complex multiple compartment cell types. We demonstrates its utility experimentally on model Escherichia coli bacteria and Saccharomyces cerevisiae budding yeast cells, and use it to address the biological question of how signals are tra…
We study a protein-DNA target search model with explicit DNA dynamics applicable to in vitro experiments. We show that the DNA dynamics plays a crucial role for the effectiveness of protein "jumps" between sites distant along the DNA contour but close in 3D space. A strongly binding protein that searches by 1D sliding and jumping alone, explores the search space less redundantly when the DNA dynamics is fast on the timescale of protein jumps than in the opposite "frozen DNA" limit. We characterize the crossover between these limits using simulations and scaling theory. We also rationalize the …
Excitation of Low-Frequency Modes and the Effects of Protein Dynamics on Spectral Densities of Bacteriochlorophyll Molecules
School of Science, Constructor University（康斯特鲁克特大学理学院）；Department of Physics and Astronomy and Thomas Young Centre, University College London（伦敦大学学院物理与天文系及托马斯·杨中心）
The mode of action of proteins is to a large extent given by their ability to adopt different conformations. This is why imaging single biomolecules at atomic resolution is one of the ultimate goals of biophysics and structural biology. The existing protein database has emerged from X-ray crystallography, NMR or cryo-TEM investigations. However, these tools all require averaging over a large number of proteins and thus over different conformations. This of course results in the loss of structural information. Likewise it has been shown that even the emergent X-FEL technique will not get away w…
The theory of elastic rods can be used to describe certain geometric and topological properties of the DNA molecules. A similar effective field theory approach was previously suggested to describe the conformations and dynamics of proteins. In this letter we report a detailed study of the basic features of a version of the proposed model, which assumes proteins to be very long continuous curves. In the most appealing case, the model is based on a potential with a pair of minima corresponding to helical and strand-like configurations of the curves. It allows to derive several predictions about …
Computational Biology Center, IBM Thomas J. Watson Research（IBM托马斯·J·沃森研究中心计算生物学中心）
Proteins in complex with small molecule ligands represent the core of structure-based drug discovery. However, three-dimensional representations are absent from most deep-learning-based generative models. We here present a graph-based generative modeling technology that encodes explicit 3D protein-ligand contacts within a relational graph architecture. The models combine a conditional variational autoencoder that allows for activity-specific molecule generation with putative contact generation that provides predictions of molecular interactions within the target binding pocket. We show that mo…
A formal analogy of fluctuating diffusivity to thermodynamics is discussed for messenger RNA molecules fluorescently fused to a protein in living cells. Regarding the average value of the fluctuating diffusivity of such RNA-protein particles as the analog of the internal energy, the analogs of the quantity of heat and work are identified. The Clausius-like inequality is shown to hold for the entropy associated with diffusivity fluctuations, which plays a role analogous to the thermodynamic entropy, and the analog of the quantity of heat. The change of the statistical fluctuation distribution i…
Protein mobility is conventionally analyzed in terms of an effective diffusion. Yet, this description often fails to properly distinguish and evaluate the physical parameters (such as the membrane friction) and the biochemical interactions governing the motion. Here, we present a method combining high-density single-molecule imaging and statistical inference to separately map the diffusion and energy landscapes of membrane proteins across the cell surface at ~100 nm resolution (with acquisition of a few minutes). When applying these analytical tools to glycine neurotransmitter receptors (GlyRs…
This paper describes a design of a molecular propagation system in molecular communication. Molecular communication is a new communication paradigm where biological and artificially-created nanomachines communicate over a short distance using molecules. A molecular propagation system in molecular communication directionally transports molecules from a sender to a receiver. In the design described in this paper, protein filaments glide over immobilized motor proteins along preconfigured microlithographic tracks, and the gliding protein filaments carry and transport molecules from a sender to a …
The low-frequency collective vibrational modes in proteins as well as the protein-water interface have been suggested as dominant factors controlling the efficiency of biochemical reactions and biological energy transport. It is thus crucial to uncover the mystery of hydration structure and dynamics as well as their coupling to collective motions of proteins in aqueous solutions. Here we report dielectric properties of aqueous bovine serum albumin protein solutions as a model system using an extremely sensitive dielectric spectrometer with frequencies spanning from megahertz to terahertz. The …
We describe simulations of Proteins and artificial pseudo-molecules interacting and shaping lipid bilayer membranes. We extract protein diffusion Parameters, membrane deformation profiles and the elastic properties of the used membrane models in preparation of calculations based on a large scale continuum model.
Here we describe the coupled standardisation of two complementary fluorescence imaging techniques and apply it to liquid-liquid phase separated condensates formed from an EGFP fluorescent reporter of Flowering Control Locus A (FCA), a protein that associates with chromosomal DNA in plants during epigenetic regulation of the flowering process. First, we use home-built single-molecule Slimfield microscopy to establish a fluorescent protein standard. This sample comprises live yeast cells expressing Mig1 protein, a metabolic regulator which localises to the nucleus under conditions of high glucos…
Here we report on the use of double-nanohole (DNH) optical tweezers as a label-free and free-solution single-molecule probe for protein-DNA interactions. Using this approach, we demonstrate the unzipping of individual 10 base pair DNA-hairpins, and quantify how tumor suppressor p53 protein delays the unzipping. From the Arrhenius behavior, we find the energy barrier to unzipping introduced by p53 to be $2\times 10^{-20}$ J, whereas cys135ser mutant p53 does not show suppression of unzipping, which gives clues to its functional inability to suppress tumor growth. This transformative approach to…
Gene regulation is one of the most important fundamental biological processes in living cells. It involves multiple protein molecules that locate specific sites on DNA and assemble gene initiation or gene repression multi-molecular complexes. While the protein search dynamics for DNA targets has been intensively investigated, the role of inter-molecular interactions during the genetic activation or repression remains not well quantified. Here we present a simple one-dimensional model of target search for two interacting molecules that can reversibly form a dimer molecular complex, which also p…
In the present work, we provide a dielectric study on two differently concentrated aqueous lysozyme solutions in the frequency range from 1 MHz to 40 GHz and for temperatures from 275 to 330 K. We analyze the three dispersion regions, commonly found in protein solutions, usually termed beta-, gamma-, and delta-relaxation. The beta-relaxation, occurring in the frequency range around 10 MHz and the gamma-relaxation around 20 GHz (at room temperature) can be attributed to the rotation of the polar protein molecules in their aqueous medium and the reorientational motion of the free water molecules…
Single molecule tracking in live cells is the ultimate tool to study subcellular protein dynamics, but it is often limited by the probe size and photostability. Due to these issues, long-term tracking of proteins in confined and crowded environments, such as intracellular spaces, remains challenging. We have developed a novel optical probe consisting of 5-nm gold nanoparticles functionalized with a small fragment of camelid antibodies that recognize widely used GFPs with a very high affinity, which we call GFP-nanobodies. These small gold nanoparticles can be detected and tracked using phototh…
Single-molecule fluorescence techniques have revolutionized our ability to study proteins. However, the presence of a fluorescent label can alter the protein structure and/or modify its reaction with other species. To avoid the need for a fluorescent label, the intrinsic autofluorescence of proteins in the ultraviolet offers the benefits of fluorescence techniques without introducing the labelling drawbacks. Unfortunately, the low autofluorescence brightness of proteins has greatly challenged single molecule detection so far. Here we introduce optical horn antennas, a dedicated nanophotonic pl…
We present an extension of the Poisson-Boltzmann model in which the solute of interest is immersed in an assembly of self-orienting Langevin water dipoles, anions, cations, and hydrophobic molecules, all of variable densities. Interactions between charges are controlled by electrostatics, while hydrophobic interactions are modeled with a Yukawa potential. We impose steric constraints by assuming that the system is represented on a cubic lattice. We also assume incompressibility, i.e. all sites of the lattice are occupied. This model, which we refer to as the Hydrophobic Dipolar Poisson Boltzma…
Electropermeabilization is a method by which molecules can be introduced in living cells through the plasma membrane. Here, we show that electropermeabilization affects the lateral mobility of Rae-1, a GPi anchored protein. Our results shows that 10 to 20 % of the membrane surface is occupied by defaults or pores. Gene electrotransfert also affects the lateral mobility of Rae-1. Furthermore, we clearly show that once inserted into the plasma membrane, DNA totaly excludes Rae-1, indicating that DNA molecules are tightly packed together to forms huge aggregates.
More than twenty years ago, scientists succeeded in pushing the limits of optical detection to single molecules using fluorescence. This breakthrough has revolutionized biophysical measurements, but restrictions in photophysics and labeling protocols have motivated many efforts to achieve fluorescence-free single-molecule sensitivity in biological studies. Although several interesting mechanisms using vibrational spectroscopy, photothermal detection, plasmonics or microcavities have been proposed for biosensing at the single-protein level, no method has succeeded in direct label-free detection…
In this review we focus on the idea of establishing connections between the mechanical properties of DNAligand complexes and the physical chemistry of DNA-ligand interactions. This type of connection is interesting because it opens the possibility of performing a robust characterization of such interactions by using only one experimental technique: single molecule stretching. Furthermore, it also opens new possibilities in comparing results obtained by very different approaches, in special when comparing single molecule techniques to ensemble-averaging techniques. We start the manuscript revie…
We developed a multiscale approach (MultiSCAAL) that integrates the potential of mean force (PMF) obtained from all-atomistic molecular dynamics simulations with a knowledge-based energy function for coarse-grained molecular simulations in better exploring the energy landscape of a small protein under chemical interference such as chemical denaturation. An excessive amount of water molecules in all-atomistic molecular dynamics simulations often negatively impacts the sampling efficiency of some advanced sampling techniques such as the replica exchange method and it makes the investigation of c…
The network paradigm is increasingly used to describe the dynamics of complex systems. Here we review the current results and propose future development areas in the assessment of perturbation waves, i.e. propagating structural changes in amino acid networks building individual protein molecules and in protein-protein interaction networks (interactomes). We assess the possibilities and critically review the initial attempts for the application of game theory to the often rather complicated process, when two protein molecules approach each other, mutually adjust their conformations via multiple…
Natively unstructured proteins defy the classical "one sequence-one structure" paradigm of protein science. Monomers of these proteins in pathological conditions can aggregate in the cell, a process that underlies socially relevant neurodegenerative diseases such as Alzheimer and Parkinson. A full comprehension of the formation and structure of the so-called misfolded intermediates from which the aggregated states ensue is still lacking. We characterized the folding and the conformational diversity of alpha-synuclein (aSyn), a natively unstructured protein involved in Parkinson disease, by mec…
Label-free Imaging of Single-Biomolecule Structure and Interaction by Stimulated Raman Photothermal Encoded Scattering
Boston University（波士顿大学）；Stowers Institute for Medical Research（斯图沃医学研究所）；University of Washington（华盛顿大学）；Northeastern University（东北大学）；Georgia Institute of Technology（佐治亚理工学院）；Emory University（埃默里大学）；University of Virginia School of Medicine（弗吉尼亚大学医学院）；Howard Hughes Medical Institute（霍华德·休斯医学研究所）
The understanding of dynamics and functioning of biological membranes and in particular of membrane embedded proteins is one of the most fundamental problems and challenges in modern biology and biophysics. In particular the impact of membrane composition and properties and of structure and dynamics of the surrounding hydration water on protein function is an upcoming hot topic, which can be addressed by modern experimental and computational techniques. Correlated molecular motions might play a crucial role for the understanding of, for instance, transport processes and elastic properties, and…
Cellular energy production depends on electron transfer (ET) between proteins. In this theoretical study, we investigate the impact of structural and conformational variations on the electronic coupling between the redox proteins methylamine dehydrogenase and amicyanin from Paracoccus denitrificans. We used molecular dynamics simulations to generate configurations over a duration of 40ns (sampled at 100fs intervals) in conjunction with an ET pathway analysis to estimate the ET coupling strength of each configuration. In the wild type complex, we find that the most frequently occurring molecula…
Super-resolution microscopy has catalyzed valuable insights into the sub-cellular, mechanistic details of many different biological processes across a wide range of cell types. Fluorescence polarization spectroscopy tools have also enabled important insights into cellular processes through identifying orientational changes of biological molecules typically at an ensemble level. Here, we combine these two biophysical methodologies in a single home-made instrument to enable the simultaneous detection of orthogonal fluorescence polarization signals from single fluorescent protein molecules used a…
I review single-molecule experiments (SME) in biological physics. Recent technological developments have provided the tools to design and build scientific instruments of high enough sensitivity and precision to manipulate and visualize individual molecules and measure microscopic forces. Using SME it is possible to: manipulate molecules one at a time and measure distributions describing molecular properties; characterize the kinetics of biomolecular reactions and; detect molecular intermediates. SME provide the additional information about thermodynamics and kinetics of biomolecular processes.…
