Conformational Changes Of Biological Macromolecules Research Articles

Resolving distance variations by single-molecule FRET and EPR spectroscopy using rotamer libraries

Förster resonance energy transfer (FRET) and electron paramagnetic resonance (EPR) spectroscopy are complementary techniques for quantifying distances in the nanometer range. Both approaches are commonly employed for probing the conformations and conformational changes of biological macromolecules based on site-directed fluorescent or paramagnetic labeling. FRET can be applied in solution at ambient temperature and thus provides direct access to dynamics, especially if used at the single-molecule level, whereas EPR requires immobilization or work at cryogenic temperatures but provides data that can be more reliably used to extract distance distributions. However, a combined analysis of the complementary data from the two techniques has been complicated by the lack of a common modeling framework. Here, we demonstrate a systematic analysis approach based on rotamer libraries for both FRET and EPR labels to predict distance distributions between two labels from a structural model. Dynamics of the fluorophores within these distance distributions are taken into account by diffusional averaging, which improves the agreement with experiment. Benchmarking this methodology with a series of surface-exposed pairs of sites in a structured protein domain reveals that the lowest resolved distance differences can be as small as ∼0.25 nm for both techniques, with quantitative agreement between experimental and simulated transfer efficiencies within a range of ±0.045. Rotamer library analysis thus establishes a coherent way of treating experimental data from EPR and FRET and provides a basis for integrative structural modeling, including studies of conformational distributions and dynamics of biological macromolecules using both techniques.

A catalytic—regulated gold nanorods etching process as a receptor with multiple readouts for protein detection

A multidimensional colorimetric sensor array was produced based on a catalytic – regulated gold nanorod (Au NR) etching process as a receptor for protein detection. With the peroxidase-like property of gold nanosphere (Au NP) and H2O2, 3,3′,5,5′-tetramethylbenzidine (TMB) (colorless) is oxidized into TMB2+ (yellow). Different concentrations of TMB2+ promote different degrees of Au NR etching, and the localized surface plasmon resonance band of the Au NR is blueshifted. The blueshift results in color displays (gray, purple, blue and pink), which can be clearly distinguished by the naked eye. When the surfaces of the Au NP are covered by proteins, the catalytic activity is influenced. On this basis, the dynamically tunable system obtains triple channel information including the plasma resonance effect, the catalytic activity of Au NP and the etching of Au NR by TMB2+ (catalytic products of TMB in the Au NP-H2O2 reaction system), which can be adjusted using different proteins. The array can correctly identify native and thermally denatured proteins. This may provide a new method for studying conformational changes in biological macromolecules. Furthermore, pure protein (different concentrations) and binary protein mixtures can be efficiently differentiated in the urine sample.

Increasing Versatility of Small Angle X‐Ray Scattering

Small Angle X‐ray Scattering (SAXS), a technique initially popular in the bio‐medical community largely due to the relative logistical ease compared to other structural biological techniques has in recent years emerged as a much more versatile biophysical tool that gives unprecedented access to a real time view of structural alterations occurring during critical processes such as protein and nucleic acid folding, enzyme‐substrate binding and other ligand induced conformational changes in biological macromolecules. Developmental efforts spanning the last decade at the Biophysics Collaborative Access Team (BioCAT) located at The Advanced Photon Source (APS), Chicago, have resulted in a time‐resolved SAXS instrument which combines a state‐of‐the‐art synchrotron beamline capable of delivering up to 1013 photons/s with microfluidic mixers that afford access to time regimes ranging from 10s of microseconds to 1.5 seconds. After being made available to the general SAXS user community the time‐resolved SAXS setup has been used to study several interesting systems of biomedical interest, which in turn have led to unprecedented insights. For instance, key aspects of protein‐folding were elucidated using the chaotic flow mixer which gives access to a time regime from ~80μs to ~95ms and mechanistic details of the Insulin Degrading Enzyme (IDE) were revealed using the laminar flow mixer which allows measurement of time‐points from ~5ms to 1.5s. Improved operational efficiency and minimal sample consumption have made these heretofore challenging experiments feasible for a wider user community.Furthermore, the equilibrium SAXS instrument, which already had an integrated liquid chromatography setup to ensure optimal sample quality, now also has in‐line MALS and DLS detectors making BioCAT a unique facility for comprehensive biophysical characterization of biological macromolecules. Software tools developed in‐house have ensured a high degree of user‐friendliness and we hope to introduce this powerful biophysical tool to a larger community of non‐expert users.Support or Funding InformationNIGMS ‐ P41 GM103622

TAPS: A traveling-salesman based automated path searching method for functional conformational changes of biological macromolecules.

Locating the minimum free energy paths (MFEPs) between two conformational states is among the most important tasks of biomolecular simulations. For example, knowledge of the MFEP is critical for focusing the effort of unbiased simulations that are used for the construction of Markov state models to the biologically relevant regions of the system. Typically, existing path searching methods perform local sampling around the path nodes in a pre-selected collective variable (CV) space to allow a gradual downhill evolution of the path toward the MFEP. Despite the wide application of such a strategy, the gradual path evolution and the non-trivial a priori choice of CVs are also limiting its overall efficiency and automation. Here we demonstrate that non-local perpendicular sampling can be pursued to accelerate the search, provided that all nodes are reordered thereafter via a traveling-salesman scheme. Moreover, path-CVs can be computed on-the-fly and used as a coordinate system, minimizing the necessary prior knowledge about the system. Our traveling-salesman based automated path searching method achieves a 5-8 times speedup over the string method with swarms-of-trajectories for two peptide systems in vacuum and solution, making it a promising method for obtaining initial pathways when investigating functional conformational changes between a pair of structures.

An efficient Bayesian kinetic lumping algorithm to identify metastable conformational states via Gibbs sampling.

Markov State Model (MSM) has become a popular approach to study the conformational dynamics of complex biological systems in recent years. Built upon a large number of short molecular dynamics simulation trajectories, MSM is able to predict the long time scale dynamics of complex systems. However, to achieve Markovianity, an MSM often contains hundreds or thousands of states (microstates), hindering human interpretation of the underlying system mechanism. One way to reduce the number of states is to lump kinetically similar states together and thus coarse-grain the microstates into macrostates. In this work, we introduce a probabilistic lumping algorithm, the Gibbs lumping algorithm, to assign a probability to any given kinetic lumping using the Bayesian inference. In our algorithm, the transitions among kinetically distinct macrostates are modeled by Poisson processes, which will well reflect the separation of time scales in the underlying free energy landscape of biomolecules. Furthermore, to facilitate the search for the optimal kinetic lumping (i.e., the lumped model with the highest probability), a Gibbs sampling algorithm is introduced. To demonstrate the power of our new method, we apply it to three systems: a 2D potential, alanine dipeptide, and a WW protein domain. In comparison with six other popular lumping algorithms, we show that our method can persistently produce the lumped macrostate model with the highest probability as well as the largest metastability. We anticipate that our Gibbs lumping algorithm holds great promise to be widely applied to investigate conformational changes in biological macromolecules.

Using Multiorder Time-Correlation Functions (TCFs) To Elucidate Biomolecular Reaction Pathways from Microsecond Single-Molecule Fluorescence Experiments.

Recent advances in single-molecule fluorescence imaging have made it possible to perform measurements on microsecond time scales. Such experiments have the potential to reveal detailed information about the conformational changes in biological macromolecules, including the reaction pathways and dynamics of the rearrangements involved in processes, such as sequence-specific DNA "breathing" and the assembly of protein-nucleic acid complexes. Because microsecond-resolved single-molecule trajectories often involve "sparse" data, that is, they contain relatively few data points per unit time, they cannot be easily analyzed using the standard protocols that were developed for single-molecule experiments carried out with tens-of-millisecond time resolution and high "data density." Here, we describe a generalized approach, based on time-correlation functions, to obtain kinetic information from microsecond-resolved single-molecule fluorescence measurements. This approach can be used to identify short-lived intermediates that lie on reaction pathways connecting relatively long-lived reactant and product states. As a concrete illustration of the potential of this methodology for analyzing specific macromolecular systems, we accompany the theoretical presentation with the description of a specific biologically relevant example drawn from studies of reaction mechanisms of the assembly of the single-stranded DNA binding protein of the T4 bacteriophage replication complex onto a model DNA replication fork.

Программа трехмерного моделирования и визуализации конформационной динамики биомакромолекул Maya-K-PDB

The concept of "active" molecular models was proposed. The Maya-K-PDB software for the creation of active molecular models in the 3D Maya environment was developed. The Maya-K-PDB provides various opportunities for dynamic visualization and modeling of conformational changes of biological macromolecules. Also many complicated processes on molecular level can be presented. The article describes the Maya-K-PDB user interface and illustrates basic examples of software implementation.

Trajectory Entropy of Continuous Stochastic Processes at Equilibrium.

We propose to quantify the trajectory entropy of a dynamic system as the information content in excess of a free-diffusion reference model. The space-time trajectory is now the dynamic variable, and its path probability is given by the Onsager-Machlup action. For the time propagation of the overdamped Langevin equation, we solved the action path integral in the continuum limit and arrived at an exact analytical expression that emerged as a simple functional of the deterministic mean force and the stochastic diffusion. This work may have direct implications in chemical and phase equilibria, bond isomerization, and conformational changes in biological macromolecules as well transport problems in general.

Small angle neutron scattering for the study of solubilised membrane proteins

Small angle neutron scattering (SANS) is a powerful technique for investigating association states and conformational changes of biological macromolecules in solution. SANS is of particular interest for the study of the multi-component systems, as membrane protein complexes, for which in vitro characterisation and structure determination are often difficult. This article details the important physical properties of surfactants in view of small angle neutron scattering studies and the interest to deuterate membrane proteins for contrast variation studies. We present strategies for the production of deuterated membrane proteins and methods for quality control. We then review some studies on membrane proteins, and focus on the strategies to overcome the intrinsic difficulty to eliminate homogeneously the detergent or surfactant signal for solubilised membrane proteins, or that of lipids for membrane proteins inserted in liposomes.

Building Markov state models with solvent dynamics

BackgroundMarkov state models have been widely used to study conformational changes of biological macromolecules. These models are built from short timescale simulations and then propagated to extract long timescale dynamics. However, the solvent information in molecular simulations are often ignored in current methods, because of the large number of solvent molecules in a system and the indistinguishability of solvent molecules upon their exchange.MethodsWe present a solvent signature that compactly summarizes the solvent distribution in the high-dimensional data, and then define a distance metric between different configurations using this signature. We next incorporate the solvent information into the construction of Markov state models and present a fast geometric clustering algorithm which combines both the solute-based and solvent-based distances.ResultsWe have tested our method on several different molecular dynamical systems, including alanine dipeptide, carbon nanotube, and benzene rings. With the new solvent-based signatures, we are able to identify different solvent distributions near the solute. Furthermore, when the solute has a concave shape, we can also capture the water number inside the solute structure. Finally we have compared the performances of different Markov state models. The experiment results show that our approach improves the existing methods both in the computational running time and the metastability.ConclusionsIn this paper we have initiated an study to build Markov state models for molecular dynamical systems with solvent degrees of freedom. The methods we described should also be broadly applicable to a wide range of biomolecular simulation analyses.

Dynamics, Flexibility and Ligand-Induced Conformational Changes in Biological Macromolecules: A Computational Approach

Biomolecules possess important dynamical properties that enable them to adapt and alternate their conformation as a response to environmental stimuli. Recent advancements in computational resources and methodology allow a higher capability to mimic in vitro conditions and open up the possibility of studying large systems over longer timescales. Here, we describe commonly used computational approaches for studying the dynamic properties of proteins. We review a selected set of simulation studies on ligand-induced changes in the chaperonin GroEL-GroES, a molecular folding machine, maltose-binding protein, a prototypical member of the periplasmic binding proteins, and the bacterial ribosomal A-site, focusing on aminoglycoside antibiotic recognition. We also discuss a recent quantitative reconstruction of the binding process of benzamidine and trypsin. These studies contribute to the understanding and further development of the medicinal regulation of large biomolecular systems.

Microfluidic Mixers for the Investigation of Rapid Protein Folding Kinetics Using Synchrotron Radiation Circular Dichroism Spectroscopy

We have developed a microfluidic mixer optimized for rapid measurements of protein folding kinetics using synchrotron radiation circular dichroism (SRCD) spectroscopy. The combination of fabrication in fused silica and synchrotron radiation allows measurements at wavelengths below 220 nm, the typical limit of commercial instrumentation. At these wavelengths, the discrimination between the different types of protein secondary structure increases sharply. The device was optimized for rapid mixing at moderate sample consumption by employing a serpentine channel design, resulting in a dead time of less than 200 micros. Here, we discuss the design and fabrication of the mixer and quantify the mixing efficiency using wide-field and confocal epi-fluorescence microscopy. We demonstrate the performance of the device in SRCD measurements of the folding kinetics of cytochrome c, a small, fast-folding protein. Our results show that the combination of SRCD with microfluidic mixing opens new possibilities for investigating rapid conformational changes in biological macromolecules that have previously been inaccessible.

FLIPDock: Docking flexible ligands into flexible receptors

Conformational changes of biological macromolecules when binding with ligands have long been observed and remain a challenge for automated docking methods. Here we present a novel protein-ligand docking software called FLIPDock (Flexible LIgand-Protein Docking) allowing the automated docking of flexible ligand molecules into active sites of flexible receptor molecules. In FLIPDock, conformational spaces of molecules are encoded using a data structure that we have developed recently called the Flexibility Tree (FT). While the FT can represent fully flexible ligands, it was initially designed as a hierarchical and multiresolution data structure for the selective encoding of conformational subspaces of large biological macromolecules. These conformational subspaces can be built to span a range of conformations important for the biological activity of a protein. A variety of motions can be combined, ranging from domains moving as rigid bodies or backbone atoms undergoing normal mode-based deformations, to side chains assuming rotameric conformations. In addition, these conformational subspaces are parameterized by a small number of variables which can be searched during the docking process, thus effectively modeling the conformational changes in a flexible receptor. FLIPDock searches the variables using genetic algorithm-based search techniques and evaluates putative docking complexes with a scoring function based on the AutoDock3.05 force-field. In this paper, we describe the concepts behind FLIPDock and the overall architecture of the program. We demonstrate FLIPDock's ability to solve docking problems in which the assumption of a rigid receptor previously prevented the successful docking of known ligands. In particular, we repeat an earlier cross docking experiment and demonstrate an increased success rate of 93.5%, compared to original 72% success rate achieved by AutoDock over the 400 cross-docking calculations. We also demonstrate FLIPDock's ability to handle conformational changes involving backbone motion by docking balanol to an adenosine-binding pocket of protein kinase A.

Bubble dynamics in DNA

The formation of local denaturation zones (bubbles) in double-stranded DNA is an important example for conformational changes of biological macromolecules. We study the dynamics of bubble formation in terms of a Fokker-Planck equation for the probability density to find a bubble of size n base pairs at time t, on the basis of the free energy in the Poland-Scheraga model. Characteristic bubble closing and opening times can be determined from the corresponding first passage time problem, and are sensitive to the specific parameters entering the model. A multistate unzipping model with constant rates recently applied to DNA breathing dynamics [G. Altan-Bonnet et al, Phys. Rev. Lett. 90, 138101 (2003)] emerges as a limiting case.

Looking behind the Beamstop: X-Ray Solution Scattering Studies of Structure and Conformational Changes of Biological Macromolecules

Publisher Summary This chapter illustrates the use of solution-scattering methods to solve problems related to the crystal structures of biological macromolecules. As a consequence, important applications that have no explicit link with crystallography, such as those related to (un)folding of proteins and nucleic acids, are hardly mentioned. Advances in X-ray crystallography have made it much easier to determine high-resolution structures of biological macromolecules, even those of large macromolecular assemblies. The perception of small-angle X-ray scattering by solutions of biological macromolecules has undergone a deep change as large facilities and increasingly powerful data analysis and modeling software have become accessible to a larger community. The value of small-angle X-ray scattering (SAXS) and the unique links it provides between high-and low-resolution structure determination methods and among structural, hydrodynamic, and other scattering methods has been amply demonstrated in many applications. A growing number of crystallographers show interest in using SAXS as a complement to crystal studies. A new generation of SAXS instruments using undulator beams has become available at several facilities.

FUS: a system to simulate conformational changes in biological macromolecules.

In order to study the dynamics of protein and nucleic acid conformations, a molecular folding-unfolding system (FUS written in Lisp) has been developed. Secondary structure features of protein and nucleic acids are graphically represented by cubes in a modified 'Blocks World' paradigm. Modeling of protein and nucleic acid unfolding (denaturation) and folding of their three-dimensional structure is possible by the use of high level 'block' operators which allow displacement of these structural features in space. Due to the flexible nature of this program, FUS is a useful tool for the rapid evaluation of user-defined rules governing conformational changes. The use of FUS to unfold three common proteins (prealbumin, flavodoxin and triose phosphate isomerase) and a tRNA is presented.

The application of perturbed directional correlation of gamma rays to the study of protein-metal interactions.

Several branches of nuclear spectroscopy—magnetic resonance and the Mossbauer effect in particular—have been the subject of considerable interest to obtain structural and motional information about biological macromolecules. Recently, a third technique— perturbed gamma-gamma directional correlation—is being tested for the study of rotational correlation times, internal rotations and conformational changes in biological macromolecules. When two gamma rays from a radioactive nucleus are emitted in a cascade and detected with a coincidence spectrometer, the coincidence counting rate depends strongly on the angle between their directions of propagation. This directional correlation can be perturbed by the interaction of the nucleus with fields existing in macromolecules. A study of such perturbed directional correlation (PDC) has the potential to provide information on protein-metal interactions. The use of a radioactive nucleus as a rotational tracer to label macromolecules offers the possibility of obtaining information on protein structure with the sensitivity and instrumental simplicity of radioactive tracer techniques. Work to date has centered on measurement of internal electric field gradients of metalloproteins, molecular correlation times and the correlation time of water associated with proteins. This paper is a brief review of the theory and experimental technique of perturbed directional correlation of gamma rays in comparison to Mossbauer spectroscopy and the methods of magnetic resonance. Particular emphasis will be placed on the isotopes which can be used in PDC especially those which may occupy sites in biological macromolecules.

Nuclear magnetic resonance and optical spectroscopic studies of poly l and poly d alanine

Nuclear magnetic resonance spectroscopy is finding increasing application to studies of conformational changes in biological macromolecules and their synthetic analogues. It has been suggested that the resonance peaks due to backbone protons in helical polymers will be broadened by non-averaging of dipole-dipole interactions and that the areas of these resonance peaks will be a measure of the random coil content of the system. In agreement with others, we do not find this to be the case for poly l and poly d alanine in trifluoracetic acid (TFA)-deuterochloroform (CDCl 3) solvent systems where the optical rotatory dispersion parameters indicate a helix content in excess of 50 per cent while the n.m.r. spectra for these solutions are still fully developed. It is suggested that the peak narrowing process operating in this system is rapid exchange between random coil and helical segments. Others suggest that, although the b 0 values for the TFA-CDCl 3 solvent systems indicate 54 per cent helix content, this o.r.d. parameter is an unreliable guide to helix content and that the polypeptide is helical; furthermore that they are observing for the first time a well developed n.m.r. spectrum for a polypeptide ‘purported to be in a helical conformation’. These conclusions are not supported by the results obtained from studies of poly l and poly d alanine in dichloracetic acid (DCA)-TFA and DCA-CDCl 3 solvent systems. In these systems it is possible to obtain appreciably higher b 0 values (up to 458°) than was observed for the TFA-CDCl 3 solvent system and these increases in b 0 are accompanied by marked broadening and loss of area of the proton resonance peaks. It is suggested therefore that when the helix content is greater than 50 per cent the motions of helical segments become too slow for the peak narrowing process of exchange between helical and random coil forms to be effective. These observations support the earlier suggestions that in fully helical macromolecules the backbone protons resonance peaks will be broadened by non-averaging of dipole-dipole interactions. It is possible to correlate the shifts in the poly l alanine resonance peaks with helix content and it has been suggested that the shifts are due to exchange of the protons between two chemical environments, i.e. the helical and random coil forms. Infra-red studies show, however, that there is a specific interaction between the carboxylic acid molecule and the helical form of poly alanine and we suggest that part of the shifts observed in the resonance peaks may result from magnetic anisotropy effects of the carboxyolic acid molecules complexed with hydrogen bonded amide groups in the helical form. In agreement with others it is suggested that the mode of interaction of the carboxylic acid molecules with poly alanine is a strong hydrogen bonding interaction and not protonation as has been suggested.

Kinetics of synthesis and/or conformational changes of biological macromolecules

AbstractThe analogy, in both the thermodynamics and the kinetics, of reversible polymerizations on templates (in the case wherein these are catalyzed by exoenzymes) to helix–coil transitions (in the case where these proceed only from one end of each macromolecular chain) is presented. A suggestion, based on this analogy, is made concerning the possible nature of biological control of synthesis of macromolecules (enzyme induction and repression). The equations governing the kinetics of these one‐dimensional cooperative processes are presented and their solutions discussed.