Computer Simulation of Aqueous Biomolecular Systems

Abstract

Computer simulation techniques are increasingly being used to predict structural and thermodynamic properties of large heterogeneous macromolecule and solvent assemblies. We discuss, with examples from our own studies, some problems we and others have experienced in using these techniques, which were originally devised for simple liquids. In particular, we consider the problems which arise from the large size and heterogeneity of macromolecule water systems, comparisons with experimental data and equilibration and sampling procedures.     

大分子的吸入治疗

随着重组DNA技术和分子生物学的发展,以蛋白质和多肽为主的大分子成为一类新型药物,并越来越受到重视,新兴的基因治疗技术使得核酸大分子也有可能成为药物。目前,绝大部分大分子药物都是通过注射途径给药,病人在医院注射费用昂贵且不方便,因而许多注射替代给药途径成为研究热门,通过肺部吸入给药就是一种很有吸引力的非侵入性给药途径。本介绍了肺吸收大分子的可能机制和大分吸入治疗的临床与基础研究以及面临的问题。     

Simulation of a fluid phase lipid bilayer membrane: Incorporation of the surface tension into system boundary conditions

Modeling membranes is not just modeling another kind of macromolecule, but modeling an entire environment for a large class of biomolecular processes. Membrane modeling poses quite a different set of technical problems and scientific isues from modeling proteins. This paper reviews some of these issues and suggests approaches that seem promising for resolving them based on work in our laboratories and that of others.     

Analyzing the normal mode dynamics of macromolecules by the component synthesis method.

This paper presents a general method for studying the harmonic dynamics of large biomolecules and molecular complexes. The performance and accuracy of the method applied to a number of molecules are also reported. The basic approach of the method is to divide a macromolecule into a number of smaller components. The local normal modes of the components are first calculated by treating individual components and the interactions between nearest neighboring components. The physical displacements of all atoms are then represented in the local normal mode space, in which a selected range of high-frequency local modes is neglected. The equation of motion of the molecule in the local normal mode space will then have a smaller dimension, and consequently the normal modes of the whole structure, particularly for large molecules, can be solved much more easily. The normal modes of two polypeptides--(Ala)6 and (Ala)12--and a double-helical DNA--d(ATATA).d(TATAT)--are analyzed with this method. Reductions on the dimensions of harmonic dynamic equations for these molecules have been made, with the fraction of the deleted high-frequency modes ranging from 1/2 to 5/6. The calculated low-frequency normal modes are found to be very accurate as compared to the exact solutions by standard procedure. The major advantage of the present approach on macromolecule harmonic dynamics is that the reduction on the dimensionality of the eigenvalue problems can be varied according to the size of molecules, so the method can be easily applied to large macromolecules with controlled accuracy.     

Characterization of a progestin-binding macromolecule in the amphibian oocyte cytosol.

A [³H]-progestin-binding macromolecule has been isolated from $\text{R}\text{.}\text{pipiens}$ oocyte cytosol and characterized using binding assays, gel filtration, DEAE-cellulose chromatography and ultracentrifugal techniques. Macromolecules present in the prophase oocyte cytosol have a high affinity and specificity for the synthetic progestin R5020 and the intact oocyte will concentrate both R5020 and progesterone 20–40 fold from the medium. This may be the first published case of a cytosol steroid binding macromolecule in a cell system in which the steroid appears to act at an extranuclear level.     

Bayesian Uncertainty Quantification for Bond Energies and Mobilities Using Path Integral Analysis

Dynamic single-molecule force spectroscopy is often used to distort bonds. The resulting responses, in the form of rupture forces, work applied, and trajectories of displacements, are used to reconstruct bond potentials. Such approaches often rely on simple parameterizations of one-dimensional bond potentials, assumptions on equilibrium starting states, and/or large amounts of trajectory data. Parametric approaches typically fail at inferring complicated bond potentials with multiple minima, while piecewise estimation may not guarantee smooth results with the appropriate behavior at large distances. Existing techniques, particularly those based on work theorems, also do not address spatial variations in the diffusivity that may arise from spatially inhomogeneous coupling to other degrees of freedom in the macromolecule. To address these challenges, we develop a comprehensive empirical Bayesian approach that incorporates data and regularization terms directly into a path integral. All experimental and statistical parameters in our method are estimated directly from the data. Upon testing our method on simulated data, our regularized approach requires less data and allows simultaneous inference of both complex bond potentials and diffusivity profiles. Crucially, we show that the accuracy of the reconstructed bond potential is sensitive to the spatially varying diffusivity and accurate reconstruction can be expected only when both are simultaneously inferred. Moreover, after providing a means for self-consistently choosing regularization parameters from data, we derive posterior probability distributions, allowing for uncertainty quantification.     

A Computational Framework for Mechanical Response of Macromolecules: Application to the Salt Concentration Dependence of DNA Bendability

A computational framework is presented for studying the mechanical response of macromolecules. The method combines a continuum mechanics (CM) model for the mechanical properties of the macromolecule with a continuum electrostatic (CE) treatment of solvation. The molecules are represented by their shape and key physicochemical characteristics such as the distribution of materials properties and charge. As a test case, we apply the model to the effect of added salt on the bending of DNA. With a simple representation of DNA, the CM/CE framework using a Debye-Hückel model leads to results that are in good agreement with both analytical theories and recent experiments, including a modified Odijk-Skolnick-Fixman theory that takes the finite length of DNA into consideration. Calculations using a more sophisticated CE model (Poisson-Boltzmann), however, suffer from convergence problems, highlighting the importance of balancing numerical accuracy in the CM and CE models when dealing with very large systems, particularly those with a high degree of symmetry.     

Assessment of morphology of asymmetrical biological macromolecules with digital techniques

Digital image processing techniques which determine the morphology of an unordered, spherical biological macromolecule are discussed. The techniques involve slicing appropriate intensity levels of the scanning transmission electron microscope image so that more accurate observation of morphology can be made. The techniques were applied to native and hybrid canine plasma high-density lipoprotein molecules. Resulting data indicated that the techniques were very useful to assess the diameters of both molecule and surface substructures of such asymmetrical macromolecules.     

Three-dimensional structure of the truncated core of the Saccharomyces cerevisiae pyruvate dehydrogenase complex determined from negative stain and cryoelectron microscopy images.

Dihydrolipoamide acyltransferase (E2), a catalytic and structural component of the three functional classes of multienzyme complexes that catalyze the oxidative decarboxylation of alpha-keto acids, forms the central core to which the other components are attached. We have imaged by negative stain and cryoelectron microscopy the truncated dihydrolipoamide acetyltransferase core (60 subunits; M(r) = 2.7 x 10(6)) of the Saccharomyces cerevisiae pyruvate dehydrogenase complex. Using icosahedral particle reconstruction techniques, we determined its structure to 25 A resolution. Although the model derived from the negative stain reconstruction was approximately 20% smaller than the model derived from the frozen-hydrated data, when corrected for the effects of the electron microscope contrast transfer functions, the reconstructions showed excellent correspondence. The pentagonal dodecahedron-shaped macromolecule has a maximum diameter, as measured along the 3-fold axis, of approximately 226 A (frozen-hydrated value), and 12 large openings (approximately 63 A in diameter) on the 5-fold axes that lead into a large solvent-accessible cavity (approximately 76-140 A diameter). The 20 vertices consist of cone-shaped trimers, each with a flattened base on the outside of the structure and an apex directed toward the center. The trimers are interconnected by 20 A thick "bridges" on the 2-fold axes. These studies also show that the highest resolution features apparent in the frozen-hydrated reconstruction are revealed in a filtered reconstruction of the stained molecule.     

Retina cognin does not bind to itself during membrane interaction in vitro

Retina cognin (R-cognin) is an intrinsic membrane protein of vertebrate retinal cells which supports tissue-specific cell adhesion and mediates cell type-specific associations during development. As a first step in understanding how R-cognin mediates specific adhesion of retinal cell membranes, we asked if cognin bound to another cognin molecule or to a different macromolecule, a possible cognin-binding protein. To do this, we constructed an affinity column with retinal cell membrane proteins (enriched for cognin) bound to the matrix. Proteins in a detergent extract of retinal cell membranes were exposed to this matrix and those which bound specifically eluted and identified by immunoelectrophoresis. Most prominent among these was a protein with an apparent mass of 64 kDa. The binding of this material to the column was blocked by cognin antibody. To eliminate possible artifacts of molecular interactions in vitro, we sought independent confirmation that 64 kDa protein actually bound R-cognin. Using a modified retina membrane vesicle system, we asked what proteins could be photoaffinity cross-linked to cognin during vesicle aggregation. Cross-linking produced a 114 kDa complex on gels which could be resolved into a 50 kDa (cognin) and a 64 kDa band under reducing conditions. Identification of a 64 kDa protein by independent techniques suggests that cognin promotes association of embryonic chick neural retina cells by binding to this macromolecule or these molecules. Identification of a second component in the mechanism should allow elucidation of cognin's role in mediating cell-cell interactions in developing neural retina.     

Crystallization of soluble proteins in vapor diffusion for x-ray crystallography

The preparation of protein single crystals represents one of the major obstacles in obtaining the detailed 3D structure of a biological macromolecule. The complete automation of the crystallization procedures requires large investments in terms of money and labor, which are available only to large dedicated infrastructures and is mostly suited for genomic-scale projects. On the other hand, many research projects from departmental laboratories are devoted to the study of few specific proteins. Here, we try to provide a series of protocols for the crystallization of soluble proteins, especially the difficult ones, tailored for small-scale research groups. An estimate of the time needed to complete each of the steps described can be found at the end of each section.     

Strategies for oral delivery of macromolecule drugs

Advances in biotechnology, gene manipulation, and protein engineering for macromolecule drugs, such as insulin, parathyroid hormone (PTH), calcitonin, human growth hormone, erythropoietin (EPO), and peptide YY (PYY) allow commercial production in large scale for diverse therapeutic uses. Other macromolecules, such as mucopolysaccharide heparin, have expanded markets through improvements in their pharmacokinetic and pharmacological effects. However, most products are available only as injectable forms and are limited to patients with no alternative therapeutic choices. Orally available macromolecule formulations are still unmet needs for improving patient compliance and expanding administration paradigms and indications. Oral delivery technologies including carrier systems, absorption enhancers, protease inhibitors, and modification by conjugating transporter or receptor recognition molecules have been developed and some are undergoing clinical studies. In this review, we discuss major obstacles for oral absorption of macromolecule drugs and summarize recent strategies to overcome the huddles related to enhancing intestinal permeation.     

Contributions to the binding free energy of ligands to avidin and streptavidin

The free energy of binding of a ligand to a macromolecule is here formally decomposed into the (effective) energy of interaction, reorganization energy of the ligand and the macromolecule, conformational entropy change of the ligand and the macromolecule, and translational and rotational entropy loss of the ligand. Molecular dynamics simulations with implicit solvation are used to evaluate these contributions in the binding of biotin, biotin analogs, and two peptides to avidin and streptavidin. We find that the largest contribution opposing binding is the protein reorganization energy, which is calculated to be from 10 to 30 kcal/mol for the ligands considered here. The ligand reorganization energy is also significant for flexible ligands. The translational/rotational entropy is 4.5-6 kcal/mol at 1 M standard state and room temperature. The calculated binding free energies are in the correct range, but the large statistical uncertainty in the protein reorganization energy precludes precise predictions. For some complexes, the simulations show multiple binding modes, different from the one observed in the crystal structure. This finding is probably due to deficiencies in the force field but may also reflect considerable ligand flexibility.     

Highly sensitive fluorescence imaging of cancer with avidin-protease probe conjugate

It is a long-term goal of cancer diagnosis to develop tumor-imaging techniques that have sufficient specificity and sensitivity to detect small tumor nodules during surgery or endoscopic surgery. Here, we introduce an avidin-conjugated fluorescence probe, Avidin-Leu-HMRG, which consists of a cancer-targeting macromolecule (avidin) and a protease-activatable probe. The conjugate has a high affinity for lectin on cancer cells and undergoes endocytosis, followed by irreversible fluorescence activation due to cleavage by lysosomal leucine aminopeptidase. In a mouse model of peritoneal ovarian metastases, the probe could detect submillimeter-sized tumor nodules with a high S/N ratio at 1 h after intraperitoneal injection.     

Recent advances in microfluidic technologies for biochemistry and molecular biologys

Advances in the fields of proteomics and genomics have necessitated the development of high-throughput screening methods (HTS) for the systematic transformation of large amounts of biological chemical data into an organized database of knowledge. Microfluidic systems are ideally suited for high-throughput biochemical experimentation since they offer high analytical throughput, consume minute quantities of expensive biological reagents, exhibit superior sensitivity and functionality compared to traditional micro-array techniques and can be integrated within complex experimental work flows. A range of basic biochemical and molecular biological operations have been transferred to chip-based microfluidic formats over the last decade, including gene sequencing, emulsion PCR, immunoassays, electrophoresis, cell-based assays, expression cloning and macromolecule blotting. In this review, we highlight some of the recent advances in the application of microfluidics to biochemistry and molecular biology.     

Kinetics of allosteric conformational transition of a macromolecule prior to ligand binding

Two fundamentally different mechanisms of ligand binding are commonly encountered in biological kinetics. One mechanism is a sequential multistep reaction in which the bimolecular binding step is followed by first-order steps. The other mechanism includes the conformational transition of the macromolecule, before the ligand binding, followed by the ligand binding process to one of the conformational states. In stopped-flow kinetic studies, the reaction mechanism is established by examining the behavior of relaxation times and amplitudes as a function of the reactant concentrations. A major diagnostic tool for detecting the presence of a conformational equilibrium of the macromolecule, before the ligand binding, is the decreasing value of one of the reciprocal relaxation times with the increasing [ligand]. The sequential mechanism cannot generate this behavior for any of the relaxation times. Such dependence is intuitively understood on the basis of approximate expressions for the relaxation times that can be comprehensively derived, using the characteristic equation of the coefficient matrix and polynomial theory. Generally, however, the used approximations may not be fulfilled. On the other hand, the two kinetic mechanisms can always be distinguished, using the approach based on the combined application of pseudo-first-order conditions, with respect to the ligand and the macromolecule. The two experimental conditions differ profoundly in the extent of the effect of the ligand on the protein conformational equilibrium. In a large excess of the ligand, the conformational equilibrium of the macromolecule, before the ligand binding, is strongly affected by the binding process. However, in a large excess of the macromolecule, ligand binding does not perturb the internal equilibrium of the macromolecule. As a result, the normal mode, affected by the conformational transition, is absent in the observed relaxation process. In the case of a sequential mechanism, the number of relaxation times is not altered by different pseudo-first-order conditions. Thus, the approach provides a strong diagnostic criterion for detecting the presence of the conformational transition of the macromolecule and establishing the correct mechanism. Application of this approach is illustrated for the binding of 3'-O-(N-methylantraniloyl)-5'-diphosphate to the E. coli DnaC protein.     

Association kinetics with coupled diffusion. An extension to coiled-chain macromolecules applied to the lac repressor-operator system.

The association of a molecule onto a specified binding site on a large chain-like macromolecule is described in the "sliding" model, where the molecule is allowed to move along the chain in a one-dimensional diffusion which is coupled to the three-dimensional diffusion in solution. The present work extends a previous one by treating the chains more generally as coiled instead of straight. The model is applied to the lac repressor-operator association. A general expression for the rate of unspecific attachment to a chain-like macromolecule is also derived.     

Mechanical degradation and changes in conformation of hydrophobically modified starch

In this paper, we study the mechanical degradation and changes in conformation of a branched ultrahigh molar mass biomacromolecule, hydrophobically modified starch, as caused by high-pressure homogenization. The characterization was performed with asymmetrical flow field-flow fractionation (AsFlFFF) with multiangle light scattering (MALS) and refractive index detection. The starch which had been chemically modified with octenyl succinate anhydride (OSA) proved to be very large and polydisperse. Upon high-pressure homogenization, the molar mass and rms radius (r(rms)) decreased, and the extent of these changes was related to the turbulent flow conditions during homogenization. The treatment also induced an increase and scaling with size in the apparent density of the macromolecules. To further study the changes in conformation, it was necessary to calculate the hydrodynamic radii (r(h)). This can be determined numerically from the elution times in the analysis and the flow conditions in the AsFlFFF channel. The results showed that the treatment can cause a dramatic decrease in the quotient between r(rms) and r(h), suggesting major conformational changes. These results together could be interpreted as degradation and "crumpling" of the macromolecule, which would give a decrease in r(rms) and an increase in apparent density, together with a "fraying" of more outer parts of the macromolecule, which could give rise to the increase in r(h).     

Diffraction imaging of single particles and biomolecules

Theory predicts that with a very short and very intense X-ray pulse, the image of a single diffraction pattern may be recorded from a large macromolecule, a virus, or a nanocluster of proteins without the need for a crystal. A three-dimensional data set can be assembled from such images when many copies of the molecule are exposed to the beam one by one in random orientations. We outline a method for structure reconstruction from such a data set in which no independent information is available about the orientation of the images. The basic requirement for reconstruction and/or signal averaging is the ability to tell whether two noisy diffraction patterns represent the same view of the sample or two different views. With this knowledge, averaging techniques can be used to enhance the signal and extend the resolution in a redundant data set. Based on statistical properties of the diffraction pattern, we present an analytical solution to the classification problem. The solution connects the number of incident X-ray photons with the particle size and the achievable resolution. The results are surprising in that they show that classification can be done with less than one photon per pixel in the limiting resolution shell, assuming Poisson-type photon noise in the image. The results can also be used to provide criteria for improvements in other image classification procedures, e.g., those used in electron tomography or diffraction.     

In vivo brain macromolecule signals in healthy and glioblastoma mouse models: 1H magnetic resonance spectroscopy,post‐processing and metabolite quantification at 14.1 T

In ¹H magnetic resonance spectroscopy, macromolecule signals underlay metabolite signals, and knowing their contribution is necessary for reliable metabolite quantification. When macromolecule signals are measured using an inversion‐recovery pulse sequence, special care needs to be taken to correctly remove residual metabolite signals to obtain a pure macromolecule spectrum. Furthermore, since a single spectrum is commonly used for quantification in multiple experiments, the impact of potential macromolecule signal variability, because of regional differences or pathologies, on metabolite quantification has to be assessed. In this study, we introduced a novel method to post‐process measured macromolecule signals that offers a flexible and robust way of removing residual metabolite signals. This method was applied to investigate regional differences in the mouse brain macromolecule signals that may affect metabolite quantification when not taken into account. However, since no significant differences in metabolite quantification were detected, it was concluded that a single macromolecule spectrum can be generally used for the quantification of healthy mouse brain spectra. Alternatively, the study of a mouse model of human glioma showed several alterations of the macromolecule spectrum, including, but not limited to, increased mobile lipid signals, which had to be taken into account to avoid significant metabolite quantification errors.