Measuring the energy landscape roughness and the transition state location of biomolecules using single molecule mechanical unfolding experiments

Single molecule mechanical unfolding experiments are beginning to provide profiles of the complex energy landscape of biomolecules. In order to obtain reliable estimates of the energy landscape characteristics it is necessary to combine the experimental measurements with sound theoretical models and simulations. Here, we show how by using temperature as a variable in mechanical unfolding of biomolecules in laser optical tweezer or AFM experiments the roughness of the energy landscape can be measured without making any assumptions about the underlying reaction oordinate. The efficacy of the formalism is illustrated by reviewing experimental results that have directly measured roughness in a protein-protein complex. The roughness model can also be used to interpret experiments on forced-unfolding of proteins in which temperature is varied. Estimates of other aspects of the energy landscape such as free energy barriers or the transition state (TS) locations could depend on the precise model used to analyze the experimental data. We illustrate the inherent difficulties in obtaining the transition state location from loading rate or force-dependent unfolding rates. Because the transition state moves as the force or the loading rate is varied it is in general difficult to invert the experimental data unless the curvature at the top of the one dimensional free energy profile is large, i.e the barrier is sharp. The independence of the TS location on force holds good only for brittle or hard biomolecules whereas the TS location changes considerably if the molecule is soft or plastic. We also comment on the usefulness of extension of the molecule as a surrogate reaction coordinate especially in the context of force-quench refolding of proteins and RNA.Comment: 44 pages, 7 figure

Do Water Molecules Displaced by Hydrophobic Interactions Stabilize Antigen-Antibody Binding? Physico-chemical background of antigen-antibody reactions analyzed by fluorescent and Fourier-transform infrared spectroscopy on FITC – anti-FITC (IgG1) model

BACKGROUND: Antigen-antibody reactions are a special field of molecular interactions. The physico-chemical nature of antigen-antibody binding and ligand-induced changes in the fine molecular structures of antigens during immunocomplex formation are less studied. However, these changes in the molecular appearance are extremely important for further molecular recognition. The major aim of this study is to clarify the physico-chemical modification of the antigen/hapten during immunobinding using model experiments. METHODS: An appropriate model system was designed for our investigations: fluorescein-iso-thiocyanate (FITC, isomer I) was used as the antigen (hapten), and its interactions with a specific antibody (monoclonal anti-FITC IgG1) were analyzed using spectrophotometry, different spectrofluorimetric methods and fluorescence polarization, and Fourier-transform infrared spectroscopic methods. RESULTS: Fluorescent polarization and infrared spectroscopic measurements detected a local decrease in the hydration degree in the submolecular area of the specific ligand between the small antigen (hapten) molecule and the hypervariable region of the specific IgG1, causing “rigidization” of molecular movements. Changes in hydration modified the molecular microenvironment, allowing them to influence further functions of both immunoglobulins and the antigen. CONCLUSION: Hydrophobic interactions with exclusion of water molecules around the binding sites seem to be thermodynamically strong enough for stable molecular binding without a covalent chemical interaction between the antigen and the antibody. The results of this study, together with data obtained in previous research, help understand the molecular dynamics of the antigen-antibody reaction better

A Bioinformatics Study of Protein Conformational Flexibility and Misfolding: a Sequence, Structure and Dynamics Approach

This PhD Thesis titled "A Bioinformatics Study of Protein Conformational Flexibility and Misfolding: a Sequence, Structure and Dynamics Approach" comprises the results and conclusions obtained by us from the study of three different but somehow related research projects, covering aspects of the phenomenon of protein local conformational instability, its relationship with protein function, evolvability and aggregation, and the effect of genetic variations on protein conformational instability related to Conformational Diseases. These projects include the prediction of putative prion proteins in complete proteomes and the study of prion biology from a genomic perspective, the prediction of conformationally unstable protein regions and the existence of a structural framework for linking conformational instability to folding and function, and the establishment of a rationale for assessing the connection among mutations and disease phenotypes in Conformational Diseases.Esta tesis doctoral comprende los resultados y conclusiones obtenidos por nosotros a partir del estudio de tres proyectos de investigación diferentes pero de alguna manera relacionados, cubriendo los aspectos del fenómeno de la inestabilidad conformacional local de la proteína, su relación con la función de la proteína, la capacidad de evolución y agregación, y el efecto de las variaciones genéticas en la inestabilidad conformacional de la proteína relacionados con las enfermedades conformacionales. Estos proyectos incluyen la predicción de presuntas proteínas priónicas en proteomas complejos y el estudio de la biología de priones desde una perspectiva genómica, la predicción de las regiones de proteínas conformacionalmente inestables y la existencia de un marco estructural para la vinculación de la inestabilidad conformacional del plegado y la función, y el establecimiento de una razón fundamental para la evaluación de la relación entre las mutaciones y fenotipos de la enfermedad en enfermedades conformacionales

Network Models for Materials and Biological Systems

abstract: The properties of materials depend heavily on the spatial distribution and connectivity of their constituent parts. This applies equally to materials such as diamond and glasses as it does to biomolecules that are the product of billions of years of evolution. In science, insight is often gained through simple models with characteristics that are the result of the few features that have purposely been retained. Common to all research within in this thesis is the use of network-based models to describe the properties of materials. This work begins with the description of a technique for decoupling boundary effects from intrinsic properties of nanomaterials that maps the atomic distribution of nanomaterials of diverse shape and size but common atomic geometry onto a universal curve. This is followed by an investigation of correlated density fluctuations in the large length scale limit in amorphous materials through the analysis of large continuous random network models. The difficulty of estimating this limit from finite models is overcome by the development of a technique that uses the variance in the number of atoms in finite subregions to perform the extrapolation to large length scales. The technique is applied to models of amorphous silicon and vitreous silica and compared with results from recent experiments. The latter part this work applies network-based models to biological systems. The first application models force-induced protein unfolding as crack propagation on a constraint network consisting of interactions such as hydrogen bonds that cross-link and stabilize a folded polypeptide chain. Unfolding pathways generated by the model are compared with molecular dynamics simulation and experiment for a diverse set of proteins, demonstrating that the model is able to capture not only native state behavior but also partially unfolded intermediates far from the native state. This study concludes with the extension of the latter model in the development of an efficient algorithm for predicting protein structure through the flexible fitting of atomic models to low-resolution cryo-electron microscopy data. By optimizing the fit to synthetic data through directed sampling and context-dependent constraint removal, predictions are made with accuracies within the expected variability of the native state.Dissertation/ThesisPh.D. Physics 201

Comparing NMR and X-ray protein structure: Lindemann-like parameters and NMR disorder

Disordered protein chains and segments are fast becoming a major pathway for our understanding of biological function, especially in more evolved species. However, the standard definition of disordered residues: the inability to constrain them in X-ray derived structures, is not easily applied to NMR derived structures. We carry out a statistical comparison between proteins whose structure was resolved using NMR and using X-ray protocols. We start by establishing a connection between these two protocols for obtaining protein structure. We find a close statistical correspondence between NMR and X-ray structures if fluctuations inherent to the NMR protocol are taken into account. Intuitively this tends to lend support to the validity of both NMR and X-ray protocols in deriving biomolecular models that correspond to in vivo conditions. We then establish Lindemann-like parameters for NMR derived structures and examine what order/disorder cutoffs for these parameters are most consistent with X-ray data and how consistent are they. Finally, we find critical value of for the best correspondence between X-ray and NMR derived order/disorder assignment, judged by maximizing the Matthews correlation, and a critical value if a balance between false positive and false negative prediction is sought. We examine a few non-conforming cases, and examine the origin of the structure derived in X-ray. This study could help in assigning meaningful disorder from NMR experiments

Global and Internal Diffusive Dynamics of Proteins in Solution Studied by Neutron Spectroscopy

Proteins are macromolecules naturally occurring in living cells and organisms, involved in a great number of processes essential for life, but they can be interesting not only from a biomedical perspective, but also for colloid physics, chemical engineering and nanotechnology, especially in the prospect of the smart production of self-assembling structures. In this thesis, the results of experiments carried out at the Institut Laue-Langevin, Grenoble, France, and at the Spallation Neutron Source at the Oak Ridge National Laboratory, Tennessee, USA, are presented. The picosecond to nanosecond (short-time) self-diffusion and internal dynamics of two model proteins in aqueous (D2O) solution is studied by neutron backscattering as a function of protein concentration, temperature and multivalent salt concentration. First, the concentration dependence of the translational diffusion of the antibody γ-globulin is rationalized in the context of colloid physics, while the protein internal dynamics is observed to slow down with increasing protein volume fraction. Second, temperature effects are studied on both the diffusion and the internal dynamics of the globular protein bovine serum albumin (BSA), below and above the denaturation temperature. A novel model is proposed to describe the dynamics of the protein side-chains, yielding a rather complete and consistent physical picture of the pico- to nanosecond dynamical changes occurring upon protein denaturation. Third, the change of the diffusion of BSA as a function of the concentration of the trivalent salt YCl3 is investigated, and a remarkably universal slowing down of the apparent diffusion coefficient of BSA molecules as a function of the number of cations per protein cs/cp in solution is found. The result is interpreted in terms of the theory of colloidal suspensions of patchy particles as a result of the semi-quantitative binding of Y3+ ions to specific sites on the protein surface leading to the formation of protein clusters with a cluster size distribution easily tunable by cs/cp

Aromatic circular dichroism in globular proteins: applications to protein structure and folding

1994 Fall.Includes bibliographical references (pages 334-342).Covers not scanned.Print version deaccessioned 2020.The exciton couplet approach was applied to estimate the circular dichroism (CD) of Trp side-chains in proteins. Calculations were performed by the origin-independent version of the matrix method, either for the indole Bb transition only or for the six lowest energy indole transitions. The dependence of the CD of a Trp pair upon its distance and geometry has been analyzed. It was predicted that mixing with far-uv transitions are as important in determining the CD intensity of the near-uv transitions as the coupling among near-uv transition. The effects of varying exposure of Trp chromophores and nearby charges on Trp CD have been examined. A survey of a large number of proteins from the Protein Data Bank reveals a number of cases where readily detectable exciton couplets are predicted to result from the exciton coupling of Trp Bb bands. The predicted CD spectra are generally couplets, often dominated by the contributions of the closest pair, but sometimes exhibit three distinct maxima. This CD depends on the distance and relative orientation of Trp pairs and thus reflects the spatial arrangement of Trp residues in the protein. It was shown that Trp side chains can make significant contributions to the CD of proteins in the far ultraviolet. The distance dependence of exciton splitting, rotational and couplet strengths of Trp pairs show general agreement with theoretical predictions. In several cases, changes in protein Trp CD can be attributed to a specific Trp pair and explained as a definite change in its conformation. Applications of the exciton couplet approach are discussed for various crystal forms of hen lysozyme, turkey and human lysozyme. Trp62 in hen and turkey lysozymes was found to be sensitive to the perturbations of the protein surface due to binding of substrate, antibodies and intermolecular contacts in the crystal. Conformational changes of Trp62 are predicted to have a strong effect on the overall Trp CD of lysozyme. Predicted Trp CD is compared with experimental results for various lysozymes, a-chymotrypsin and chymotrypsinogen A, concanavalin, dihydrofolate reductase and ribonuclease from Bacillus intermedius 7P (binase). The calculated near-uv CD for hen lysozyme matches the experimental amplitude. Correlation of conformational changes in proteins with Trp CD is shown for a-chymotrypsin and chymotrypsinogen A. We found that the exciton couplet approach might be useful in relating Trp CD and changes in protein structure upon local mutations and conformational changes involved in enzyme activation. Small globular proteins are usually composed of a single structural domain and undergo cooperative denaturation. We have demonstrated that a protein with a single structural domain, binase, and a protein with multiple structural domains, porcine pepsin, contain fewer cooperative regions (energetic domains) under the conditions optimal for their functional activity. The study was performed by combining a CD analysis of the structural changes in the proteins during thermal denaturation and under various solvent conditions with thermodynamic properties observed by scanning microcalorimetry. Estimates of secondary structure were obtained from CD spectra, taking side-chain CD into account. It was found that neither of the proteins show any changes in secondary structure or local environment of aromatic amino acids upon separation of the energetic domains. The structural regions in binase corresponding to energetic domains were identified. It was shown that binase is converted from a single cooperative system into two separate energetic domains when ion pairs are disrupted, whereas the size of cooperative units in pepsin decrease as the electrostatic repulsion between regions in the molecule increases