Computational protein design with backbone plasticity

The computational algorithms used in the design of artificial proteins have become increasingly sophisticated in recent years, producing a series of remarkable successes. The most dramatic of these is the de novo design of artificial enzymes. The majority of these designs have reused naturally occurring protein structures as “scaffolds” onto which novel functionality can be grafted without having to redesign the backbone structure. The incorporation of backbone flexibility into protein design is a much more computationally challenging problem due to the greatly increase search space but promises to remove the limitations of reusing natural protein scaffolds. In this review, we outline the principles of computational protein design methods and discuss recent efforts to consider backbone plasticity in the design process

Evaluating experimental and theoretical measures of protein conformational dynamics

Molecular biologists have traditionally interpreted the B-factor data of a protein crystal structure as a reflection of the protein's conformational flexibility. Crystallographers, in contrast, are wary of assigning too much significance to B-factors since they can also be attributed to processes unrelated to conformational dynamics such as experimental imprecision; crystal imperfections; or rigid body motion. In this study, the usefulness of both isotropic and anisotropic B-factors as measures of conformational dynamics were evaluated using high resolution structures. Alpha-carbon B-factor values were analysed in relation to structural properties generally accepted to be correlates of conformational variability. The influence of secondary structure, amino acid type, surface exposure, distance to the centre of mass and packing density were investigated. The results support the argument that B-factors measure conformational variability by demonstrating that atoms with the highest B-factors are typically located in regions expected to have a high degree of conformational freedom. Nevertheless, the results also highlight some of the limitations of crystallographic data. Despite using high quality crystal structures, only very general qualitative trends between B-factors values and the properties investigated could be established. Thus, B-factors appear to be influenced, to a significant degree, by the numerous sources of error in a crystallographic experiment. By considering proteins with multiple published crystal structures, the existence of consensus B-factor profiles were identified. These consensus profiles were hypothesised to represent the dynamics within the crystal with a high degree of accuracy since much of the variation between individual experiments would be eliminated. However, when compared against measurements derived from molecular dynamic simulations, these consensus profiles only weakly correlated with the predictions of the computer models. Therefore, although there is some evidence to suggest that B-factors reflect conformational variability, B-factors cannot be assumed to be reliable descriptors of the internal dynamics of a protein within a crystal

Minimizing the overlap problem in protein NMR: a computational framework for precision amino acid labeling

Motivation: Recent advances in cell-free protein expression systems allow specific labeling of proteins with amino acids containing stable isotopes (¹⁵N, ¹³C and ²H), an important feature for protein structure determination by nuclear magnetic resonance (NMR) spectroscopy. Given this labeling ability, we present a mathematical optimization framework for designing a set of protein isotopomers, or labeling schedules, to reduce the congestion in the NMR spectra. The labeling schedules, which are derived by the optimization of a cost function, are tailored to a specific protein and NMR experiment. Results: For 2D ¹⁵N-¹H HSQC experiments, we can produce an exact solution using a dynamic programming algorithm in under 2 h on a standard desktop machine. Applying the method to a standard benchmark protein, calmodulin, we are able to reduce the number of overlaps in the 500 MHZ HSQC spectrum from 10 to 1 using four samples with a true cost function, and 10 to 4 if the cost function is derived from statistical estimates. On a set of 448 curated proteins from the BMRB database, we are able to reduce the relative percent congestion by 84.9% in their HSQC spectra using only four samples. Our method can be applied in a high-throughput manner on a proteomic scale using the server we developed. On a 100-node cluster, optimal schedules can be computed for every protein coded for in the human genome in less than a month. Availability: A server for creating labeling schedules for ¹⁵N-¹H HSQC experiments as well as results for each of the individual 448 proteins used in the test set is available at http://nmr.proteomics.ics.uci.edu

Multiscale Molecular Dynamics Simulations of Histidine Kinase Activity

Zweikomponentensysteme (TCS), bestehend aus einer Sensorhistidinkinase (HK) und einem Antwortregulationsprotein, sind Schlüsselbausteine in bakteriellen Signaluebertragungsmechanismen. Die Fähigkeit von Bakterien auf eine breite Vielfalt von chemischen und physikalischen Stimuli angemessen zu reagieren ist ausschlaggebend für ihr Überleben. Es ist daher nicht überraschend, dass TCS zu den meistuntersuchten bakteriellen Proteinsystemen gehört. Sensorhistidinkinasen sind typischerweise in die Zellmembran integrierte, homodimere Proteine bestehend aus mehreren Domänen. Reizwahrnehmung an der Sensordomäne von HK löst eine Reihe von großskaligen Konformationsübergangen entlang der Domänen aus. Während sich die strukturellen Eigenschaften von verschiedenen HKs unterscheiden können, erhalten sie alle einen katalytischen ATP-bindenden Kern (CA) und dimerisierende Histidinphosphotransferdomänen (DHp). Während der Signalkaskade nimmt der Kern eine asymmetrische Konformation an, sodass die Kinase an einem der Protomere aktiv ist und die der anderen inaktiv. Das ermöglicht es dem ATP in einer der CA-Domänen seine $\gamma$-Phosphatgruppe an das Histidin der DHp abzugeben. Diese Phosphorylgrouppe wird anschließend an das Antwortregulationsprotein weitergegeben, die eine angemessene Reaktion der Zelle veranlasst. In der vorliegenden Arbeit untersuche ich die Konformationsdynamik des Kinasekerns mithilfe von Molekulardynamiksimulationen (MD). Der Fokus der Arbeit liegt auf zwei verschiedenen HKs: WalK und CpxA. Wegen der Größe der Systeme und den erforderlichen biologischen Zeitskalen, ist es nicht möglich die relevanten Konformationsübergänge in klassischen MD-Simulationen zu berechnen. Um dieses Problem zu umgehen, verwende ich ein strukturbasiertes Modell mit paarweisen harmonischen Potentialen. Diese Näherung erlaubt es, den Übergang zwischen dem inaktiven und den aktiven Zustand mit wesentlich geringerem rechnerischen Aufwand zu untersuchen. Nachdem ich das System mithilfe dieses vereinfachten Modells erkundet habe, benutze ich angereicherte Stichprobenverfahren mit atomistischen Modellen um detailliertere Einsichten in die Dynamik zu gewinnen. Die Ergebnisse in dieser Arbeit legen nahe, dass das Verhalten der einzelnen Unterdomänen des Kinasekerns eng miteinander gekoppelt ist

Molecular Distance Geometry Approach to solve Alpha Carbon Trace Problem

To structural researchers, predicting protein structures currently remains a challenging task. During the past decades, different methodologies have been developed to address this issue. One such protein structure prediction problem is the Alpha Carbon Trace Problem. The Alpha Carbon trace problem is to determine the 3-D coordinates of the main chain atoms(C, N, and O) from just the CA carbon coordinates. This master\u27s thesis presents a novel approach for solving the CA trace problem by using a molecular distance geometry approach. The current approach uses the algorithms which are used to solve the Molecular Distance Geometry Problem to nd the coordinates of the atoms in the peptide plane of a given protein. Once, the coordinates of the atoms(CA, C, N, and O) in the single peptide plane are computed, the two CA atoms are aligned with the first two CA atoms in the CA trace by finding the appropriate rotation and translation. The same rotation and translation are applied to all the other atoms in the peptide plane(C, N, and O). The process is then repeated for the entire trace, and the coordinates of all the atoms in the main chain of the protein are retrieved. In order to predict the side-chain atoms from the main Chain, SCWRL4.0 is used. The output generated by SCWRL4.0 is then subjected to LBFGS energy minimizer using a tool called MESHI. The key advantage of using our approach is that it eliminates the building and searching for a huge protein fragment library. Experiments show that our approach is highly comparable to other approaches such as BBQ, PD2Main, and PULCHRA for solving the CA trace problem

Revisiting the mechanism of coagulation factor XIII activation and regulation from a structure/functional perspective

The activation and regulation of coagulation Factor XIII (FXIII) protein has been the subject of active research for the past three decades. Although discrete evidence exists on various aspects of FXIII activation and regulation a combinatorial structure/functional view in this regard is lacking. In this study, we present results of a structure/function study of the functional chain of events for FXIII. Our study shows how subtle chronological submolecular changes within calcium binding sites can bring about the detailed transformation of the zymogenic FXIII to its activated form especially in the context of FXIIIA and FXIIIB subunit interactions. We demonstrate what aspects of FXIII are important for the stabilization (first calcium binding site) of its zymogenic form and the possible modes of deactivation (thrombin mediated secondary cleavage) of the activated form. Our study for the first time provides a structural outlook of the FXIIIA 2 B 2 heterotetramer assembly, its association and dissociation. The FXIIIB subunits regulatory role in the overall process has also been elaborated upon. In summary, this study provides detailed structural insight into the mechanisms of FXIII activation and regulation that can be used as a template for the development of future highly specific therapeutic inhibitors targeting FXIII in pathological conditions like thrombosis