Internal Protein Cavities Research Articles

PBCAVE: Program for exact classification of the mesh points of a protein with possible internal cavities and its application to Poisson–Boltzmann equation solution

The main contribution of the program is in the analytic approach to the setting of dielectric constant for all discretization points of a grid in the computational domain according to their locations with respect to the molecular surface allowing also taking into account possible cavities. Assigned values of dielectric constant are used in a solver of the linear Poisson–Boltzmann equation for a protein–water system using a finite difference method. Program summaryProgram Title: PBCAVEProgram Files doi:http://dx.doi.org/10.17632/d77n7yskmc.1Licensing provisions: MITProgramming language: C++11 (or more recent)Nature of problem: A key problem in the solution of the Poisson–Boltzmann equation for protein using a discretization of the domain consists in the assignment of proper dielectric constants at the individual mesh points of the discretization. A widely used approach is to check if a given point is inside some atom and then its dielectric constant is set to the one of an internal point while if it is not, then its constant is set to the value of the solvent (usually water). While this approach is sufficient for most of the points, there are still many points which do not belong to these two groups, for instance because they are close to the surface of the protein, but are not accessible yet by the solvent.Solution method: To secure proper dielectric constant values, we use our previous work on the calculation of the volume and the accessible volume of a protein (see, e.g., [1–3]). A prerequisite for setting dielectric constants is to load the user provided protein. Then the so-called enveloping triangulation [4] is built with the aim to identify the cavities inside the protein and to make a list of the points close to the exterior part of triangles forming this triangulation. After this we conduct a series of tests for each mesh point and determine its proper dielectric constant with respect to the molecular surface. The tests allow us to define four types of points. Based on analytic calculations verifying the fulfillment of inequality systems or calculating the total oriented spherical angle of a point with respect of the enveloping triangulation. Now we have all dielectric constants set and we can solve the Poisson–Boltzmann equation. To solve this equation we have used the SOR method over the Gauss–Seidel iterative finite differences scheme with parameter choice according to [5]. Since the main focus of our research was the classification of the mesh points, we have not studied other potentially more suitable methods of solving this equation. Also, we used only homogeneous Dirichlet conditions while the program allows the user for implementing his own boundary conditions.Additional comments including restrictions and unusual features: While we have built our program to comply with C++17, it also compiles without problems with older standards (C++11). We have used CMake to make installation and transfer of the program easier between various computers/operating systems but it is also possible to compile the whole project by hand as well in the case of missing CMake.[1] J. Buša, I. Pokorný, E. A. Hayryan, J. Skřivánek, Identification of Internal Points of Macromolecular System for the Definition of the Parameters of a Poisson–Boltzmann Equation, Bulletin of PFUR Series Mathematics. Information Sciences. Physics. 2 (2) (2010) 70–75. http://vestnik-miph.sci.pfu.edu.ru/webfm_send/32.[2] J. Buša, S. Hayryan, C.-K. Hu, J. Skřivánek, M.-C. Wu, CAVE: A package for detection and quantitative analysis of internal cavities in a system of overlapping balls: Application to proteins, Computer Physics Communications 181 (12) (2010) 2116–2125. http://dx.doi.org/10.1016/j.cpc.2010.08.032.[3] J. Buša Jr, J. Buša, S. Hayryan, C.-K. Hu, M.-C. Wu, CAVE-CL: An OpenCL version of the package for detection and quantitative analysis of internal cavities in a system of overlapping balls: Application to proteins, Computer Physics Communications 190 (2015) 224–227. http://dx.doi.org/10.1016/j.cpc.2014.12.017.[4] J. Buša, S. Hayryan, C.-K. Hu, J. Skřivánek, M.-C. Wu, Enveloping triangulation method for detecting internal cavities in proteins and algorithm for computing their surface areas and volumes, Journal of Computational Chemistry 30 (3) (2009) 346–357. http://dx.doi.org/10.1002/jcc.21060.[5] B. A. Carré, The determination of the optimum accelerating factor for successive over-relaxation, The Computer Journal 4 (1) (1961) 73–78. https://doi.org/10.1093/comjnl/4.1.73.

Nuclear magnetic resonance-based determination of dioxygen binding sites in protein cavities.

The location and ligand accessibility of internal cavities in cysteine-free wild-type T4 lysozyme was investigated using O2 gas-pressure NMR spectroscopy and molecular dynamics (MD) simulation. Upon increasing the concentration of dissolved O2 in solvent to 8.9 mM, O2 -induced paramagnetic relaxation enhancements (PREs) to the backbone amide and side chain methyl protons were observed, specifically around two cavities in the C-terminal domain. To determine the number of O2 binding sites and their atomic coordinates from the 1/r6 distance dependence of the PREs, we established an analytical procedure using Akaike's Information Criterion, in combination with a grid-search. Two O2 -accessible sites were identified in internal cavities: One site was consistent with the xenon-binding site in the protein in crystal, and the other site was established to be a novel ligand-binding site. MD simulations performed at 10 and 100 mM O2 revealed dioxygen ingress and egress as well as rotational and translational motions of O2 in the cavities. It is therefore suggested that conformational fluctuations within the ground-state ensemble transiently develop channels for O2 association with the internal protein cavities.

Analysis of O2-binding Sites in Proteins Using Gas-Pressure NMR Spectroscopy: Outer Surface Protein A

Internal cavities in proteins produce conformational fluctuations and enable the binding of small ligands. Here, we report a NMR analysis of O2-binding sites by O2-induced paramagnetic relaxation enhancements (PREs) on amide groups of proteins in solution. Outer surface protein A contains a nonglobular single-layer β-sheet that connects the N- and C-terminal globular domains. Several cavities have been observed in both domains of the crystallized protein structure. The receptor-binding sites are occluded and line the largest cavity of the C-terminal domain. We observed significant O2-induced PREs for amide protons located around the largest cavity and at the central β-sheet. We suggested three potential O2-accessible sites in the protein based on the 1/r6 distance dependence of the PRE. Two sites were in or close to the largest cavity and the third site was in the surface crevice of the central β-sheet. These results provide, to our knowledge, the first evidence of ligand binding to the surface crevice and cavity of the protein in solution. Because O2 generally binds more specifically to hydrophobic rather than hydrophilic cavities within a protein, the results also indicated that the receptor-binding sites lining the largest cavity were in the hydrophobic environment in the ground-state conformation. Molecular dynamics simulations permitted the visualization of the rotational and translational motions of O2 within the largest cavity, egress of O2 from the cavity, and ingress of O2 in the surface crevice of the β-sheet. These molecular dynamics simulation results qualitatively explained the O2-induced changes in NMR observations. Exploring cavities that are sufficiently dynamic to enable access by small molecules can be a useful strategy for the design of stable proteins and their ligands.

Computationally Efficient Multiscale Reactive Molecular Dynamics to Describe Amino Acid Deprotonation in Proteins.

An important challenge in the simulation of biomolecular systems is a quantitative description of the protonation and deprotonation process of amino acid residues. Despite the seeming simplicity of adding or removing a positively charged hydrogen nucleus, simulating the actual protonation/deprotonation process is inherently difficult. It requires both the explicit treatment of the excess proton, including its charge defect delocalization and Grotthuss shuttling through inhomogeneous moieties (water and amino residues), and extensive sampling of coupled condensed phase motions. In a recent paper (J. Chem. Theory Comput.2014, 10, 2729−273725061442), a multiscale approach was developed to map high-level quantum mechanics/molecular mechanics (QM/MM) data into a multiscale reactive molecular dynamics (MS-RMD) model in order to describe amino acid deprotonation in bulk water. In this article, we extend the fitting approach (called FitRMD) to create MS-RMD models for ionizable amino acids within proteins. The resulting models are shown to faithfully reproduce the free energy profiles of the reference QM/MM Hamiltonian for PT inside an example protein, the ClC-ec1 H+/Cl– antiporter. Moreover, we show that the resulting MS-RMD models are computationally efficient enough to then characterize more complex 2-dimensional free energy surfaces due to slow degrees of freedom such as water hydration of internal protein cavities that can be inherently coupled to the excess proton charge translocation. The FitRMD method is thus shown to be an effective way to map ab initio level accuracy into a much more computationally efficient reactive MD method in order to explicitly simulate and quantitatively describe amino acid protonation/deprotonation in proteins.

QM/MM Methods: Recent Developments and Application to Membrane Proteins and Molecular Motors

I'll briefly discuss recent developments of QM/MM methods in our lab, with an emphasis on methods that allow an efficient sampling of at least local motions coupled to the biochemical process of interest. Next, I'll discuss the application of these methods to address specific mechanistic questions in proton pumps and biomolecular motors. The applications highlight that calibrated QM/MM methods are valuable because they provide not only energetic/kinetic information for the relevant biochemical driving forces (e.g., ATP hydrolysis) in these biomolecular machines but also spectroscopic observables that can be compared directly to experiments. Another feature that emerges from these applications is that changing hydration level of internal protein cavities may play an important role in modulating the proton affinity of key groups and thus the timing of key chemical events. In short, the discussions highlight the advantages of an efficient QM/MM framework based on an approximate DFT method (DFTB3) and the diverse roles of water molecules in biomolecular functions.

Exploring cavity dynamics in biomolecular systems

BackgroundThe internal cavities of proteins are dynamic structures and their dynamics may be associated with conformational changes which are required for the functioning of the protein. In order to study the dynamics of these internal protein cavities, appropriate tools are required that allow rapid identification of the cavities as well as assessment of their time-dependent structures.ResultsIn this paper, we present such a tool and give results that illustrate the applicability for the analysis of molecular dynamics trajectories. Our algorithm consists of a pre-processing step where the structure of the cavity is computed from the Voronoi diagram of the van der Waals spheres based on coordinate sets from the molecular dynamics trajectory. The pre-processing step is followed by an interactive stage, where the user can compute, select and visualize the dynamic cavities. Importantly, the tool we discuss here allows the user to analyze the time-dependent changes of the components of the cavity structure. An overview of the cavity dynamics is derived by rendering the dynamic cavities in a single image that gives the cavity surface colored according to its time-dependent dynamics.ConclusionThe Voronoi-based approach used here enables the user to perform accurate computations of the geometry of the internal cavities in biomolecules. For the first time, it is possible to compute dynamic molecular paths that have a user-defined minimum constriction size. To illustrate the usefulness of the tool for understanding protein dynamics, we probe the dynamic structure of internal cavities in the bacteriorhodopsin proton pump.

The Interaction of Polyphenol Flavonoids with β-lactoglobulin: Molecular Docking and Molecular Dynamics Simulation Studies

The interaction of quercetin, quercitrin, and rutin, as natural polyphenolic compounds, with β-lactoglobulin (BLG) using molecular docking and molecular dynamics simulation methods was examined. Molecular docking studies showed that quercetin and quercitrin were bounded to the internal cavity of protein, while rutin was bounded to the entrance of the cavity because of its large structural volume. It was found that there were one-, three-, and four-hydrogen bond interactions between BLG and quercetin, quercitrin, and rutin respectively. This showed that with an increase in the number of OH groups in the flavonoid structure, there was an increase in the number of hydrogen bond interactions. The binding constants for the binding of quercetin, quercitrin, and rutin to BLG were 1.2 × 106, 1.9 × 106, and 7.4 × 104 M−1 respectively. The results of molecular dynamics simulation showed that the root mean square deviation (RMSD) of non-liganded BLG and BLG–ligand complexes reached equilibration after 3500 ps. The study of the radius of gyration revealed that BLG and BLG–ligand complexes were stabilized around 2500 ps, and unlike the two other complexes, there was no conformational change for BLG–quercetin. Finally, analyzing the RMS fluctuations suggested that the structure of the ligand binding site remained approximately rigid during simulation.

CAVE: A package for detection and quantitative analysis of internal cavities in a system of overlapping balls: Application to proteins

We developed a software package ( CAVE) in Fortran language to detect internal cavities in proteins which can be applied also to an arbitrary system of balls. The volume, the surface area and other quantitative characteristics of the cavities can be calculated. The code is based on the recently suggested enveloping triangulation algorithm [J. Buša et al., J. Comp. Chem. 30 (2009) 346] for computing volume and surface area of the cavity by analytical equations. Different standard sets of atomic radii can be used. The PDB compatible file containing the atomic coordinates must be stored on the disk in advance. Testing of the code on different proteins and artificial ball systems showed efficiency and accuracy of the algorithm. The program is fast. It can handle a system of several thousands of balls in the order of seconds on contemporary PC's. The code is open source and free. Program summary Program title: CAVE Catalogue identifier: AEHC_v1_0 Program summary URL: http://cpc.cs.qub.ac.uk/summaries/AEHC_v1_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: Standard CPC licence, http://cpc.cs.qub.ac.uk/licence/licence.html No. of lines in distributed program, including test data, etc.: 8670 No. of bytes in distributed program, including test data, etc.: 100 131 Distribution format: tar.gz Programming language: Fortran Computer: PC Pentium and Core Operating system: Linux system and Windows XP system Classification: 16.1 Nature of problem: Molecular structure analysis. Solution method: Analytical method for cavities detection, and numerical algorithm for volume and surface area calculation based on the analytical formulas, after using the stereographic transformation. Running time: Depends on the size of the molecule under consideration. The test example included in the distribution takes about 1 minute to run.

The Effect of Non-Coordinated Water in the Heme Pocket on the Ligand Binding Dynamics of Heme Proteins

Water molecules in internal protein cavities play fundamental roles in satisfying the H-bonding potentials of main chain atoms in turns, coils, and loops, determining the stability and rigidity of proteins, shifting the pKa values of buried ionizable residues, and modulating dynamical processes such as folding, catalysis, and proton transfers. Detecting these internal water molecules is sometimes obscured in x-ray crystallography due to positional disorder. We have developed a spectrokinetic assay that accurately detects the presence of a non-coordinated water molecule in the distal heme pocket of myoglobin and in a series of distal pocket mutants, including many where this water molecule is positionally disordered. We also have shown that this water plays a major role in determining the observed bimolecular recombination rate constant. We show that 1) this water molecule modulates the ligand binding dynamics of a series of H64, L29 and V68 mutants; 2) it plays the major role in the observed pH dependence of the CO recombination kinetics between pH 4 and 7 with the protonation of the distal histidine acting as a switch to change water occupancy; and 3) it may also modulate ligand binding dynamics in isolated hemoglobin chains, with the occupancy being larger in the alpha chains. Accurately measuring water occupancy in heme proteins answers crucial questions about water in apolar or slightly polar protein cavities and clarifies the role internal water molecules play in modulating protein function.

Recognition and Discrimination of Gases by the Signal Transducers HemAT

The recognition and discrimination of diatomic gases that affect biological processes through the perturbation of protein function is a formidable challenge towards our understanding of the molecular mechanisms involved in signal transduction by heme-based sensor proteins (1). There is a general consensus now that internal cavities in proteins are involved in controlling the dynamics and reactivity of the protein reactions with small ligands, such as O2, CO, and NO usually through ligand accommodation. These cavities serve as a local storehouse for ligands near the active site, thereby increasing the effective concentration of the ligand many times. In addition to having functional role in ligand binding they are also important for determining relative affinities. In the heme-based oxygen sensors such as HemAT the recognition and discrimination of the specific gas leads to either activation or inhibition of a regulated domain. The dynamic coupling between two distinct binding sites as the underlying allosteric mechanism for gas-recognition/ discrimination that triggers a conformational switch for signaling by the oxygen sensor protein HemAT will be presented (2-3).

Enveloping triangulation method for detecting internal cavities in proteins and algorithm for computing their surface areas and volumes

Detection and quantitative characterization of the internal cavities in proteins remain an important topic in studying protein structure and function. Here we propose a new analytical method for detecting the existence of cavities in proteins. The method is based on constructing the special enveloping triangulation enclosing the cavities. Based on this method, we develop an algorithm and a fortran package, CAVE, for computing volumes and surface areas of cavities in proteins. We first test our method and algorithm in some artificial systems of spheres and find that the calculated results are consistent with exact results. Then we apply the package to compute volumes and surface areas of cavities for some protein structures in the Protein Data Bank. We compare our calculated results with those obtained by some other methods and find that our approach is reliable.

Role of Flexibility and Polarity as Determinants of the Hydration of Internal Cavities and Pockets in Proteins

Molecular dynamics simulations of Staphylococcal nuclease and of 10 variants with internal polar or ionizable groups were performed to investigate systematically the molecular determinants of hydration of internal cavities and pockets in proteins. In contrast to apolar cavities in rigid carbon structures, such as nanotubes or buckeyballs, internal cavities in proteins that are large enough to house a few water molecules will most likely be dehydrated unless they contain a source of polarity. The water content in the protein interior can be modulated by the flexibility of protein elements that interact with water, which can impart positional disorder to water molecules, or bias the pattern of internal hydration that is stabilized. This might explain differences in the patterns of hydration observed in crystal structures obtained at cryogenic and room temperature conditions. The ability of molecular dynamics simulations to determine the most likely sites of water binding in internal pockets and cavities depends on its efficiency in sampling the hydration of internal sites and alternative protein and water conformations. This can be enhanced significantly by performing multiple molecular dynamics simulations as well as simulations started from different initial hydration states.

Disentangling Ligand Migration and Heme Pocket Relaxation in Cytochrome P450cam

In this work we show that ligand migration and active site conformational relaxation can occur independently of each other in hemoproteins. The complicated kinetics of carbon monoxide rebinding with cytochrome P450cam display up to five distinct processes between 77K and 300K. They were disentangled by using a combination of three approaches: 1), the competition of the ligand with xenon for the occupation of internal protein cavities; 2), the modulation of the amount of distal steric hindrance within the heme pocket by varying the nature of the substrate; and 3), molecular mechanics calculations to support the proposed heme-substrate relaxation mechanism and to seek internal cavities. In cytochrome P450cam, active site conformational relaxation results from the displacement of the substrate toward the heme center upon photodissociation of the ligand. It is responsible for the long, puzzling bimodal nature of the rebinding kinetics observed down to 77K. The relaxation rate is strongly substrate-dependent. Ligand migration is slower and is observed only above 135K. Migration and return rates are independent of the substrate.

Inhalational Anesthetic-binding Proteins in Rat Neuronal Membranes

Molecular targets of inhaled anesthetics must be represented in the group that specifically bind these drugs, but the paucity of direct binding data has limited the number of candidates for further evaluation. To find candidate targets, we used a combination of photolabeling, two-dimensional gel electrophoresis, and mass spectrometry to identify halothane-binding targets in rat neuronal membranes. Of the 265 spots detected on the two-dimensional gels, 90 were labeled by [(14)C]halothane, and 34 were identified. Mitochondrial proteins, especially respiratory complex and voltage-dependent anion channels, dominated the labeled group, and there were several examples of subunit- and state-dependent binding. A significant correlation was found between internal protein cavities and binding in a group of proteins with high resolution structures. Therefore, in addition to identifying novel neuronal targets, these data suggest a general molecular feature, the buried cavity, as a dominant attribute of volatile anesthetic-binding sites found in a limited number of neuronal membrane proteins.

Infrared Study of Carbon Monoxide Migration among Internal Cavities of Myoglobin Mutant L29W.

Myoglobin, a small globular heme protein that binds gaseous ligands such asO(2), CO and NO reversibly at the heme iron, provides an excellent modelsystem for studying structural and dynamic aspects of protein reactions. Flashphotolysis experiments, performed over wide ranges in time and temperature, reveal a complex ligand binding reaction with multiple kinetic intermediates, resulting from protein relaxation and movements of the ligand within the protein. Our recent studies of carbonmonoxy-myoglobin (MbCO) mutant L29W, using time-resolved infrared spectroscopy in combination with x-ray crystallography, have correlated kinetic intermediates with photoproduct structures that are characterized by the CO residing in different internal protein cavities, so-called xenon holes. Here we have used Fourier transform infrared temperature derivative spectroscopy (FTIR-TDS) to further examine the role of internal cavities in the dynamics. Different cavities can be accessed by the CO ligands at different temperatures, and characteristic infrared absorption spectra have been obtained for the different locations of the CO ligand within the protein, enabling us to monitor ligand migration through the protein as well as conformational changes of the protein.

Internal cavities and buried waters in globular proteins.

A fast algorithm that detects internal cavities in proteins and predicts the positions of buried water molecules is described. The cavities are characterized in terms of volume, surface area, polarity, and the presence of bound waters. The algorithm is applied to 12 proteins whose structures are known to high resolution and successfully predicts the locations of over 80% of internal water molecules. Most proteins are found to have a number of internal cavities ranging in volume from 10 to 180 A3. Some of these cavities contain water and some do not, with the probability of containing a buried water increasing with cavity size. However, many large cavities are found to be empty (i.e., they do not contain a crystallographically determined water). For multidomain proteins over half of the total cavity volume is at the interdomain interface. Possible implications for the energetics of cavity formation and for the functional role of internal cavities are discussed.