Crystal contact-free conformation of an intrinsically flexible loop in protein crystal: Tim21 as the case study

BackgroundIn protein crystals, flexible loops are frequently deformed by crystal contacts, whereas in solution, the large motions result in the poor convergence of such flexible loops in NMR structure determinations. We need an experimental technique to characterize the structural and dynamic properties of intrinsically flexible loops of protein molecules.MethodsWe designed an intended crystal contact-free space (CCFS) in protein crystals, and arranged the flexible loop of interest in the CCFS. The yeast Tim 21 protein was chosen as the model protein, because one of the loops (loop 2) is distorted by crystal contacts in the conventional crystal.ResultsYeast Tim21 was fused to the MBP protein by a rigid α-helical linker. The space created between the two proteins was used as the CCFS. The linker length provides adjustable freedom to arrange loop 2 in the CCFS. We re-determined the NMR structure of yeast Tim21, and conducted MD simulations for comparison. Multidimensional scaling was used to visualize the conformational similarity of loop 2. We found that the crystal contact-free conformation of loop 2 is located close to the center of the ensembles of the loop 2 conformations in the NMR and MD structures.ConclusionsLoop 2 of yeast Tim21 in the CCFS adopts a representative, dominant conformation in solution.General significanceNo single powerful technique is available for the characterization of flexible structures in protein molecules. NMR analyses and MD simulations provide useful, but incomplete information. CCFS crystallography offers a third route to this goal.     

Automatic prediction of protein interactions with large scale motion

Proteins often change their conformation upon binding to other molecules. Taking these conformational changes into account in docking is an extremely difficult task: the larger the scale of the motion the harder it is to predict the structure of the association complex. Here, we present a fully automated method for flexible docking with large scale motion in one of the docked molecules. The method automatically identifies hinge regions and rigid parts and then docks the input molecules while explicitly considering the hinges and possible protein motions.     

Flexible protein alignment and hinge detection

Here we present a novel technique for the alignment of flexible proteins. The method does not require an a priori knowledge of the flexible hinge regions. The FlexProt algorithm simultaneously detects the hinge regions and aligns the rigid subparts of the molecules. Our technique is not sensitive to insertions and deletions. Numerous methods have been developed to solve rigid structural comparisons. Unlike FlexProt, all previously developed methods designed to solve the protein flexible alignment require an a priori knowledge of the hinge regions. The FlexProt method is based on 3-D pattern-matching algorithms combined with graph theoretic techniques. The algorithm is highly efficient. For example, it performs a structural comparison of a pair of proteins with 300 amino acids in about 7 s on a 400-MHz desktop PC. We provide experimental results obtained with this algorithm. First, we flexibly align pairs of proteins taken from the database of motions. These are extended by taking additional proteins from the same SCOP family. Next, we present some of the results obtained from exhaustive all-against-all flexible structural comparisons of 1329 SCOP family representatives. Our results include relatively high-scoring flexible structural alignments between the C-terminal merozoite surface protein vs. tissue factor; class II aminoacyl-tRNA synthase, histocompatibility antigen vs. neonatal FC receptor; tyrosine-protein kinase C-SRC vs. haematopoetic cell kinase (HCK); tyrosine-protein kinase C-SRC vs. titine protein (autoinhibited serine kinase domain); and tissue factor vs. hormone-binding protein. These are illustrated and discussed, showing the capabilities of this structural alignment algorithm, which allows un-predefined hinge-based motions.     

hdANM: a new comprehensive dynamics model for protein hinges

Hinge motions are essential for many protein functions, and their dynamics are important to understand underlying biological mechanisms. The ways that these motions are represented by various computational methods differ significantly. By focusing on a specific class of motion, we have developed a new hinge-domain anisotropic network model (hdANM) that is based on the prior identification of flexible hinges and rigid domains in the protein structure and the subsequent generation of global hinge motions. This yields a set of motions in which the relative translations and rotations of the rigid domains are modulated and controlled by the deformation of the flexible hinges, leading to a more restricted, specific view of these motions. hdANM is the first model, to our knowledge, that combines information about protein hinges and domains to model the characteristic hinge motions of a protein. The motions predicted with this new elastic network model provide important conceptual advantages for understanding the underlying biological mechanisms. As a matter of fact, the generated hinge movements are found to resemble the expected mechanisms required for the biological functions of diverse proteins. Another advantage of this model is that the domain-level coarse graining makes it significantly more computationally efficient, enabling the generation of hinge motions within even the largest molecular assemblies, such as those from cryo-electron microscopy. hdANM is also comprehensive as it can perform in the same way as the well-known protein dynamics models (anisotropic network model, rotations-translations of blocks, and nonlinear rigid block normal mode analysis), depending on the definition of flexible and rigid parts in the protein structure and on whether the motions are extrapolated in a linear or nonlinear fashion. Furthermore, our results indicate that hdANM produces more realistic motions as compared to the anisotropic network model. hdANM is an open-source software, freely available, and hosted on a user-friendly website.     

Conformational ensemble of an intrinsically flexible loop in mitochondrial import protein Tim21 studied by modeling and molecular dynamics simulations

BackgroundTim21, a subunit of a highly dynamic translocase of the inner mitochondrial membrane (TIM23) complex, translocates proteins by interacting with subunits in the translocase of the outer membrane (TOM) complex and Tim23 channel in the TIM23 complex. A loop segment in Tim21, which is in close proximity of the binding site of Tim23, has different conformations in X-ray, NMR and new crystal contact-free space (CCFS) structures. MD simulations can provide information on the structure and dynamics of the loop in solution.MethodsThe conformational ensemble of the loop was characterized using loop modeling and molecular dynamics (MD) simulations.ResultsMD simulations confirmed mobility of the loop. Multidimensional scaling and clustering were used to characterize the dynamic conformational ensemble of the loop. Free energy landscape showed that the CCFS crystal structure occupied a low energy region as compared to the conventional X-ray crystal structure. Analysis of crystal packing indicates that the CCFS provides larger conformational space for the motions of the loop.ConclusionsOur work reported the conformational ensemble of the loop in solution, which is in agreement with the structure obtained from CCFS approach. The combination of the experimental techniques and computational methods is beneficial for studying highly flexible regions of proteins.General significanceComputational methods, such as loop modeling and MD simulations, have proved to be useful for studying conformational flexibility of proteins. These methods in integration with experimental techniques such as CCFS has the potential to transform the studies on flexible regions of proteins.     

Occluded molecular surface: Analysis of protein packing

We describe a novel method to calculate the packing interactions in protein structural models. The method calculates the interatomic occluded surface areas for each atom in the protein model. The identification of, and degree of interaction with, neighboring atoms is accomplished by extending surface normal from a dot surface of each atom to the point of intersection with neighboring atoms. The combined occluded and non-occluded surface areas may be normalized for the amino acid composition of the protein providing a single parameter, the normalized protein surface ratio, which is diagnostic for native-like Structures. Individual residues in the model which are in infrequent occluded surface environments may be identified. The method provides a means to explicitly describe packing densities and packing environments of individual atoms in a protein model. Finally, the method allows estimation of the complementarity between any interacting molecules, for example a ligand binding to a receptor.     

HingeProt: automated prediction of hinges in protein structures

Proteins are highly flexible molecules. Prediction of molecular flexibility aids in the comprehension and prediction of protein function and in providing details of functional mechanisms. The ability to predict the locations, directions, and extent of molecular movements can assist in fitting atomic resolution structures to low-resolution EM density maps and in predicting the complex structures of interacting molecules (docking). There are several types of molecular movements. In this work, we focus on the prediction of hinge movements. Given a single protein structure, the method automatically divides it into the rigid parts and the hinge regions connecting them. The method employs the Elastic Network Model, which is very efficient and was validated against a large data set of proteins. The output can be used in applications such as flexible protein-protein and protein-ligand docking, flexible docking of protein structures into cryo-EM maps, and refinement of low-resolution EM structures. The web server of HingeProt provides convenient visualization of the results and is available with two mirror sites at http://www.prc.boun.edu.tr/appserv/prc/HingeProt3 and http://bioinfo3d.cs.tau.ac.il/HingeProt/.     

The interpretation of protein structures: total volume, group volume distributions and packing density

Following the general procedure of Bernal &; Finney (1967) using Voronoi polyhedra, volumes occupied by all the atoms, or groups of atoms, in lysozyme and ribonuclease S have been estimated from the atomic co-ordinates provided by crystal structure studies. The average packing density for the interior of both proteins is close to 0.75, which is in the middle of the range found for crystals of most small organic molecules. For all atom types the mean packing densities fall between 0.7 and 0.8 with standard deviations between ± 0.1 and ± 0.2. It is suggested that simple geometrical packing considerations may provide useful criteria in guiding and evaluating trial structures in theoretical studies of protein folding, especially the association of distant parts of a peptide chain.Packing densities averaged over a relatively small number of atoms (5 to 15) appear to vary substantially in different parts of the same protein. Low densities representing packing defects may permit relatively easy motions, for example in an active site. Surrounding areas of high density may serve as relatively incompressible regions which transmit or correlate motions over considerable distances.The total volume of each of these two proteins as derived from the co-ordinate list appears to be larger than that estimated from the partial specific volume by 7 to 10%. If this volume difference is attributed to a change in the packing of water in a monolayer surrounding the protein, it would correspond to an average decrease, relative to bulk water, of 1 to 2 ų per water molecule in this monolayer. Such a change is about half of the decrease that occurs on the melting of ice.     

Practical aspects of the 2D 15N-[1h]-NOE experiment

The heteronuclear ¹⁵N-NOE experiment was extensively tested with respect to statistical and systematic experimental error. The dependence of signal intensity in the NOE experiment and in the reference experiment on the saturation and relaxation time was experimentally investigated. The statistics of the experimental values were accessed by numerous repetitions of identical set-ups. As a model system a protein of typical size for NMR studies was chosen, i.e., a 120 amino acid residues containing fragment of the F-actin binding gelation factor (ABP-120). The fragment exhibits fast dynamics that are accessible with the ¹⁵N-NOE experiment with various amplitudes. The results of this study show that commonly used parameters are only adequate for accurate measurement of motions with moderate amplitude. Highly flexible parts require longer delay times and thus more experimental time than commonly used. On the other hand, a qualitative or semi-quantitative assessment of a protein's mobility on fast times scales can be obtained from rapidly recorded experiments with unusual short delay times. The findings of this study are of equal importance for highly accurate measurement of the ¹⁵N-NOE as well as for quick identification of mobile or even unstructured residues/parts of a protein.     

Determining and visualizing flexibility in protein structures

How to compare the structures of an ensemble of protein conformations is a fundamental problem in structural biology. As has been previously observed, the widely used RMSD measure due to Kabsch, in which a rigid‐body superposition minimizing the least‐squares positional deviations is performed, has its drawbacks when comparing and visualizing a set of flexible protein structures. Here, we develop a method, fleximatch, of protein structure comparison that takes flexibility into account. Based on a distance matrix measure of flexibility, a weighted superposition of distance matrices rather than of atomic coordinates is performed. Subsequently, this allows a consistent determination of (a) a superposition of structures for visualization, (b) a partitioning of the protein structure into rigid molecular components (core atoms), and (c) an atomic mobility measure. The method is suitable for highlighting both particularly flexible and rigid parts of a protein from structures derived from NMR, X‐ray diffraction or molecular simulation. Proteins 2015; 83:820–826. © 2015 Wiley Periodicals, Inc.     

Motions and structural variability within toxins: implication for their use as scaffolds for protein engineering

Animal toxins are small proteins built on the basis of a few disulfide bonded frameworks. Because of their high variability in sequence and biologic function, these proteins are now used as templates for protein engineering. Here we report the extensive characterization of the structure and dynamics of two toxin folds, the "three-finger" fold and the short alpha/beta scorpion fold found in snake and scorpion venoms, respectively. These two folds have a very different architecture; the short alpha/beta scorpion fold is highly compact, whereas the "three-finger" fold is a beta structure presenting large flexible loops. First, the crystal structure of the snake toxin alpha was solved at 1.8-A resolution. Then, long molecular dynamics simulations (10 ns) in water boxes of the snake toxin alpha and the scorpion charybdotoxin were performed, starting either from the crystal or the solution structure. For both proteins, the crystal structure is stabilized by more hydrogen bonds than the solution structure, and the trajectory starting from the X-ray structure is more stable than the trajectory started from the NMR structure. The trajectories started from the X-ray structure are in agreement with the experimental NMR and X-ray data about the protein dynamics. Both proteins exhibit fast motions with an amplitude correlated to their secondary structure. In contrast, slower motions are essentially only observed in toxin alpha. The regions submitted to rare motions during the simulations are those that exhibit millisecond time-scale motions. Lastly, the structural variations within each fold family are described. The localization and the amplitude of these variations suggest that the regions presenting large-scale motions should be those tolerant to large insertions or deletions.     

Recapitulation of protein family divergence using flexible backbone protein design

We use flexible backbone protein design to explore the sequence and structure neighborhoods of naturally occurring proteins. The method samples sequence and structure space in the vicinity of a known sequence and structure by alternately optimizing the sequence for a fixed protein backbone using rotamer based sequence search, and optimizing the backbone for a fixed amino acid sequence using atomic-resolution structure prediction. We find that such a flexible backbone design method better recapitulates protein family sequence variation than sequence optimization on fixed backbones or randomly perturbed backbone ensembles for ten diverse protein structures. For the SH3 domain, the backbone structure variation in the family is also better recapitulated than in randomly perturbed backbones. The potential application of this method as a model of protein family evolution is highlighted by a concerted transition to the amino acid sequence in the structural core of one SH3 domain starting from the backbone coordinates of an homologous structure.     

An analysis of the influence of protein intrinsic dynamical properties on its thermal unfolding behavior

The influence of the protein topology-encoded dynamical properties on its thermal unfolding motions was studied in the present work. The intrinsic dynamics of protein topology was obtained by the anisotropic network model (ANM). The ANM has been largely used to investigate protein collective functional motions, but it is not well elucidated if this model can also reveal the preferred large-scale motions during protein unfolding. A small protein barnase is used as a typical case study to explore the relationship between protein topology-encoded dynamics and its unfolding motions. Three thermal unfolding simulations at 500 K were performed for barnase and the entire unfolding trajectories were sampled and partitioned into several windows. For each window, the preferred unfolding motions were investigated by essential dynamics analysis, and then associated with the intrinsic dynamical properties of the starting conformation in this window, which is detected by ANM. The results show that only a few slow normal modes imposed by protein structure are sufficient to give a significant overlap with the preferred unfolding motions. Especially, the large amplitude unfolding movements, which imply that the protein jumps out of a local energy basin, can be well described by a single or several ANM slow modes. Besides the global motions, it is also found that the local residual fluctuations encoded in protein structure are highly correlated with those in the protein unfolding process. Furthermore, we also investigated the relationship between protein intrinsic flexibility and its unfolding events. The results show that the intrinsic flexible regions tend to unfold early. Several early unfolding events can be predicted by analysis of protein structural flexibility. These results imply that protein structure-encoded dynamical properties have significant influences on protein unfolding motions.     

In situ observation of elementary growth processes of protein crystals by advanced optical microscopy

To start systematically investigating the quality improvement of protein crystals, the elementary growth processes of protein crystals must be first clarified comprehensively. Atomic force microscopy (AFM) has made a tremendous contribution toward elucidating the elementary growth processes of protein crystals and has confirmed that protein crystals grow layer by layer utilizing kinks on steps, as in the case of inorganic and low-molecular-weight compound crystals. However, the scanning of the AFM cantilever greatly disturbs the concentration distribution and solution flow in the vicinity of growing protein crystals. AFM also cannot visualize the dynamic behavior of mobile solute and impurity molecules on protein crystal surfaces. To compensate for these disadvantages of AFM, in situ observation by two types of advanced optical microscopy has been recently performed. To observe the elementary steps of protein crystals noninvasively, laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM) was developed. To visualize individual mobile protein molecules, total internal reflection fluorescent (TIRF) microscopy, which is widely used in the field of biological physics, was applied to the visualization of protein crystal surfaces. In this review, recent progress in the noninvasive in situ observation of elementary steps and individual mobile protein molecules on protein crystal surfaces is outlined.     

Macromolecular impurities and disorder in protein crystals.

The mechanisms by which macromolecular impurities degrade the diffraction properties of protein crystals have been investigated using X-ray topography, high-resolution diffraction line shape measurements, crystallographic data collection, chemical analysis, and two-photon excitation fluorescence microscopy. Hen egg-white lysozyme crystals grown from solutions containing a structurally unrelated protein (ovotransferrin) and a related protein (turkey egg-white lysozyme) can exhibit significantly broadened mosaicity due to formation of cracks and dislocations but have overall B factors and diffraction resolutions comparable to those of crystals grown from uncontaminated lysozyme. Direct fluorescence imaging of the three-dimensional impurity distribution shows that impurities incorporate with different densities in sectors formed by growth on different crystal faces, and that impurity densities in the crystal core and along boundaries between growth sectors can be much larger than in other parts of the crystal. These nonuniformities create stresses that drive formation of the defects responsible for the mosaic broadening. Our results provide a rationale for the use of seeding to obtain high-quality crystals from heavily contaminated solutions and have implications for the use of crystallization for protein purification. Proteins 1999;36:270-281.     

The effects of cosolutes on protein dynamics: the reversal of denaturant-induced protein fluctuations by trimethylamine N-oxide

The protein stabilizing effects of the small molecule osmolyte, trimethylamine N-oxide, against chemical denaturant was investigated by NMR spin-relaxation measurements and model-free analysis. In the presence of 0.7 M guanidine hydrochloride increased picosecond-nanosecond dynamics are observed in the protein ribonuclease A. These increased fluctuations occur throughout the protein, but the most significant increases in flexibility occur at positions believed to be the first to unfold. Addition of 0.35 M trimethylamine N-oxide to this destabilized form of ribonuclease results in significant rigidification of the protein backbone as assessed by (1)H-(15)N order parameters. Statistically, these order parameters are the same as those measured in native ribonuclease indicating that TMAO reduces the amplitude of backbone fluctuations in a destabilized protein. These data suggest that TMAO restricts the bond vector motions on the protein energy landscape to resemble those motions that occur in the native protein and points to a relation between stability and dynamics in this enzyme.     

Modification of protein crystal packing by systematic mutations of surface residues: Implications on biotemplating and crystal porosity

Bioinspired nano‐scale biotemplating for the development of novel composite materials has recently culminated in several demonstrations of nano‐structured hybrid materials. Protein crystals, routinely prepared for the elucidation of protein 3D structures by X‐ray crystallography, present an ordered and highly accurate 3D array of protein molecules. Inherent to the 3D arrangement of the protein “building blocks” in the crystal, a complementary 3D array of interconnected cavities—voids array, exhibiting highly ordered porosity is formed. The porous arrays of protein crystal may serve as a nano‐structured, accurate biotemplate by a “filling” process. These cavities arrays are shaped by the mode of protein packing throughout the crystallization process. Here we propose and demonstrate feasibility of targeting site specific mutations to modify protein's surface to affect protein crystal packing, enabling the generation of a series of protein crystal “biotemplates” all originating from same parent protein. The selection of these modification sites was based on in silico analysis of protein–protein interface contact areas in the parent crystal. The model protein selected for this study was the N‐terminal type II cohesin from the cellulosomal scaffold in ScaB subunit of Acetivibrio cellulolyticus and mutations were focused on lysine residues involved in protein packing as prime target. The impact of systematically mutating these lysine residues on protein packing and its resulting interconnected cavities array were found to be most significant when surface lysine residues were substituted to tryptophan residues. Our results demonstrate the feasibility of using pre‐designed site directed mutations for the generation of a series of protein crystal biotemplates from a “parent” protein. Biotechnol. Bioeng. 2009; 104: 444–457 © 2009 Wiley Periodicals, Inc.     

FlexProt: alignment of flexible protein structures without a predefinition of hinge regions.

FlexProt is a novel technique for the alignment of flexible proteins. Unlike all previous algorithms designed to solve the problem of structural comparisons allowing hinge-bending motions, FlexProt does not require an a priori knowledge of the location of the hinge(s). FlexProt carries out the flexible alignment, superimposing the matching rigid subpart pairs, and detects the flexible hinge regions simultaneously. A large number of methods are available to handle rigid structural alignment. However, proteins are flexible molecules, which may appear in different conformations. Hence, protein structural analysis requires algorithms that can deal with molecular flexibility. Here, we present a method addressing specifically a flexible protein alignment task. First, the method efficiently detects maximal congruent rigid fragments in both molecules. Transforming the task into a graph theoretic problem, our method proceeds to calculate the optimal arrangement of previously detected maximal congruent rigid fragments. The fragment arrangement does not violate the protein sequence order. A clustering procedure is performed on fragment-pairs which have the same 3-D rigid transformation regardless of insertions and deletions (such as loops and turns) which separate them. Although the theoretical worst case complexity of the algorithm is O(n(6)), in practice FlexProt is highly efficient. It performs a structural comparison of a pair of proteins 300 amino acids long in about seven seconds on a standard desktop PC (400 MHz Pentium II processor with 256MB internal memory). We have performed extensive experiments with the algorithm. An assortment of these results is presented here. FlexProt can be accessed via WWW at bioinfo3d.cs.tau.ac.il/FlexProt/.     

High-resolution spot-scan electron microscopy of microcrystals of an alpha-helical coiled-coil protein

We describe the electron microscopy of a crystalline assembly of an alpha-helical coiled-coil protein extracted from the ootheca of the praying mantis. Electron diffraction patterns of unstained crystals show crystal lattice sampling of the coiled-coil molecular transform to a resolution beyond 1.5 A. Using a "spot-scan" method of electron imaging, micrographs of unstained crystals have been obtained that visibly diffract laser light from crystal spacings as small as 4.3 A. A projection map was calculated to 4 A using electron diffraction amplitudes and phases from computer-processed images. The projection map clearly shows modulations in density arising from the 5.1 A alpha-helical repeat, the first time this type of modulation has been revealed by electron microscopy. The crystals have p2 plane group symmetry with a = 92.4 A, b = 150.7 A, y = 92.4 degrees. Examination of tilted specimens shows that c is approximately 18 A, indicating that the unit cell is only one molecule thick. A preliminary interpretation shows tightly packed molecules some 400 A long lying with their long axes in the plane of the projection. The molecules have a coiled-coil configuration for most of their length. The possible modes of packing of the molecules in three dimensions are discussed.     

Flexible structural comparison allowing hinge-bending, swiveling motions

We present an efficient method for flexible comparison of protein structures, allowing swiveling motions. In all currently available methodologies developed and applied to the comparisons of protein structures, the molecules are considered to be rigid objects. The method described here extends and generalizes current approaches to searches for structural similarity between molecules by viewing proteins as objects consisting of rigid parts connected by rotary joints. During the matching, the rigid subparts are allowed to be rotated with respect to each other around swiveling points in one of the molecules. This technique straightforwardly detects structural motifs having hinge(s) between their domains. Whereas other existing methods detect hinge-bent motifs by initially finding the matching rigid parts and subsequently merging these together, our method automatically detects recurring substructures, allowing full 3 dimensional rotations about their swiveling points. Yet the method is extremely fast, avoiding the time-consuming full conformational space search. Comparison of two protein structures, without a predefinition of the motif, takes only seconds to one minute on a workstation per hinge. Hence, the molecule can be scanned for many potential hinge sites, allowing practically all C(alpha) atoms to be tried as swiveling points. This algorithm provides a highly efficient, fully automated tool. Its complexity is only O(n2), where n is the number of C(alpha) atoms in the compared molecules. As in our previous methodologies, the matching is independent of the order of the amino acids in the polypeptide chain. Here we illustrate the performance of this highly powerful tool on a large number of proteins exhibiting hinge-bending domain movements. Despite the motions, known hinge-bent domains/motifs which have been assembled and classified, are correctly identified. Additional matches are detected as well. This approach has been motivated by a technique for model based recognition of articulated objects originating in computer vision and robotics.