EXTRACT: A program to extract three-dimensional coordinates from stereo diagrams of proteins

The program EXTRACT has been developed to extract accurate three-dimensional coordinates from published stereo α-carbon diagrams of protein structures. The approach is based on the display of scanned images of the left and right eye views of the diagram on a stereo-equipped workstation, allowing construction of a molecular model using the diagram as a guide. A number of structural checks assess the building, including probability maps derived for α-carbon geometry in protein structures. The procedure has also been extended to produce less accurate models from mono images.     

A Bayesian network model for protein fold and remote homologue recognition

MOTIVATION: The Bayesian network approach is a framework which combines graphical representation and probability theory, which includes, as a special case, hidden Markov models. Hidden Markov models trained on amino acid sequence or secondary structure data alone have been shown to have potential for addressing the problem of protein fold and superfamily classification. RESULTS: This paper describes a novel implementation of a Bayesian network which simultaneously learns amino acid sequence, secondary structure and residue accessibility for proteins of known three-dimensional structure. An awareness of the errors inherent in predicted secondary structure may be incorporated into the model by means of a confusion matrix. Training and validation data have been derived for a number of protein superfamilies from the Structural Classification of Proteins (SCOP) database. Cross validation results using posterior probability classification demonstrate that the Bayesian network performs better in classifying proteins of known structural superfamily than a hidden Markov model trained on amino acid sequences alone.     

Three-dimensional in vivo imaging of green fluorescent protein-expressing T cells in mice with noncontact fluorescence molecular tomography

Given that optical tomography is capable of quantitatively imaging the distribution of several important chromophores and fluorophores in vivo, there has been a great deal of interest in developing optical imaging systems with increased numbers of measurements under optimal experimental conditions. In this article, we present a novel system that enables three-dimensional imaging of fluorescent probes in whole animals using a noncontact setup, in parallel with a three-dimensional surface reconstruction algorithm. This approach is directed toward the in vivo imaging of fluorophore or fluorescent protein concentration in small animals. The system consists of a rotating sample holder and a lens-coupled charge-coupled device camera in combination with a fiber-coupled laser scanning device. By measuring multiple projections, large data sets can be obtained, thus improving the accuracy of the inversion models used for quantitative three-dimensional reconstruction of fluorochrome distribution, as well as facilitating a higher spatial resolution. In this study, the system was applied to determining the distribution of green fluorescent protein (GFP)-expressing T lymphocytes in a transgenic mouse model, thus demonstrating the potential of the system for studying immune system function. The technique was used to image and reconstruct fluorescence originating from 32 x 10(6) T cells in the thymus and 3 x 10(5) T cells in the spleen.     

Three-dimensional response of a lumped parameter head-neck model due to impact and impulsive loading

A numerical procedure developed previously for predicting sagittal-plane motion of the human head-neck system due to impact and impulsive loading has been extended to three dimensions. In both situations, a lumped parameter approach is employed, but the current model lumps the mechanical response of each intervertebral joint into a single force-deformation relation evaluated from mechanical properties assembled by various investigators. Computations were performed to obtain the response of the model to a two-dimensional case of flexion whiplash, to one three-dimensional case of side impact to the skull and to another involving base acceleration normal to the sagittal plane. Agreement of the kinematic variables with the results of both the previous two-dimensional analysis and experimental data from a volunteer run is satisfactory, but somewhat poorer correspondence was found for the three-dimensional predictions upon comparison with data obtained from a physical model and from a volunteer when subjected to the prescribed loading. The differences in response are attributed to higher stiffness of facet separation of the model relative both to the structure and the volunteer, to insufficient damping, as well as to substantial differences in the mechanical deformation characteristic of the components of the prototypes and the numerical model.     

Bioinformatics methods to predict protein structure and function. A practical approach

Protein structure prediction by using bioinformatics can involve sequence similarity searches, multiple sequence alignments, identification and characterization of domains, secondary structure prediction, solvent accessibility prediction, automatic protein fold recognition, constructing three-dimensional models to atomic detail, and model validation. Not all protein structure prediction projects involve the use of all these techniques. A central part of a typical protein structure prediction is the identification of a suitable structural target from which to extrapolate three-dimensional information for a query sequence. The way in which this is done defines three types of projects. The first involves the use of standard and well-understood techniques. If a structural template remains elusive, a second approach using nontrivial methods is required. If a target fold cannot be reliably identified because inconsistent results have been obtained from nontrivial data analyses, the project falls into the third type of project and will be virtually impossible to complete with any degree of reliability. In this article, a set of protocols to predict protein structure from sequence is presented and distinctions among the three types of project are given. These methods, if used appropriately, can provide valuable indicators of protein structure and function.     

Reactivity-based analysis of domain structures in native replication protein A

Replication protein A (RPA) is an essential heterotrimeric ssDNA binding protein that participates in DNA repair, replication, and recombination. Though X-ray and NMR experiments have been used to determine three-dimensional structure models of the protein's domain fragments, a complete RPA structural model has not been reported. To test whether the fragment structures faithfully represent the same portions in the native solution-state protein, we have examined the structure of RPA under biologically relevant conditions. We have probed the location of multiple amino acids within the native RPA three-dimensional structure using reactivity of these amino acids toward proteolytic and chemical modification reagents. In turn, we evaluated different structural models by comparing the observed native RPA reactivities with anticipated reactivities based on candidate structural models. Our results show that our reactivity analysis approach is capable of critically assessing structure models and can be a basis for selecting the most relevant from among alternate models of a protein structure. Using this analytical approach, we verified the relevance of RPA fragment models to the native protein structure. Our results further indicate several important features of native RPA's structure in solution, such as flexibility at specific locations in RPA, particularly in the C-terminal region of RPA70. Our findings are consistent with reported DNA-free structural models and support the role of conformational change in the ssDNA binding mechanism of RPA.     

Modeling of protein binary complexes using structural mass spectrometry data

In this article, we describe a general approach to modeling the structure of binary protein complexes using structural mass spectrometry data combined with molecular docking. In the first step, hydroxyl radical mediated oxidative protein footprinting is used to identify residues that experience conformational reorganization due to binding or participate in the binding interface. In the second step, a three-dimensional atomic structure of the complex is derived by computational modeling. Homology modeling approaches are used to define the structures of the individual proteins if footprinting detects significant conformational reorganization as a function of complex formation. A three-dimensional model of the complex is constructed from these binary partners using the ClusPro program, which is composed of docking, energy filtering, and clustering steps. Footprinting data are used to incorporate constraints-positive and/or negative-in the docking step and are also used to decide the type of energy filter-electrostatics or desolvation-in the successive energy-filtering step. By using this approach, we examine the structure of a number of binary complexes of monomeric actin and compare the results to crystallographic data. Based on docking alone, a number of competing models with widely varying structures are observed, one of which is likely to agree with crystallographic data. When the docking steps are guided by footprinting data, accurate models emerge as top scoring. We demonstrate this method with the actin/gelsolin segment-1 complex. We also provide a structural model for the actin/cofilin complex using this approach which does not have a crystal or NMR structure.     

Three-dimensional measurement of rearfoot motion during running

Excessive ranges of motion during running have been speculated to be connected to injuries to the lower extremities. Movement of the foot and lower leg has commonly been studied with two-dimensional techniques. However, differences in the alignment of the longitudinal axis of the foot with the camera axis will produce measurement errors for projected angles of the lower extremities. A three-dimensional approach would not have this limitation. The purpose of this study is to present a three-dimensional model for calculation of angles between lower leg and foot, lower leg and ground, and foot and ground, and to compare results from treadmill running derived from this model with results derived from a two-dimensional model for different alignment angles between foot axis and camera axis. A two camera Selspot system was used to obtain three-dimensional information on motion of the studied segments. It was found that several two-dimensional variables measured from a posterior view are very sensitive to the alignment angle between the foot and the camera axis. Some variables change as much as 1 degrees for every 2 degrees of change of the alignment angle. The large influence of rotations other than the measured one in two-dimensional measurements makes advisable the use of a three-dimensional model when studying motion between foot and lower leg during running.     

The three-dimensional solution structure of NaD1, a new floral defensin from Nicotiana alata and its application to a homology model of the crop defense protein alfAFP

NMR spectroscopy and simulated annealing calculations have been used to determine the three-dimensional structure of NaD1, a novel antifungal and insecticidal protein isolated from the flowers of Nicotiana alata. NaD1 is a basic, cysteine-rich protein of 47 residues and is the first example of a plant defensin from flowers to be characterized structurally. Its three-dimensional structure consists of an alpha-helix and a triple-stranded antiparallel beta-sheet that are stabilized by four intramolecular disulfide bonds. NaD1 features all the characteristics of the cysteine-stabilized alphabeta motif that has been described for a variety of proteins of differing functions ranging from antibacterial insect defensins and ion channel-perturbing scorpion toxins to an elicitor of the sweet taste response. The protein is biologically active against insect pests, which makes it a potential candidate for use in crop protection. NaD1 shares 31% sequence identity with alfAFP, an antifungal protein from alfalfa that confers resistance to a fungal pathogen in transgenic potatoes. The structure of NaD1 was used to obtain a homology model of alfAFP, since NaD1 has the highest level of sequence identity with alfAFP of any structurally characterized antifungal defensin. The structures of NaD1 and alfAFP were used in conjunction with structure-activity data for the radish defensin Rs-AFP2 to provide an insight into structure-function relationships. In particular, a putative effector site was identified in the structure of NaD1 and in the corresponding homology model of alfAFP.     

Building native protein conformation from NMR backbone chemical shifts using Monte Carlo fragment assembly

We have been analyzing the extent to which protein secondary structure determines protein tertiary structure in simple protein folds. An earlier paper demonstrated that three-dimensional structure can be obtained successfully using only highly approximate backbone torsion angles for every residue. Here, the initial information is further diluted by introducing a realistic degree of experimental uncertainty into this process. In particular, we tackle the practical problem of determining three-dimensional structure solely from backbone chemical shifts, which can be measured directly by NMR and are known to be correlated with a protein's backbone torsion angles. Extending our previous algorithm to incorporate these experimentally determined data, clusters of structures compatible with the experimentally determined chemical shifts were generated by fragment assembly Monte Carlo. The cluster that corresponds to the native conformation was then identified based on four energy terms: steric clash, solvent-squeezing, hydrogen-bonding, and hydrophobic contact. Currently, the method has been applied successfully to five small proteins with simple topology. Although still under development, this approach offers promise for high-throughput NMR structure determination.     

Prediction of protein structure from ideal forms

For many years it has been accepted that the sequence of a protein can specify its three-dimensional structure. However, there has been limited progress in explaining how the sequence dictates its fold and no attempt to do this computationally without the use of specific structural data has ever succeeded for any protein larger than 100 residues. We describe a method that can predict complex folds up to almost 200 residues using only basic principles that do not include any elements of sequence homology. The method does not simulate the folding chain but generates many thousands of models based on an idealized representation of structure. Each rough model is scored and the best are refined. On a set of five proteins, the correct fold score well and when tested on a set of larger proteins, the correct fold was ranked highest for some proteins more than 150 residues, with others being close topological variants. All other methods that approach this level of success rely on the use of templates or fragments of known structures. Our method is unique in using a database of ideal models based on general packing rules that, in spirit, is closer to an ab initio approach.     

Determination of three-dimensional protein structures from nuclear magnetic resonance data using fragments of known structures

A method to build a three-dimensional protein model from nuclear magnetic resonance (NMR) data using fragments from a data base of crystallographically determined protein structures is presented. The interproton distances derived from the nuclear Overhauser effect (NOE) data are compared to the precalculated distances in the known protein structures. An efficient search algorithm is used, which arranges the distances in matrices akin to a C alpha diagonal distance plot, and compares the NOE distance matrices for short sequential zones of the protein to the data base matrices. After cluster analysis of the fragments found in this way, the structure is built by aligning fragments in overlapping zones. The sequentially long-range NOEs cannot be used in the initial fragments search but are vital to discriminate between several possible combinations of different groups of fragments. The method has been tested on one simulated NOE data set derived from a crystal structure and one experimental NMR data set. The method produces models that have good local structure, but may contain larger global errors. These models can be used as the starting point for further refinement, e.g., by restrained molecular dynamics or interactive graphics.     

Abundance of intrinsic disorder in SV-IV, a multifunctional androgen-dependent protein secreted from rat seminal vesicle

The potent immunomodulatory, anti-inflammatory and procoagulant properties of protein no. 4 secreted from the rat seminal vesicle epithelium (SV-IV) have previously been found to be modulated by a supramolecular monomer-trimer equilibrium. More structural details that integrate experimental data into a predictive framework have recently been reported. Unfortunately, homology modelling and fold-recognition strategies were not successful in creating a theoretical model of the structural organization of SV-IV. It was inferred that the global structure of SV-IV is not similar to that of any protein of known three-dimensional structure. Reversing the classical approach to the sequence-structure-function paradigm, in this paper we report novel information obtained by comparing the physicochemical parameters of SV-IV with two datasets composed of intrinsically unfolded and ideally globular proteins. In addition, we analyse the SV-IV sequence by several publicly available disorder-oriented predictors. Overall, disorder predictions and a re-examination of existing experimental data strongly suggest that SV-IV needs large plasticity to efficiently interact with the different targets that characterize its multifaceted biological function, and should therefore be better classified as an intrinsically disordered protein.     

Characterization of protein fold by wide-angle X-ray solution scattering

Wide-angle X-ray solution scattering (WAXS) patterns contain substantial information about the three-dimensional structure of a protein. Although WAXS data have far less information than is required for determination of a full three-dimensional structure, the actual amount of information contained in a WAXS pattern has not been carefully quantified. Here we carry out an analysis of the amount of information that can be extracted from a WAXS pattern and demonstrate that it is adequate to estimate the secondary-structure content of a protein and to strongly limit its possible tertiary structures. WAXS patterns computed from the atomic coordinates of a set of 498 protein domains representing all of known fold space were used as the basis for constructing a multidimensional space of all corresponding WAXS patterns (‘WAXS space’). Within WAXS space, each scattering pattern is represented by a single vector. A principal components analysis was carried out to identify those directions in WAXS space that provide the greatest discrimination among patterns. The number of dimensions that provide significant discrimination among protein folds agrees well with the number of independent parameters estimated from a naïve Shannon sampling theorem approach. Estimates of the relative abundances of secondary structures were made using training/test sets derived from this data set. The average error in the estimate of α-helical content was 11%, and of β-sheet content was 9%. The distribution of proteins that are members of the four structure classes, α, β, α/β and α+β, are well separated in WAXS space when data extending to a spacing of 2.2 Å are used. Quantification of the information embedded within a WAXS pattern indicates that these data can be used as a powerful constraint in homology modeling of protein structures.     

Xylanase homology modeling using the inverse protein folding approach.

Xylanase has been used in wood pulp bleaching in an effort to reduce chlorine release into the environment and pollution associated with paper production. The three-dimensional structure of xylanase is important to enable better understanding of the enzyme mechanism and to help design a more thermostable xylanase mutant. At the time this work was begun, there was no sequence homologous protein available for traditional sequence-based homology modeling. In order to circumvent this problem, the inverse protein folding approach was undertaken to find a suitable template structure. Model structures of Bacillus circulans xylanase were built based on the data-base search results of related proteins. The model structures were refined and compared to the recently solved xylanase X-ray crystal structure. The overall structural similarity between the theoretical model and experimental structure demonstrate the usefulness of this approach. Disagreement in folding topology, however, warrants further research into the inverse protein folding approach.     

Assembly of a polytopic membrane protein structure from the solution structures of overlapping peptide fragments of bacteriorhodopsin.

Three-dimensional structures of only a handful of membrane proteins have been solved, in contrast to the thousands of structures of water-soluble proteins. Difficulties in crystallization have inhibited the determination of the three-dimensional structure of membrane proteins by x-ray crystallography and have spotlighted the critical need for alternative approaches to membrane protein structure. A new approach to the three-dimensional structure of membrane proteins has been developed and tested on the integral membrane protein, bacteriorhodopsin, the crystal structure of which had previously been determined. An overlapping series of 13 peptides, spanning the entire sequence of bacteriorhodopsin, was synthesized, and the structures of these peptides were determined by NMR in dimethylsulfoxide solution. These structures were assembled into a three-dimensional construct by superimposing the overlapping sequences at the ends of each peptide. Onto this construct were written all the distance and angle constraints obtained from the individual solution structures along with a limited number of experimental inter-helical distance constraints, and the construct was subjected to simulated annealing. A three-dimensional structure, determined exclusively by the experimental constraints, emerged that was similar to the crystal structure of this protein. This result suggests an alternative approach to the acquisition of structural information for membrane proteins consisting of helical bundles.     

Structural insights into the beta-mannosidase from T. reesei obtained by synchrotron small-angle X-ray solution scattering enhanced by X-ray crystallography

A molecular envelope of the beta-mannosidase from Trichoderma reesei has been obtained by combined use of solution small-angle X-ray scattering (SAXS) and protein crystallography. Crystallographic data at 4 A resolution have been used to enhance informational content of the SAXS data and to obtain an independent, more detailed protein shape. The phased molecular replacement technique using a low resolution SAXS model, building, and refinement of a free atom model has been employed successfully. The SAXS and crystallographic free atom models exhibit a similar globular form and were used to assess available crystallographic models of glycosyl hydrolases. The structure of the beta-galactosidase, a member of a family 2, clan GHA glycosyl hydrolases, shows an excellent fit to the experimental molecular envelope and distance distribution function of the beta-mannosidase, indicating gross similarities in their three-dimensional structures. The secondary structure of beta-mannosidase quantified by circular dichroism measurements is in a good agreement with that of beta-galactosidase. We show that a comparison of distance distribution functions in combination with 1D and 2D sequence alignment techniques was able to restrict the number of possible structurally homologous proteins. The method could be applied as a general method in structural genomics and related fields once protein solution scattering data are available.     

Three-dimensional localization of serine 2808, a phosphorylation site in cardiac ryanodine receptor

Type 2 ryanodine receptor (RyR2) is the major calcium release channel in cardiac muscle. Phosphorylation of RyR2 by cAMP-dependent protein kinase A and by calmodulin-dependent protein kinase II modulates channel activity. Hyperphosphorylation at a single amino acid residue, Ser-2808, has been proposed to directly disrupt the binding of a 12.6-kDa FK506-binding protein (FKBP12.6) to RyR2, causing a RyR2 malfunction that triggers cardiac arrhythmias in human heart failure. To determine the structural basis of the interaction between Ser-2808 and FKBP12.6, we have employed two independent approaches to map this phosphorylation site in RyR2 by three-dimensional cryo-electron microscopy. In one approach, we inserted a green fluorescent protein (GFP) after amino acid Tyr-2801, and mapped the GFP three-dimensional location in the RyR2 structure. In another approach, the binding site of monoclonal antibody 34C was mapped in the three-dimensional structure of skeletal muscle RyR1. The epitope of antibody 34C has been mapped to amino acid residues 2,756 through 2,803 of the RyR1 sequence, corresponding to residues 2,722 through 2,769 of the RyR2 sequence. These locations of GFP insertion and antibody binding are adjacent to one another in domain 6 of the cytoplasmic clamp region. Importantly, the three-dimensional location of the Ser-2808 phosphorylation site is 105-120 A distance from the FKBP12.6 binding site mapped previously, indicating that Ser-2808 is unlikely to be directly involved in the binding of FKBP12.6 to RyR2, as had been proposed previously.