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Four mutated hemoglobin (Hb) variants and wild type hemoglobin as a reference have been investigated using density functional theory methods focusing on oxygen binding. Dispersion-corrected B3LYP functional is used and found to provide reliable oxygen binding energies. It also correctly reproduces the spin distribution of both bound and free heme groups as well as provides correct geometries at their close vicinity. Mutations in hemoglobin are not only an intrigued biological problem and it is also highly important to understand their effects from a clinical point of view. This study clearly shows how even small structural differences close to the heme group can have a significant effect in reducing the oxygen binding of mutated hemoglobins and consequently affecting the health condition of the patient suffering from the mutations. All of the studied mutated Hb variants did exhibit much weaker binding of molecular oxygen compared to the wild type of hemoglobin.
Assuntos
Compostos Férricos/química , Hemoglobinas/química , Histidina/química , Mutação , Modelos MolecularesRESUMO
Glucose oxidase (GOx) is a flavoenzyme having applications in food and medical industries. However, GOx, as many other enzymes when extracted from the cells, has relatively short operational lifetimes. Several recent studies (both experimental and theoretical), carried out on small proteins (or small fractions of large proteins), show that a detailed knowledge of how the breakdown process starts and proceeds on molecular level could be of significant help to artificially improve the stability of fragile proteins. We have performed extended molecular dynamics (MD) simulations to study the denaturation of GOx (a protein dimer containing nearly 1200 amino acids) to identify weak points in its structure and in this way gather information to later make it more stable, for example, by mutations. A denaturation of a protein can be simulated by increasing the temperature far above physiological temperature. We have performed a series of MD simulations at different temperatures (300, 400, 500, and 600 K). The exit from the protein's native state has been successfully identified with the clustering method and supported by other methods used to analyze the simulation data. A common set of amino acids is regularly found to initiate the denaturation, suggesting a moiety where the enzyme could be strengthened by a suitable amino acid based modification.
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Glucose Oxidase/química , Simulação de Dinâmica Molecular , Penicillium/enzimologia , Desnaturação Proteica , Dobramento de Proteína , Temperatura Alta , Modelos Moleculares , Conformação ProteicaRESUMO
MOTIVATION: The precise prediction of one-dimensional (1D) protein structure as represented by the protein secondary structure and 1D string of discrete state of dihedral angles (i.e. Shape Strings) is a prerequisite for the successful prediction of three-dimensional (3D) structure as well as protein-protein interaction. We have developed a novel 1D structure prediction method, called Frag1D, based on a straightforward fragment matching algorithm and demonstrated its success in the prediction of three sets of 1D structural alphabets, i.e. the classical three-state secondary structure, three- and eight-state Shape Strings. RESULTS: By exploiting the vast protein sequence and protein structure data available, we have brought secondary-structure prediction closer to the expected theoretical limit. When tested by a leave-one-out cross validation on a non-redundant set of PDB cutting at 30% sequence identity containing 5860 protein chains, the overall per-residue accuracy for secondary-structure prediction, i.e. Q3 is 82.9%. The overall per-residue accuracy for three- and eight-state Shape Strings are 85.1 and 71.5%, respectively. We have also benchmarked our program with the latest version of PSIPRED for secondary structure prediction and our program predicted 0.3% better in Q3 when tested on 2241 chains with the same training set. For Shape Strings, we compared our method with a recently published method with the same dataset and definition as used by that method. Our program predicted at 2.2% better in accuracy for three-state Shape Strings. By quantitatively investigating the effect of data base size on 1D structure prediction we show that the accuracy increases by approximately 1% with every doubling of the database size.
Assuntos
Biologia Computacional/métodos , Estrutura Secundária de Proteína , Proteínas/química , Bases de Dados de Proteínas , Modelos Moleculares , Análise de Sequência de Proteína/métodosRESUMO
The structure of a complicated quasicrystal approximant epsilon(16) was predicted from a known and related quasicrystal approximant epsilon(6) by the strong-reflections approach. Electron-diffraction studies show that in reciprocal space, the positions of the strongest reflections and their intensity distributions are similar for both approximants. By applying the strong-reflections approach, the structure factors of epsilon(16) were deduced from those of the known epsilon(6) structure. Owing to the different space groups of the two structures, a shift of the phase origin had to be applied in order to obtain the phases of epsilon(16). An electron-density map of epsilon(16) was calculated by inverse Fourier transformation of the structure factors of the 256 strongest reflections. Similar to that of epsilon(6), the predicted structure of epsilon(16) contains eight layers in each unit cell, stacked along the b axis. Along the b axis, epsilon(16) is built by banana-shaped tiles and pentagonal tiles; this structure is confirmed by high-resolution transmission electron microscopy (HRTEM). The simulated precession electron-diffraction (PED) patterns from the structure model are in good agreement with the experimental ones. Epsilon(16) with 153 unique atoms in the unit cell is the most complicated approximant structure ever solved or predicted.
RESUMO
After a hundred years with no solution, the seemingly simple but actually very complex mineral kaliophilite, KAlSiO4, is finally revealed by electron crystallography.
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MOTIVATION: Motivated by the abundance, importance and unique functionality of zinc, both biologically and physiologically, we have developed an improved method for the prediction of zinc-binding sites in proteins from their amino acid sequences. RESULTS: By combining support vector machine (SVM) and homology-based predictions, our method predicts zinc-binding Cys, His, Asp and Glu with 75% precision (86% for Cys and His only) at 50% recall according to a 5-fold cross-validation on a non-redundant set of protein chains from the Protein Data Bank (PDB) (2727 chains, 235 of which bind zinc). Consequently, our method predicts zinc-binding Cys and His with 10% higher precision at different recall levels compared to a recently published method when tested on the same dataset. AVAILABILITY: The program is available for download at www.fos.su.se/~nanjiang/zincpred/download/
Assuntos
Algoritmos , Metaloproteínas/química , Modelos Químicos , Modelos Moleculares , Mapeamento de Interação de Proteínas/métodos , Análise de Sequência de Proteína/métodos , Zinco/química , Sequência de Aminoácidos , Sítios de Ligação , Simulação por Computador , Dados de Sequência Molecular , Ligação ProteicaRESUMO
Crystallography of nanocrystalline materials has witnessed a true revolution in the past 10 years, thanks to the introduction of protocols for 3D acquisition and analysis of electron diffraction data. This method provides single-crystal data of structure solution and refinement quality, allowing the atomic structure determination of those materials that remained hitherto unknown because of their limited crystallinity. Several experimental protocols exist, which share the common idea of sampling a sequence of diffraction patterns while the crystal is tilted around a noncrystallographic axis, namely, the goniometer axis of the transmission electron microscope sample stage. This Outlook reviews most important 3D electron diffraction applications for different kinds of samples and problematics, related with both materials and life sciences. Structure refinement including dynamical scattering is also briefly discussed.
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Different methods for describing and comparing the structures of the tens of thousands of proteins that have been determined by X-ray crystallography are reviewed. Such comparisons are important for understanding the structures and functions of proteins and facilitating structure prediction, as well as assessing structure prediction methods. We summarize methods in this field emphasizing ways of representing protein structures as one-dimensional geometrical strings. Such strings are based on the shape symbols of clustered regions of phi/Psi dihedral angle pairs of the polypeptide backbones as described by the Ramachandran plot. These one-dimensional expressions are as compact as secondary structure description but contain more information in loop regions. They can be used for fast searching for similar structures in databases and for comparing similarities between proteins and between the predicted and native structures.
Assuntos
Modelos Moleculares , Conformação Proteica , Proteínas/química , Sequência de Aminoácidos , Cristalografia por Raios X , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
The study of crystals at atomic level by electrons - electron crystallography - is an important complement to X-ray crystallography. There are two main advantages of structure determinations by electron crystallography compared to X-ray diffraction: (i) crystals millions of times smaller than those needed for X-ray diffraction can be studied and (ii) the phases of the crystallographic structure factors, which are lost in X-ray diffraction, are present in transmission-electron-microscopy (TEM) images. In this paper, some recent developments of electron crystallography and its applications, mainly on inorganic crystals, are shown. Crystal structures can be solved to atomic resolution in two dimensions as well as in three dimensions from both TEM images and electron diffraction. Different techniques developed for electron crystallography, including three-dimensional reconstruction, the electron precession technique and ultrafast electron crystallography, are reviewed. Examples of electron-crystallography applications are given. There is in principle no limitation to the complexity of the structures that can be solved by electron crystallography.
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Recent developments of novel electron diffraction techniques have shown to be powerful for determination of atomic resolution structures from micron- and nano-sized crystals, too small to be studied by single-crystal X-ray diffraction. In this work, the structure of a rare lysozyme polymorph is solved and refined using continuous rotation MicroED data and standard X-ray crystallographic software. Data collection was performed on a standard 200 kV transmission electron microscope (TEM) using a highly sensitive detector with a short readout time. The data collection is fast (â¼3 min per crystal), allowing multiple datasets to be rapidly collected from a large number of crystals. We show that merging data from 33 crystals significantly improves not only the data completeness, overall I/σ and the data redundancy, but also the quality of the final atomic model. This is extremely useful for electron beam-sensitive crystals of low symmetry or with a preferred orientation on the TEM grid.
Assuntos
Cristalografia/estatística & dados numéricos , Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Microscopia Eletrônica de Transmissão/estatística & dados numéricos , Muramidase/química , Animais , Galinhas , Cristalização , Cristalografia/métodos , Conjuntos de Dados como Assunto , Clara de Ovo/química , Microscopia Eletrônica de Transmissão/instrumentação , Microscopia Eletrônica de Transmissão/métodos , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , SoftwareRESUMO
As membrane transporter proteins, VGLUT1-3 mediate the uptake of glutamate into synaptic vesicles at presynaptic nerve terminals of excitatory neural cells. This function is crucial for exocytosis and the role of glutamate as the major excitatory neurotransmitter in the central nervous system. The three transporters, sharing 76% amino acid sequence identity in humans, are highly homologous but differ in regional expression in the brain. Although little is known regarding their three-dimensional structures, hydropathy analysis on these proteins predicts 12 transmembrane segments connected by loops, a topology similar to other members in the major facilitator superfamily, where VGLUT1-3 have been phylogenetically classified. In this work, we present a three-dimensional model for the human VGLUT1 protein based on its distant bacterial homolog in the same superfamily, the glycerol-3-phosphate transporter from Escherichia coli. This structural model, stable during molecular dynamics simulations in phospholipid bilayers solvated by water, reveals amino acid residues that face its pore and are likely to affect substrate translocation. Docking of VGLUT1 substrates to this pore localizes two different binding sites, to which inhibitors also bind with an overall trend in binding affinity that is in agreement with previously published experimental data.
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Ácido Glutâmico/metabolismo , Conformação Proteica , Proteínas Vesiculares de Transporte de Glutamato/antagonistas & inibidores , Proteínas Vesiculares de Transporte de Glutamato/química , Proteínas de Transporte Vesicular/fisiologia , Sequência de Aminoácidos , Sítios de Ligação , Simulação por Computador , Sequência Conservada , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Propriedades de Superfície , Vesículas Transportadoras/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/antagonistas & inibidores , Proteína Vesicular 1 de Transporte de Glutamato/química , Proteína Vesicular 2 de Transporte de Glutamato/antagonistas & inibidores , Proteína Vesicular 2 de Transporte de Glutamato/química , Proteínas Vesiculares de Transporte de Glutamato/metabolismoRESUMO
A practical, offline method for experimental detection and correction for projector lens distortion in the transmission electron microscope (TEM) operating in high-resolution (HR) and selected area electron diffraction (SAED) modes is described. Typical TEM works show that, in the simplest case, the distortion transforms on the recording device, which would be a circle into an ellipse. The first goal of the procedure described here is to determine the elongation and orientation of the ellipse. The second goal is to correct for the distortion using an ordinary graphic program. The same experimental data set may also be used to determine the actual microscope magnification and the rotation between SAED patterns and HR images. The procedure may be helpful in several quantitative applications of electron diffraction and HR imaging, for instance while performing accurate lattice parameter determination, or while determining possible metrical deviations (cell edges and angles) from a given symmetry.
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Antifreeze proteins (AFP) are responsible for the survival of several species, ranging from bacteria to fish, that encounter subzero temperatures in their living environment. AFPs have been divided into two main families, moderately and hyperactive, depending on their thermal hysteresis activity. We have studied one protein from both families, the AFP from the snow flea (sfAFP) and from the winter flounder (wfAFP), which belong to the hyperactive and moderately active family, respectively. On the basis of molecular dynamics simulations, we have estimated the thickness of the water/ice interface for systems both with and without the AFPs attached onto the ice surface. The calculation of the diffusion profiles along the simulation box allowed us to measure the interface width for different ice planes. The obtained widths clearly show a different influence of the two AFPs on the ice/water interface. The different impact of the AFPs here studied on the interface thickness can be related to two AFPs properties: the protein hydrophobic surface and the number of hydrogen bonds that the two AFPs faces form with water molecules.
Assuntos
Proteínas Anticongelantes/química , Água/química , Animais , Proteínas de Artrópodes/química , Artrópodes , Simulação por Computador , Difusão , Proteínas de Peixes/química , Linguado , Congelamento , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Solventes/química , Propriedades de Superfície , Ubiquitina/químicaRESUMO
Phase identification and structure determination are important and widely used techniques in chemistry, physics and materials science. Recently, two methods for automated three-dimensional electron diffraction (ED) data collection, namely automated diffraction tomography (ADT) and rotation electron diffraction (RED), have been developed. Compared with X-ray diffraction (XRD) and two-dimensional zonal ED, three-dimensional ED methods have many advantages in identifying phases and determining unknown structures. Almost complete three-dimensional ED data can be collected using the ADT and RED methods. Since each ED pattern is usually measured off the zone axes by three-dimensional ED methods, dynamic effects are much reduced compared with zonal ED patterns. Data collection is easy and fast, and can start at any arbitrary orientation of the crystal, which facilitates automation. Three-dimensional ED is a powerful technique for structure identification and structure solution from individual nano- or micron-sized particles, while powder X-ray diffraction (PXRD) provides information from all phases present in a sample. ED suffers from dynamic scattering, while PXRD data are kinematic. Three-dimensional ED methods and PXRD are complementary and their combinations are promising for studying multiphase samples and complicated crystal structures. Here, two three-dimensional ED methods, ADT and RED, are described. Examples are given of combinations of three-dimensional ED methods and PXRD for phase identification and structure determination over a large number of different materials, from Ni-Se-O-Cl crystals, zeolites, germanates, metal-organic frameworks and organic compounds to intermetallics with modulated structures. It is shown that three-dimensional ED is now as feasible as X-ray diffraction for phase identification and structure solution, but still needs further development in order to be as accurate as X-ray diffraction. It is expected that three-dimensional ED methods will become crucially important in the near future.
RESUMO
Protein folding starts before the whole polypeptide has been synthesized by the ribosome. No matter how long the polypeptide is or how intricate the fold, both ends of the chain always end up on the surface. From a topological point of view, this is surprising; one would have expected to find the starting (N-terminal) end inside the core of the folded protein, just as in a ball of yarn. We suggest here that the reason for this apparent paradox is that the first amino acid of the emerging polypeptide chain is gripped during protein synthesis, perhaps by the ribosome, and is not released until the whole polypeptide has been synthesized. This binding would greatly decrease the degrees of freedom for the protein-folding process and could also explain why knots are so rare in proteins. Gripping would also guarantee that the N-terminal is accessible on the protein surface as required for binding of ubiquitin, which regulates the natural degradation of proteins and avoids buildup of protein aggregates, such as those found in Huntington's, Alzheimer's, Parkinson's, and other neurodegenerative diseases.
Assuntos
Dobramento de Proteína , Proteínas/química , Proteínas/metabolismo , Alquil e Aril Transferases/química , Alquil e Aril Transferases/metabolismo , Sequência de Aminoácidos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica , SoftwareRESUMO
The crystal structure of Ti(2)P has been analysed using electron diffraction and high-resolution electron-microscopy techniques. A new unit cell was found, the compound is hexagonal with a = 19.969 (1) and c = 3.4589 (1) A. The structure was first solved in space group P-62m in projection using direct methods on electron diffraction data from the [001] zone axis. A three-dimensional solution was obtained using again direct methods but on a three-dimensional set of electron diffraction data recorded with the precession technique. Ti(2)P is a distorted Fe(2)P structure and, based on high-resolution images, it is possible to explain that the tripling of the unit cell is due to the ordering of P vacancies that reduces the symmetry to P-6.
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A program system-Trice-for reconstructing the 3D reciprocal lattice from an electron diffraction tilt series is described. The unit-cell parameters can be determined from electron diffraction patterns directly by Trice. The unit cell can be checked and the lattice type and crystal system can be determined from the 3D reciprocal lattice. Trice can be applied to all crystal systems and lattice types.
RESUMO
Antifreeze proteins (AFP) allow different life forms, insects as well as fish and plants, to survive in subzero environments. AFPs prevent freezing of the physiological fluids. We have studied, through molecular dynamics simulations, the behavior of the small isoform of the AFP found in the snow flea (sfAFP), both in water and at the ice/water interface, of four different ice planes. In water at room temperature, the structure of the sfAFP is found to be slightly unstable. The loop between two polyproline II helices has large fluctuations as well as the C-terminus. Torsional angle analyses show a decrease of the polyproline II helix area in the Ramachandran plots. The protein structure instability, in any case, should not affect its antifreeze activity. At the ice/water interface the sfAFP triggers local melting of the ice surface. Bipyramidal, secondary prism, and prism ice planes melt in the presence of AFP at temperatures below the melting point of ice. Only the basal plane is found to be stable at the same temperatures, indicating an adsorption of the sfAFP on this ice plane as confirmed by experimental evidence.
Assuntos
Proteínas Anticongelantes/química , Gelo , Proteínas de Insetos/química , Animais , Congelamento , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Isoformas de Proteínas/química , Estabilidade Proteica , Estrutura Secundária de Proteína , Sifonápteros , Solventes/química , Propriedades de Superfície , TemperaturaRESUMO
Implementation of a computer program package for automated collection and processing of rotation electron diffraction (RED) data is described. The software package contains two computer programs: RED data collection and RED data processing. The RED data collection program controls the transmission electron microscope and the camera. Electron beam tilts at a fine step (0.05-0.20°) are combined with goniometer tilts at a coarse step (2.0-3.0°) around a common tilt axis, which allows a fine relative tilt to be achieved between the electron beam and the crystal in a large tilt range. An electron diffraction (ED) frame is collected at each combination of beam tilt and goniometer tilt. The RED data processing program processes three-dimensional ED data generated by the RED data collection program or by other approaches. It includes shift correction of the ED frames, peak hunting for diffraction spots in individual ED frames and identification of these diffraction spots as reflections in three dimensions. Unit-cell parameters are determined from the positions of reflections in three-dimensional reciprocal space. All reflections are indexed, and finally a list with hkl indices and intensities is output. The data processing program also includes a visualizer to view and analyse three-dimensional reciprocal lattices reconstructed from the ED frames. Details of the implementation are described. Data collection and data processing with the software RED are demonstrated using a calcined zeolite sample, silicalite-1. The structure of the calcined silicalite-1, with 72 unique atoms, could be solved from the RED data by routine direct methods.
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A structure projection reconstruction method based on contrast transfer function correction of through-focus series of high-resolution transmission electron microscopy images is presented. In this method, defocus values are determined by evaluating phase similarities of the pixels on the Fourier transforms of the images after correction using trial defocus values. Two-fold astigmatism is also determined, by measuring focus variation along different directions. Each image in the series is corrected for the effects of contrast transfer function and then combined into a structure projection image. The method works for both crystalline and non-crystalline objects. Test results with experimental images are presented. Influences of experimental parameters for imaging and effects of crystal thickness on reconstruction are discussed.