1,721,100 research outputs found
β-Hairpin stability and folding: Molecular dynamics studies of the first β-hairpin of tendamistat
No full textThe stability and (un)folding of the 19-residue peptide, SCVTLYQSWRYSQADNGCA, corresponding to the first β-hairpin (residues 10 to 28) of the α-amylase inhibitor tendamistat (PDB entry 3AIT) has been studied by molecular dynamics simulations in explicit water under periodic boundary conditions at several temperatures (300 K, 360 K and 400 K), starting from various conformations for simulation lengths, ranging from 10 to 30 ns. Comparison of trajectories of the reduced and oxidized native peptides reveals the importance of the disulphide bridge closing the β-hairpin in maintaining a proper turn conformation, thereby insuring a proper side-chain arrangement of the conserved turn residues. This allows rationalization of the conservation of those cysteine residues among the family of α-amylase inhibitors. High temperature simulations starting from widely different initial configurations (native β-hairpin, α and left-handed helical and extended conformations) begin sampling similar regions of the conformational space within tens of nanoseconds, and both native and non-native β-hairpin conformations are recovered. Transitions between conformational clusters are accompanied by an increase in energy fluctuations, which is consistent with the increase in heat capacity measured experimentally upon protein folding. The folding events observed in the various simulations support a model for β-hairpin formation in which the turn is formed first, followed by hydrogen bond formation closing the hairpin, and subsequent stabilization by side-chain hydrophobic interactions. (C) 2000 Academic Press
Atomic insight into the CD4 binding-induced conformational changes in HIV-1 gp120
No full textThe entry of HIV-1 into a target cell requires gp120 and receptor CD4 as well as coreceptor CCR5/CXCR4 recognition events associated with conformational changes of the involved proteins. The binding of CD4 to gp120 is the initiation step of the whole process involving structural rearrangements that are crucial for subsequent pathways. Despite the wealth of knowledge about the gp120/CD4 interactions, details of the conformational changes occurring at this stage remain elusive. We have performed molecular dynamics simulations in explicit solvent based on the gp120/CD4/CD4i crystal structure in conjunction with modeled V3 and V4 loops to gain insight into the dynamics of the binding process. Three differentiated interaction modes between CD4 and gp120 were found, which involve electrostatics, hydrogen bond and van der Waals networks. A binding funnel model is proposed based on the dynamical nature of the binding interface together with a CD4-attraction gradient centered in gp120 at the CD4-Phe43-binding cavity. Distinct dynamical behaviors of free and CD4-bound gp120 were monitored, which likely represent the ground and pre-fusogenic states, respectively. The transition between these states revealed concerted motions in gp120 leading to: i) loop contractions around the CD4-Phe43-insertion cavity; ii) stabilization of the four-stranded bridging sheet structure; and iii) translocation and clustering of the V3 loop and the bridging sheet leading to the formation of the coreceptor binding site. Our results provide new insight into the dynamic of the underlying molecular recognition mechanism that complements the biochemical and structural studies
Various strategies of using residual dipolar couplings in NMR-driven protein docking: application to Lys48-linked di-ubiquitin and validation against 15N-relaxation data
No full textExpression screening, protein purification and NMR analysis of human protein domains for structural genomics
No full textStructural genomics, the determination of protein structures on a genome-wide scale, is still in its infancy for eukaryotes due to the number and size of their genes. Low protein expression and solubility of eukaryotic geneproducts are the major bottlenecks in high-throughput (HTP) recombinant protein production with the E. coli expression systems. To circumvent this problem we decided to focus on separate protein domains. We describe here a fast microtiterplate based, expression and solubility screening procedure, using a combination of in vitro and in vivo expression, and purification with nickel-NTA magnetic beads. All steps are optimized for automatic HTP processing using a liquid handling station. Furthermore, large-scale expression and protein purification conditions are optimized, permitting the purification of 24 protein samples per week. We further show that results obtained from the expression screening can be extrapolated to the production of protein samples for NMR. Starting with 81 cloned human protein domains, in vivo expression was detected in 54 cases, and from 28 of those milligrams of protein were purified. An informative HSQC spectrum was recorded for 18 proteins (22%), half of which were indicative of a folded protein. The success rate and quality of the HSQC spectra suggest that the domain approach holds promise for human proteins
Toward an integrated model of protein-DNA recognition as inferred from NMR studies on the Lac repressor system
No full textSequence-specific protein-DNA interactions are responsible for the regulation of key biological functions such as replication of the genome, initiation of transcription, and repair of damaged DNA. All of these regulatory pathways are built on the foundation that proteins are able to bind selectively to a particular DNA site in the genome. The most challenging issue for specific protein-DNA recognition is that the target sequence is immersed in a huge molar excess of nonspecific DNA sequences, which also compete for the same protein. The distribution of protein between regulatory and nonspecific sequences determines the occupancy of the target site in the cell and, hence, the transcriptional activity of the corresponding promoter. The problem is much more acute than it is for other protein-ligand interactions, because obviously the protein cannot differentiate between these sites on the basis of their size or shape.1 For repressor molecules, such as the lactose (lac) repressor, the problem of finding the correct site is even more severe, as only a few copies ( 10) of the protein exist in the cell. Thus, to compete with the large excess of nonspecific genomic DNA, these proteins have evolved to bind with a very high specificity ratio ( 107)
Side chain dynamics monitored by 13C-13C cross-relaxation
No full textA method to measure 13C-13C cross-relaxation rates in a fully 13C labeled protein has been developed that can give information about the mobility of side chains in proteins. The method makes use of the (H)CCH-NOESY pulse sequence and includes a suppression scheme for zero-quantum (ZQ) coherences that allows the extraction of initial rates from NOE buildup curves. The method has been used to measure 13C-13C cross-relaxation rates in the 269-residue serine-protease PB92. We focused on Cα-Cβ cross-relaxation rates, which could be extracted for 64% of all residues, discarding serine residues because of imperfect ZQ suppression, and methyl 13C-13C cross-relaxation rates, which could be extracted for 47% of the methyl containing C-C pairs. The Cα-Cβ cross-relaxation rates are on average larger in secondary structure elements as compared to loop regions, in agreement with the expected higher rigidity in these elements. The cross-relaxation rates for methyl containing C-C pairs show a general decrease of rates further into the side chain, indicating more flexibility with increasing separation from the main chain. In the case of leucine residues also long-range Cβ-Cδ cross-peaks are observed. Surprisingly, for most of the leucines a cross-peak with only one of the methyl Cδ carbons is observed, which correlates well with the χ2 torsion-angle and can be explained by a difference in mobility for the two methyl groups due to an anisotropic side chain motion
Refinement of protein structures in explicit solvent
No full textWe present a CPU efficient protocol for refinement of protein structures in a thin layer of explicit solvent and energy parameters with completely revised dihedral angle terms. Our approach is suitable for protein structures determined by theoretical (e.g., homology modeling or threading) or experimental methods (e.g., NMR). In contrast to other recently proposed refinement protocols, we put a strong emphasis on consistency with widely accepted covalent parameters and computational efficiency. We illustrate the method for NMR structure calculations of three proteins: interleukin- 4, ubiquitin, and crambin. We show a comparison of their structure ensembles before and after refinement in water with and without a force field energy term for the dihedral angles; crambin was also refined in DMSO. Our results demonstrate the significant improvement of structure quality by a short refinement in a thin layer of solvent. Further, they show that a dihedral angle energy term in the force field is beneficial for structure calculation and refinement. We discuss the optimal weight for the energy constant for the backbone angle omega and include an extensive discussion of meaning and relevance of the calculated validation criteria, in particular root mean square Z scores for covalent parameters such as bond lengths. Proteins 2003;50: 496–506
Use of very long-distance NOEs in a fully deuterated protein: an approach for rapid protein fold determination
No full textThe high sensitivity of modern NMR instrumentation, in combination with full deuteration, enabled the measurement of long-range NOEs between amide protons in a fully deuterated protein corresponding to distances up to 8 Å. These are beyond the limit normally observed in protonated samples. Such long-distance NOEs could be observed using long mixing times, which became possible due to reduced spin diffusion and T1 relaxation of the amide protons in the fully deuterated sample. This information was used in combination with secondary structure restraints derived from secondary chemical shifts for structure calculations. With these backbone amide proton NOEs only, a unique fold could be obtained with positional root mean square deviations from the average of 1.30 and 2.25 Å for backbone and heavy atoms, respectively. Despite the low density of restraints, no mirror image problems were observed. Addition of sidechain NOE information increased the precision of the ensemble and in particular of the core packing. The structures obtained in this way were close to the published crystal structure. NOE completeness analysis revealed that the cumulative completeness is still more than 80% for an 8.0 Å cut-off distance
Structural properties of the promiscuous VP16 activation domain
No full textHerpes simplex virion protein 16 (VP16) contains two strong activation regions that can independently and cooperatively activate transcription in vivo. We have identified the regions and residues involved in the interaction with the human transcriptional coactivator positive cofactor 4 (PC4) and the general transcription factor TFIIB. NMR and biochemical experiments revealed that both VP16 activation regions are required for the interaction and undergo a conformational transition from random coil to α-helix upon binding to its target PC4. The interaction is strongly electrostatically driven and the binding to PC4 is enhanced by the presence of its amino-terminal domain. We propose models for binding of VP16 to the core domains of PC4 and TFIIB that are based on two independent docking approaches using NMR chemical shift changes observed in titration experiments. The models are consistent with results from site-directed mutagenesis and provide an explanation for the contribution of both acidic and hydrophobic residues for transcriptional activation by VP16. Both intrinsically unstructured activation domains are attracted to their interaction partner by electrostatic interactions, and adopt an α-helical conformation around the important hydrophobic residues. The models showed multiple distinct binding surfaces upon interaction with various partners, providing an explanation for the promiscuous properties, cooperativity, and the high activity of this activation domain
The precision of NMR structure ensembles revisited.
No full textContains fulltext :
79477.pdf (Publisher’s version ) (Closed access)Biomolecular structures provide the basis for many studies in research areas such as structure-based drug design and homology modeling. In order to use molecular coordinates it is important that they are reliable in terms of accurate description of the experimental data and in terms of the overall and local geometry. Besides these primary quality criteria an indication is needed for the uncertainty in the atomic coordinates that may arise from the dynamic behavior of the considered molecules as well as from experimental- and computational procedures.In contrast to the crystallographic B-factor, a good measure for the uncertainty in NMR-derived atomic coordinates is still not available. It has become clear in recent years that the widely used atomic Root Mean Square Deviation (RMSD), which is a measure for the precision of the data, overestimates the accuracy of NMR structure ensembles and therefore is a problematic measure for the uncertainty in the atomic coordinates.In this study we report a method that yields a more realistic estimate of the uncertainty in the atomic coordinates by maximizing the RMSD of an ensemble of structures, while maintaining the accordance with the experimentally derived data. The results indicate that the RMSD of most NMR structure ensembles can be significantly increased compromising neither geometric quality nor NMR data. This maximized RMSD therefore seems a better estimate of the true uncertainty in the atomic coordinates
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