Biomedical subjects

Peter J Steinbach

Publications and source records attributed to Peter J Steinbach.

11 recordsLinked to original sources

PubMed2007

Prediction of side-chain conformations on protein surfaces.

An approach is described that improves the prediction of the conformations of surface side chains in crystal structures, given the main-chain conformation of a protein. A key element of the methodology involves the use of the colony energy. This phenomenological term favors conformations found in frequently sampled regions, thereby approximating entropic effects and serving to smooth the potential energy surface. Use of the colony energy significantly improves prediction accuracy for surface side chains with little additional computational cost. Prediction accuracy was quantified as the percentage of side-chain dihedral angles predicted to be within 40 degrees of the angles measured by X-ray diffraction. Use of the colony energy in predictions for single side chains improved the prediction accuracy for chi(1) and chi(1+2) from 65 and 40% to 74 and 59%, respectively. Several other factors that affect prediction of surface side-chain conformations were also analyzed, including the extent of conformational sampling, details of the rotamer library employed, and accounting for the crystallographic environment. The prediction of conformations for polar residues on the surface was generally found to be more difficult than those for hydrophobic residues, except for polar residues participating in hydrogen bonds with other protein groups. For surface residues with hydrogen-bonded side chains, the prediction accuracy of chi(1) and chi(1+2) was 79 and 63%, respectively. For surface polar residues, in general (all side-chain prediction), the accuracy of chi(1) and chi(1+2) was only 73 and 56%, respectively. The most accurate results were obtained using the colony energy and an all-atom description that includes neighboring molecules in the crystal (protein chains and hetero atoms). Here, the accuracy of chi(1) and chi(1+2) predictions for surface side chains was 82 and 73%, respectively. The root mean square deviations obtained for hydrogen-bonding surface side chains were 1.64 and 1.81 A, with and without consideration of crystal packing effects, respectively.

Crystallography, X-Ray↗

PubMed2006

Biological effect of a novel mutation in the third leucine-rich repeat of human luteinizing hormone receptor.

A novel heterozygous mutation A340T leading to the substitution of Phe for the conserved amino acid Ile114 was identified by nucleotide sequencing of the human LH/chorionic gonadotropin receptor (hLHR) of a patient with Leydig cell hypoplasia. This mutation is located in the third leucine-rich repeat in the ectodomain of the hLHR. In vitro expression studies demonstrated that this mutation results in reduced ligand binding and signal transduction of the receptor. Studies of hLHR constructs in which various amino acids were substituted for the conserved Ile114 showed that receptor activity is sensitive to changes in size, shape, and charge of the side chain. A homology model of the wild-type hLHR ectodomain was made, illustrating the packing of conserved hydrophobic side chains in the protein core. Substitution of Ile114 by Phe might disrupt intermolecular contacts between hormone and receptor. This mutation might also affect an LHR-dimer interaction. Thus, the I114F mutation reduces ligand binding and signal transduction by the hLHR, and it is partially responsible for Leydig cell hypoplasia in the patient.

Base Sequence↗

PubMed2005

Eukaryotic RNases H1 act processively by interactions through the duplex RNA-binding domain.

Ribonucleases H have mostly been implicated in eliminating short RNA primers used for initiation of lagging strand DNA synthesis. Escherichia coli RNase HI cleaves these RNA-DNA hybrids in a distributive manner. We report here that eukaryotic RNases H1 have evolved to be processive enzymes by attaching a duplex RNA-binding domain to the RNase H region. Highly conserved amino acids of the duplex RNA-binding domain are required for processivity and nucleic acid binding, which leads to dimerization of the protein. The need for a processive enzyme underscores the importance in eukaryotic cells of processing long hybrids, most of which remain to be identified. However, long RNA-DNA hybrids formed during immunoglobulin class-switch recombination are potential targets for RNase H1 in the nucleus. In mitochondria, where RNase H1 is essential for DNA formation during embryogenesis, long hybrids may be involved in DNA replication.

Amino Acid Sequence↗

PubMed2005

Polyproline and the "spectroscopic ruler" revisited with single-molecule fluorescence.

To determine whether Forster resonance energy transfer (FRET) measurements can provide quantitative distance information in single-molecule fluorescence experiments on polypeptides, we measured FRET efficiency distributions for donor and acceptor dyes attached to the ends of freely diffusing polyproline molecules of various lengths. The observed mean FRET efficiencies agree with those determined from ensemble lifetime measurements but differ considerably from the values expected from Forster theory, with polyproline treated as a rigid rod. At donor-acceptor distances much less than the Forster radius R(0), the observed efficiencies are lower than predicted, whereas at distances comparable to and greater than R(0), they are much higher. Two possible contributions to the former are incomplete orientational averaging during the donor lifetime and, because of the large size of the dyes, breakdown of the point-dipole approximation assumed in Forster theory. End-to-end distance distributions and correlation times obtained from Langevin molecular dynamics simulations suggest that the differences for the longer polyproline peptides can be explained by chain bending, which considerably shortens the donor-acceptor distances.

Fluorescence↗

PubMed2005

Computer simulation of protein-ligand interactions: challenges and applications.

The accurate modeling of protein-ligand interactions, like any prediction of macromolecular structure, requires an energy function of sufficient detail to account for all relevant interactions and a conformational search method that can reliably find the energetically favorable conformations of a heterogeneous system. Both of these prerequisites represent daunting challenges. Consequently, the routine docking of small molecules or peptides to proteins in their correct binding modes, and the reliable ranking of binding affinities remain unsolved problems. Nonetheless, computational techniques are continually evolving so as to broaden the range of feasible applications, and the accuracy of predictions and theoretical approaches can often be of great help in guiding and interpreting experiments. We discuss the energetics of protein-ligand systems and survey conformational searching techniques. We illustrate how molecular modeling of a protein-ligand complex sheds light on the observed resistance of a mutant dihydrofolate reductase to the antibiotic trimethoprim. In another example, we show that relaxation of side chains in different crystal structures of the same complex, benzamidine bound to trypsin, is needed to draw sensible conclusions from the calculations. The results of these relatively simple conformational searches underscore the importance of incorporating protein flexibility in simulations of protein-ligand interactions, even in the context of relatively rigid binding pockets.

Animals↗

PubMed2004

Exploring peptide energy landscapes: a test of force fields and implicit solvent models.

A biased Monte Carlo-minimization/annealing conformational search was used to characterize five descriptions of the energy landscape for each of three model systems: the 20-residue "trp-cage" miniprotein, the 20-residue "BS1" peptide, and the 17-residue "U(1-17)T9D" peptide. The EEF1 and SASA energy landscapes were studied as well as those defined by using the GB/ACE implicit water model with one of three protein force fields: CHARMM19, CHARMM22, and CHARMM22/CMAP. The lowest-energy structures of the trp-cage and BS1 peptides found for the EEF1 landscape have main-chain root-mean-square deviations (rmsds) from the respective NMR structures of less than 2 A; for U(1-17)T9D, the deviation is less than 3 A using EEF1. The main-chain rmsd of the minimum-energy trp-cage conformation obtained for the GB/ACE/CHARMM22/CMAP landscape is less than 1 A. However, this energy function strongly favored helical structures for the two peptides shown by NMR to form beta-sheet structures. Brief annealing of the system following main-chain conformational changes was found to enhance the exploration of low-energy states. The thousands of simulations reported here suggest that the prediction of protein structure might be improved by the simultaneous use of a CMAP-like description of the main chain and an EEF1-like description of the solvent.

Computer Simulation↗

PubMed2004

Mutation in the leucine-rich repeat C-flanking region of platelet glycoprotein Ib beta impairs assembly of von Willebrand factor receptor.

We describe a syndrome of thrombocytopenia, bleeding episodes, congenital heart disease and facial dysmorphism in a newborn infant, and trace the cause to mutations on chromosome 22 that involve the gene for platelet glycoprotein Ib beta (GPIb beta, Human Genome Organisation gene symbol GPIBB), a critical component of the von Willebrand factor (vWF) receptor. Fluorescence in situ hybridization in transformed lymphoblasts revealed hemizygous microdeletion of 22q11.2 containing the GP1BB locus. DNA sequencing revealed a C to T transition in the patient's remaining GP1BB allele, predicting a novel proline to serine substitution (Pro96Ser) in the carboxyterminal flanking domain of a leucine-rich repeat. We characterized the mutant GP1BB allele by expression in a cell line (CHO alpha IX) that stably expresses two other components of the vWF receptor, GPIb alpha and GPIX. Flow cytometry and confocal imaging of transfected CHO alpha IX cells demonstrated that P96S GPIb beta abrogates surface assembly of the complex, consistent with platelet flow cytometry studies in the patient. Based on sequence homology to the known crystal structures of two other leucine-rich repeat proteins, the human Nogo receptor and GPIb alpha, we propose a new structural model of GPIb beta. The model refutes earlier assumptions about cysteine-cysteine interactions in the amino-terminal region of GPIb beta, and predicts a hydrophobic patch the burial of which may contribute to proper conformation of the fully assembled vWF receptor complex.

Abnormalities, Multiple↗

PubMed2002

Redesign of a four-helix bundle protein by phage display coupled with proteolysis and structural characterization by NMR and X-ray crystallography.

To test whether it is practical to use phage display coupled with proteolysis for protein design, we used this approach to convert a partially unfolded four-helix bundle protein, apocytochrome b(562), to a stably folded four-helix bundle protein. Four residues expected to form a hydrophobic core were mutated. One residue was changed to Trp to provide a fluorescence probe for studying the protein's physical properties and to partially fill the void left by the heme. The other three positions were randomly mutated. In addition, another residue in the region to be redesigned was substituted with Arg to provide a specific cutting site for protease Arg-c. This library of mutants was displayed on the surface of phage and challenged with protease Arg-c to select stably folded proteins. The consensus sequence that emerged from the selection included hydrophobic residues at only one of the three positions and non-hydrophobic residues at the other two. Nevertheless, the selected proteins were thermodynamically very stable. The structure of a selected protein was characterized using multi-dimensional NMR. All four helices were formed in the structure. Further, site-directed mutagenesis was used to change one of the two non-hydrophobic residues to a hydrophobic residue, which increased the stability of the protein, indicating that the selection result was not based solely on the protein's global stability and that local structural characteristics may also govern the selection. This conclusion is supported by the crystal structure of another mutant that has two hydrophobic residues substituted for the two non-hydrophobic residues. These results suggest that the hydrophobic interactions in the core are not sufficient to dictate the selection and that the location of the cutting site of the protease also influences the selection of structures.

Amino Acid Sequence↗

PubMed2002

Carboxypeptidase E, a prohormone sorting receptor, is anchored to secretory granules via a C-terminal transmembrane insertion.

Carboxypeptidase E (CPE) is a sorting receptor that directs the prohormone pro-opiomelanocortin (POMC) to the regulated secretory pathway, and is also a prohormone processing enzyme in neuro/endocrine cells. It has been suggested that the 25 C-terminal amino acids are necessary for the binding of CPE to secretory granule membranes, but its orientation in the membrane is not known. In this study, we examined the structure and orientation of the membrane-binding domain at the C-terminus of CPE. In vitro experiments using model membranes demonstrated that the last 22 amino acids of CPE (CP peptide) insert in a shallow orientation into lipid bilayers at low pH. Circular dichroism analysis indicated that the CP peptide adopts a partial alpha-helical configuration at low pH, and helix content increases when it is bound to lipid. Protease protection experiments, immunolabeling, and immunoisolation of intact secretory granules with a C-terminal antibody revealed a cytoplasmic domain in CPE, consistent with a transmembrane orientation of this protein. We conclude that the membrane-binding domain of CPE must adopt an alpha-helical configuration to bind to lipids, and that CPE may require another integral membrane "chaperone" protein to insert through the lipid bilayer in a transmembrane fashion.

Acrylamide↗

PubMed2002

Analysis of kinetics using a hybrid maximum-entropy/nonlinear-least-squares method: application to protein folding.

A hybrid analysis that combines the maximum entropy method (MEM) with nonlinear least squares (NLS) fitting has been developed to interpret a general time-dependent signal. Data that include processes of opposite sign and a slow baseline drift can be inverted to obtain both a continuous distribution of lifetimes and a sum of discrete exponentials. Fits by discrete exponentials are performed with initial parameters determined from the distribution of lifetimes obtained with the MEM. The regularization of the parameter space achieved by the MEM stabilizes the introduction of each successive exponential in the NLS fits. This hybrid approach is particularly useful when fitting by a large number of exponentials. Revision of the MEM "prior" based on features in the data can improve the lifetime distribution obtained. Standard errors in the mean are estimated automatically for raw data. The results presented for simulated data and for fluorescence measurements of protein folding illustrate the utility and accuracy of the hybrid algorithm. Analysis of the folding of dihydrofolate reductase reveals six kinetic processes, one more than previously reported.

Algorithms↗

PubMedDate not supplied

Inferring lifetime distributions from kinetics by maximizing entropy using a bootstrapped model.

A bootstrapped model is used to improve the lifetime distribution recovered using the maximum entropy method from kinetics that involves overlapping exponential and distributed phases. The model defaulted to in the limit of low signal-to-noise is iteratively derived from the data to counter the tendency of regularization methods to over-smooth sharp features while under-smoothing broad ones. Upon each revision, some of the lifetime distribution is focused and the rest is blurred. This differential blurring can produce distributions that are virtually free of artifacts. The change in the result obtained upon a reasonable change in the default model provides a useful measure of the uncertainty in the lifetime distribution. In particular, the widths of peaks may not be well determined.

Journal Article↗