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S C Harvey

Publications and source records attributed to S C Harvey.

At least 91 records · Page 5Linked to original sources
PubMed1994

Use of photoaffinity crosslinking and molecular modeling to analyze the global architecture of ribonuclease P RNA.

Bacterial ribonuclease P (RNase P), an endonuclease involved in tRNA maturation, is a ribonucleoprotein containing a catalytic RNA. The secondary structure of this ribozyme is well established, but comparatively little is understood about its 3-D structure. In this analysis, orientation and distance constraints between elements within the Escherichia coli RNase P RNA-pre-tRNA complex were determined by intra- and intermolecular crosslinking experiments. A molecular mechanics-based RNA structure refinement protocol was used to incorporate the distance constraints indicated by crosslinking, along with the known secondary structure of RNase P RNA and the tertiary structure of tRNA, into molecular models. Seven different structures that satisfy the constraints equally well were generated and compared by superposition to estimate helix positions and orientations. Manual refinement within the range of conformations indicated by the molecular mechanics analysis was used to derive a model of RNase P RNA with bound substrate pre-tRNA that is consistent with the crosslinking results and the available phylogenetic comparisons.

Affinity Labels↗
PubMed1994

A quantitative model of the Escherichia coli 16 S RNA in the 30 S ribosomal subunit.

We use a computer-based protocol for automated structure refinement of large RNAs and ribonucleoproteins to propose a three-dimensional model for the Escherichia coli 16 S RNA in the 30 S ribosomal subunit along with the first quantitative estimates of the uncertainties in the model. Our models are based on the 16 S RNA secondary structure, the small angle neutron scatter map of 30 S proteins, tertiary RNA-RNA and RNA-protein contacts as suggested by cross-linking, chemical footprinting and other experimental studies, and electron microscopy data for the shape of the 30 S subunit and placement of 16 S RNA fragments, along with known motifs in RNA structure. In addition, some data on the interaction of the tRNAs/mRNA with the 16 S RNA were used to localize the active site. Since there are not enough structural data to derive a unique three-dimensional folding of the 16 S RNA, several different conformations can be generated to satisfy the experimental data. A set of seven models was refined to survey the range of acceptable conformations. These models were analyzed to deduce probable positions and orientations of the different helical segments that comprise the 16 S RNA in the Escherichia coli small subunit, and one consensus model from this set is presented here. An estimate of the reliability of our predicted structure is made using the variations between the models, and about 75% of 16 S RNA helical segments are localized to 15 A or less in their position in the small subunit. Our models show a distinct separation of the three major domains of the 16 S RNA. The 5' major domain and the central domain are clustered in the body of the 30 S subunit, whereas the 3' major domain is localized in the head of the subunit. Our modeling results are compared with models of the 16 S RNA proposed by other researchers, and are seen to be similar to the manually built models by Stern et al. and Brimacombe et al. with a few significant differences. The position of nucleotides implicated by footprinting and crosslinking data in tRNA and mRNA binding, and in subunit association are examined, and many of these sites are seen to lie along the 30 S subunit neck, cleft and the platform.

Base Sequence↗
PubMed1994

Lagrangian molecular dynamics using selected conformational degrees of freedom, with application to the pseudorotation dynamics of furanose rings.

Using internal conformational degrees of freedom for biopolymers as natural variables, and introducing a Lagrangian dynamics approach, one can simulate time-dependent processes over a much longer time scale than in classical Newtonian molecular dynamics (MD) techniques. Two factors contribute to this: a substantial reduction in the number of degrees of freedom and a very large increase in the size of the time step. We present the Lagrangian equations of motion for repuckering transitions in model furanose (F), ribose (R), and 2'-deoxyribose (dR) ring systems using the pseudorotation phase angle as the single dynamic variable. As in most Lagrangian analyses, the effective masses for the R and dR models are dependent on conformation, and we test the behavior of this variable mass (VM) model. Since the variation in effective mass is small, the VM model is compared with a simplified constant mass (CM) model, which is shown to be an excellent approximation. The equations of motion for the CM and VM models are integrated with the leapfrog and the iterative leapfrog algorithms, respectively. The Lagrangian dynamics approach reduces the number of degrees of freedom from about 40 to 1, and allows the use of time steps on the order of 20 fs, about an order of magnitude greater than is used in conventional MD simulations.

Biopolymers↗
PubMed1994

Langevin dynamics of the choline head group in a membrane environment.

Computer simulations of dipalmitoylphosphatidylcholine (DPPC) have been performed using Langevin dynamics and a Marcelja-type mean field. This work has focused on the dynamics of the choline head group to parameterize the empirical constraints against phosphorus-carbon dipolar couplings (Dp-c) as measured by nuclear magnetic resonance (13C-NMR). The results show good agreement with experimental values at constraints equivalent to the choline tilt observed in joint refinement of x-ray diffraction and neutron diffraction scatterings. Quadrupolar splittings for the alpha and beta positions are also calculated and compared with 2H-NMR experiments. The model predicts torsional transition rates around the alpha-beta bonds and for the two C-O-P-O torsions. It also predicts T1 relaxation times for the alpha and beta carbons.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1994

Modeling large RNAs and ribonucleoprotein particles using molecular mechanics techniques.

There is a growing body of low-resolution structural data that can be utilized to devise structural models for large RNAs and ribonucleoproteins. These models are routinely built manually. We introduce an automated refinement protocol to utilize such data for building low-resolution three-dimensional models using the tools of molecular mechanics. In addition to specifying the positions of each nucleotide, the protocol provides quantitative estimates of the uncertainties in those positions, i.e., the resolution of the model. In typical applications, the resolution of the models is about 10-20 A. Our method uses reduced representations and allows us to refine three-dimensional structures of systems as big as the 16S and 23S ribosomal RNAs, which are about one to two orders of magnitude larger than nucleic acids that can be examined by traditional all-atom modeling methods. Nonatomic resolution structural data--secondary structure, chemical cross-links, chemical and enzymatic footprinting patterns, protein positions, solvent accessibility, and so on--are combined with known motifs in RNA structure to predict low-resolution models of large RNAs. These structural constraints are imposed on the RNA chain using molecular mechanics-type potential functions with parameters based on the quality of experimental data. Surface potential functions are used to incorporate shape and positional data from electron microscopy image reconstruction experiments into our models. The structures are optimized using techniques of energy refinement to get RNA folding patterns. In addition to providing a consensus model, the method finds the range of models consistent with the data, which allows quantitative evaluation of the resolution of the model. The method also identifies conflicts in the experimental data. Although our protocol is aimed at much larger RNAs, we illustrate these techniques using the tRNA structure as an example and test-bed.

Anticodon↗
PubMed1993

Conformational transitions using molecular dynamics with minimum biasing.

The molecular dynamics algorithm (MD), which simulates intramolecular motions on the subnanosecond timescale, has been modified to allow the investigation of slow conformational transitions that do not necessarily occur spontaneously in MD simulations. The method is designated CONTRA MD (CONformational TRAnsitions by Molecular Dynamics with minimum biasing). The method requires the prior definition of a single conformational variable that is required to vary monotonically from an initial conformation to a final target conformation. The simulation is broken up into a series of short free MD segments, and we determine, after each segment of MD, whether or not the system has evolved toward the final conformation. Those segments that do not move the system in that direction are deleted. Those that do move it toward the final conformation are patched together sequentially to generate a single representative trajectory along the transition pathway. The CONTRA MD method is demonstrated first by application to the simultaneous C2'-endo to C3'-endo repucker and anti to syn N-glycosidic torsion transitions in 2'-deoxyadenosine and then to the large-scale bending in phenylalanine transfer RNA.

Monte Carlo Method↗
PubMed1993

Langevin dynamics studies of unsaturated phospholipids in a membrane environment.

Computer simulations of three unsaturated phospholipids in a membrane environment have been carried out using Langevin dynamics and a mean-field based on the Marcelja model. The applicability of the mean-field to model unsaturated lipids was judged by comparison to available experimental NMR data. The results show that the mean-field methodology and the parameters developed for saturated lipids are applicable in simulations of unsaturated molecules, indicating that these simulations have good predictive capabilities. Single molecule simulations, each 100 ns in length, of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-elaidoyl-sn-glycero-3-phosphocholine (PEPC), and 1-palmitoyl-2-isolinoleoyl-sn-glycero-3-phosphocholine (PiLPC) reveal similarities between PEPC and DPPC. The presence of the trans double bond in PEPC has a minimum impact on the structural and dynamic properties of the molecule, which is probably the reason that isolated trans double bonds are rare in biological lipids. POPC exhibits different behavior, especially in the calculated average interchain distances, because of the cis double bond. The position of the two double bonds in PiLPC imparts special properties to the molecule.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1992

A comprehensive classification of nucleic acid structural families based on strand direction and base pairing.

We propose a classification of DNA structures formed from 1 to 4 strands, based only on relative strand directions, base to strand orientation and base pairing geometries. This classification and its associated notation enable all nucleic acids to be grouped into structural families and bring to light possible structures which have not yet been observed experimentally. It also helps in understanding transitions between families and can assist in the design of multistrand structures.

Base Composition↗
PubMed1992

Analyzing the normal mode dynamics of macromolecules by the component synthesis method.

This paper presents a general method for studying the harmonic dynamics of large biomolecules and molecular complexes. The performance and accuracy of the method applied to a number of molecules are also reported. The basic approach of the method is to divide a macromolecule into a number of smaller components. The local normal modes of the components are first calculated by treating individual components and the interactions between nearest neighboring components. The physical displacements of all atoms are then represented in the local normal mode space, in which a selected range of high-frequency local modes is neglected. The equation of motion of the molecule in the local normal mode space will then have a smaller dimension, and consequently the normal modes of the whole structure, particularly for large molecules, can be solved much more easily. The normal modes of two polypeptides--(Ala)6 and (Ala)12--and a double-helical DNA--d(ATATA).d(TATAT)--are analyzed with this method. Reductions on the dimensions of harmonic dynamic equations for these molecules have been made, with the fraction of the deleted high-frequency modes ranging from 1/2 to 5/6. The calculated low-frequency normal modes are found to be very accurate as compared to the exact solutions by standard procedure. The major advantage of the present approach on macromolecule harmonic dynamics is that the reduction on the dimensionality of the eigenvalue problems can be varied according to the size of molecules, so the method can be easily applied to large macromolecules with controlled accuracy.

DNA↗
PubMed1992

Teaching macromolecular modeling.

Training newcomers to the field of macromolecular modeling is as difficult as is training beginners in x-ray crystallography, nuclear magnetic resonance, or other methods in structural biology. In one or two lectures, the most that can be conveyed is a general sense of the relationship between modeling and other structural methods. If a full semester is available, then students can be taught how molecular structures are built, manipulated, refined, and analyzed on a computer. Here we describe a one-semester modeling course that combines lectures, discussions, and a laboratory using a commercial modeling package. In the laboratory, students carry out prescribed exercises that are coordinated to the lectures, and they complete a term project on a modeling problem of their choice. The goal is to give students an understanding of what kinds of problems can be attacked by molecular modeling methods and which problems are beyond the current capabilities of those methods.

Biochemistry↗
PubMed1992

Molecular modeling to predict the structural and biological effects of mutations in a highly conserved histone mRNA loop sequence.

The 3'-end of histone mRNAs contains a highly conserved sequence motif which is believed to form a 6 base pair stem and a 4 base loop. These sequences are involved in both the efficiency of 3'-end formation and stability of the mature histone mRNA. We have modeled four stem basepairs and the loop portion of this structure using the wildtype sequences and several mutant sequences. A structure for the wildtype stem-loop is proposed that is based on energy minimization using a representative wildtype sequence and comparison with structures obtained using naturally occurring mutations which do not alter loop function. A wildtype structure is proposed in which the top basepair of the stem is broken, forming a six base loop. Mutant sequences with altered bases in the loop and in the stem were also modeled. The effect of these mutations on the proposed wildtype structure is discussed and possible biological consequences considered.

Base Sequence↗
PubMed1991

Mean field stochastic boundary molecular dynamics simulation of a phospholipid in a membrane.

Computer simulations of phospholipid membranes have been carried out by using a combined approach of molecular and stochastic dynamics and a mean field based on the Marcelja model. First, the single-chain mean field simulations of Pastor et al. [(1988) J. Chem. Phys. 89, 1112-1127] were extended to a complete dipalmitoylphosphatidylcholine molecule; a 102-ns Langevin dynamics simulation is presented and compared with experiment. Subsequently, a hexagonally packed seven-lipid array was simulated with Langevin dynamics and a mean field at the boundary and with molecular dynamics (and no mean field) in the center. This hybrid method, mean field stochastic boundary molecular dynamics, reduces bias introduced by the mean field and eliminates the need for periodic boundary conditions. As a result, simulations extending to tens of nanoseconds may be carried out by using a relatively small number of molecules to model the membrane environment. Preliminary results of a 20-ns simulation are reported here. A wide range of motions, including overall reorientation with a nanosecond decay time, is observed in both simulations, and good agreement with NMR, IR, and neutron diffraction data is found.

Computer Simulation↗
PubMed1990

Nuclear magnetic resonance and molecular modeling studies on O-beta-D-galactopyranosyl-(1----4)-O-beta-D-xylopyranosyl-(1----0)-L-se rine, a carbohydrate-protein linkage region fragment from connective tissue proteoglycans.

The solution conformation of O-beta-D-galactopyranosyl-(1----4)-O-beta-D-xylopyranosyl-(1----0)-L-ser ine (GXS), a carbohydrate-protein linkage region fragment from connective tissue proteoglycans, was investigated by two-dimensional NMR spectroscopy and molecular modeling calculations. Specifically, the 1H and 13C resonances were assigned by 2D-COSY and by 1H-13C heteronuclear correlation spectroscopy methods. 2D-NOESY was used to generate distance constraints between the galactose and xylose and between the xylose and serine residues. The 1H vicinal coupling constants for the sugars and the serine were also determined. A general molecular modeling methodology suitable for complex carbohydrates was developed. This methodology employed molecular dynamics and energy minimization procedures together with the application of inter-residue spatial constraints across the linkages derived from 2D-NOESY. The first step in this methodology is the generation of a wide variety of starting conformations that span the (phi, psi) space for each linkage. In the present study, nine such conformations were constructed for each linkage using the torsion angles phi and psi corresponding to the gauche+, gauche-, and trans configurations across each of the two bonds constituting the linkage. These conformations were subjected to a combined molecular dynamics/energy minimization refinement using the NOESY derived constraints as pseudoenergy functions. Families of conformations for the whole molecule were then constructed from the structures derived for each linkage. Characterization of GXS using this methodology identified a single family of conformations that are consistent with the solution phase NMR data on this molecule.

Computer Graphics↗
PubMed1990

Electrical potential of transfer RNAs: codon-anticodon recognition.

Calculations of the electrostatic potentials were made around yeast elongator phenylalanine, aspartate tRNAs, and yeast initiator methionine tRNA in aqueous solution at physiological ionic strength. The calculations were carried out with a finite difference algorithm for solving the nonlinear Poisson-Boltzmann equation that incorporates the screening effects of the electrolyte, the exclusion of ions by the molecule, the molecular shape, and the different polarizabilities of the solvent and the tRNA. The initiator tRNA is surrounded by uniformly spaced contours of negative potential. The elongator tRNAs are also surrounded by a similar contour pattern except in the anticodon region where there is a pronounced "hole" in the potential surface. This hole is caused by an invagination of the potential contours, which also results in an increase in the local field strength. The effect of this hole is that the anticodon region in the elongator tRNAs is the least negative, or conversely the most positive, region of the molecule. This hole, which is not found when simple Coulombic potentials are used, is due both to the structure of the elongator tRNA anticodon loops and to the different polarizabilities of the solvent and tRNA. The existence of the potential hole in elongator tRNAs may account in part for their ability to associate with other negatively charged macromolecules, in particular mRNA. Moreover, it suggests that the anticodon loop of elongator tRNAs is the energetically most favorable point of approach of mRNA to tRNA.

Anticodon↗
PubMed1990

Prediction of the three-dimensional structure of Escherichia coli 30S ribosomal subunit: a molecular mechanics approach.

We introduce a computer-assisted procedure for folding large RNA chains into three-dimensional conformations consistent with their secondary structure and other known experimental constraints. The RNA chain is modeled using pseudoatoms at different levels of detail--from a single pseudoatom per helix to a single pseudoatom for each nucleotide. A stepwise procedure is used, starting with a simple representation of the macromolecule that is refined and then extrapolated into higher resolution for further refinement. The procedure is capable of folding different random-walk chains by using energy minimization, allowing generation of a range of conformations consistent with given experimental data. We use this procedure to generate several possible conformations of the 16S RNA in the 30S ribosomal subunit of Escherichia coli by using secondary structure and the neutron-scattering map of the 21 proteins in the small subunit. The RNA chain is modeled using a single pseudoatom per helix. RNA-RNA and RNA-protein crosslinks, reported in current literature, are included in our model. Footprinting data for different ribosomal proteins in the 16S RNA are also used. Several conformations of the 16S RNA are generated and compared to predict gross structural features of the 30S subunit as well as to identify regions of the 16S RNA that cannot be well-defined with current experimental data.

Escherichia coli↗