Peptide structural analysis by solid-state NMR spectroscopy.

Solid-state nmr spectroscopy provides a robust method for investigating polypeptides that have been prepared by chemical synthesis and that are immobilized by strong interactions with solid surfaces or large macroscopic complexes. Solid-state nmr spectroscopy has been widely used to investigate membrane polypeptides or peptide aggregates such as amyloid fibrils. Whereas magic angle spinning solid-state nmr spectroscopy allows one to measure distances and dihedral angles with high accuracy, static membrane samples that are aligned with respect to the magnetic field direction allow one to determine the secondary structure of bound polypeptides and their orientation with respect to the bilayer normal. Peptide dynamics and the effect of polypeptides on the macroscopic phase preference of phospholipid membranes have been investigated in nonoriented samples. Investigations of the structure and topology of membrane channels, peptide antibiotics, signal sequences as well as model systems that allow one to dissect the interaction contributions in phospholipid membranes will be presented in greater detail.     

Acetabular morphology and resurfacing design

The bony surfaces of 18 archaeological hemipelves were scanned using a 3D laser surface scanner and CyDir™ software on a Silicon Graphics workstation. The acetabular area was selected and point data from the approximately spherical bone surface saved. These data were input to a MATLAB routine that calculated the radius and centre of the best-fit sphere. The goodness of fit was estimated using the mean and standard deviation of the distance of the bone surface points from the sphere surface. Eight points, at approximately equal distances around the acetabular rim, were selected with reference to bony landmarks. A plane containing three of these points served as an orientation reference plane. The vectors joining the eight rim points to the centre of the best-fit sphere were found. The angles between these vectors and the normal to the reference plane were calculated. Paired angles were summed to give the angle subtended by the acetabular rim in four directions. The overall mean angle was 158° (range of mean angles 145°–173°). The largest individual angles, some exceeding 180°, were in the superior–inferior direction, while the mean angle in the anterior–posterior direction, i.e. that controlling flexion-extension, was 152°. Males had larger subtended angles than females, although the difference was not statistically significant. Simulated reaming increased all angles by approximately 10°. The subtended angles are important parameters in the design of the acetabular component of a hip replacement and particularly important in resurfacing hip replacement when the volume available is tightly constrained.     

The structure of the double-stranded RNA pentamer 5'(CACAG) . 5'(CUGUG) determined by nuclear Overhauser enhancement measurements: interproton distance determination and structure refinement on the basis of X-ray coordinates

The structure in solution of the duplex RNA pentamer 5'(CACAG) . 5'(CUGUG), comprising the stem of the T psi C loop of yeast tRNAPhe, has been investigated by means of one- and two-dimensional nuclear Overhauser enhancement measurements. All non-exchangeable base and sugar proton resonances with the exception of the H5'/H5" sugar resonances are assigned in a sequential manner. From the relative intensities of the cross-peaks obtained in the pure-phase absorption two-dimensional nuclear Overhauser enhancement spectra at several mixing times, it is deduced that the RNA pentamer adopts an A-type conformation in solution. Cross-relaxation rates and interproton distances are determined from the time dependence of the nuclear Overhauser effects, principally by one-dimensional measurements. The structure of the RNA pentamer is then refined by restrained least-squares minimization on the basis of both distance and planarity restraints using fibre diffraction data as an initial model. The refined structure of the RNA pentamer is of the A type but exhibits local structural variations in glycosidic bond and backbone torsion angles as well as in propeller twist, base roll and base tilt angles.     

Refinement of the solution structure of the DNA decamer 5'd(CTGGATCCAG)2: combined use of nuclear magnetic resonance and restrained molecular dynamics

The solution structure of the self-complementary DNA decamer 5'd(CTGGATCCAG)2 comprising the specific target site for the restriction endonuclease BamH1 is investigated by using nuclear magnetic resonance sectroscopy and restrained molecular dynamics. With the exception of the H5'/H5" sugar proton resonances, all the nonexchangeable proton resonances are assigned sequentially by using pure-phase absorption two-dimensional nuclear Overhauser enhancement spectroscopy. From the time dependence of the nuclear Overhauser effects a set of 160 approximate interproton distances is determined and used as the basis of a structure refinement employing restrained molecular dynamics in which the interproton distances are incorporated into the total energy function of the system in the form of an effective potential term. Two restrained dynamics simulations are carried out, starting from classical B- and A-DNA [atomic root mean square (rms) difference 5.7 A]. In both cases convergence is achieved to very similar B-type structures with an atomic rms difference of 0.9 A which is comparable to the rms fluctuations of the atoms about their average positions. In addition, the rms difference between the experimental and calculated values of the interproton distances for both average restrained dynamics structures is approximately 0.3 A. These results suggest that the converged restrained molecular dynamics structures represent reasonable approximations of the solution structure. The average restrained dynamics structures exhibit clear sequence-dependent variations of torsion angles and helical parameters. In addition, the structures exhibit a small bend of around 10-20 degrees at the second (TpG) and eighth (CpA) base pair steps. This can be attributed to the positive base roll angles and large base pair slide values at the two Pyr-Pur steps. The central core of the decamer comprising the six-base recognition site for BamH1 (GGATCC), however, is straight.     

The geometry of interactions between catalytic residues and their substrates

The process of deducing the catalytic mechanism of an enzyme from its structure is highly complex and requires extensive experimental work to validate a proposed mechanism. As one step towards improving the reliability of this process, we have gathered statistics describing the typical geometry of catalytic residues with regard to the substrate and one another. In order to analyse residue-substrate interactions, we have assembled a dataset of structures of enzymes of known mechanism bound to substrate, product, or a substrate analogue. Despite the challenges presented in obtaining such experimental data, we were able to include 42 enzyme structures. We have also assembled a separate dataset of catalytic residues which act upon other catalytic residues, using a set of 60 enzyme structures. For both datasets, we have extracted the distances between residues with a given catalytic function and their target moieties. The geometry of residues whose function involves the transfer or sharing of hydrogens (either with substrate or another residue) was analysed more closely. The results showed that the geometry for such productive interactions (prior to the transition state) closely resembles that seen in non-catalytic hydrogen bonds, with distances and angles in the normal expected range. Such statistics provide limits on "expected geometries" for catalytic residues, which will help to identify these residues and elucidate enzyme mechanisms.     

The solution structure of a B-DNA undecamer comprising a portion of the specific target site for the cAMP receptor protein in the gal operon. Refinement on the basis of interproton distance data.

A restrained least squares refinement of the solution structure of the double-stranded DNA undecamer 5'd(AAGTGT-GACAT).5'd(ATGTCACACTT) comprising a portion of the specific target site of the cAMP receptor protein in the gal operon is presented. The structure is refined on the basis of both distance and planarity restraints, 2331 in all. The distance restraints comprise 150 interproton distances determined from pre-steady state nuclear Overhauser enhancement measurements and 2159 other interatomic distances derived from idealized geometry (i.e., distances between covalently bonded atoms, between atoms defining fixed bond angles, and between atoms defining hydrogen bonding in AT and GC base pairs). Two refinements were carried out and in both cases the final RMS difference between the experimental and calculated interproton distances was 0.2 A. The difference between the two refined structures is small (overall RMS difference of 0.23 A) and represents the error in the refined coordinates. Although the refined structures have an overall B-type conformation there are large variations in many of the local conformational parameters including backbone and glycosidic bond torsion angles, helical twist and propellor twist, base roll and base tilt angles.     

Wobble dG X dT pairing in right-handed DNA: solution conformation of the d(C-G-T-G-A-A-T-T-C-G-C-G) duplex deduced from distance geometry analysis of nuclear Overhauser effect spectra

We report below on features of the three-dimensional structure of the d(C-G-T-G-A-A-T-T-C-G-C-G) self-complementary duplex (designated 12-mer GT) containing symmetrical G X T mismatches in the interior of the helix. The majority of the base and sugar protons in the 12-mer GT duplex were assigned by two-dimensional nuclear Overhauser effect (NOESY) spectra in H2O and D2O solution. A set of 92 short (less than 4.5-A) proton-proton distances defined by lower and upper bounds for one symmetrical half of the 12-mer GT duplex were estimated from NOESY data sets recorded as a function of mixing time. These experimental distances combined with nucleotide bond length parameters were embedded into Cartesian space; several trial structures were refined to minimize bond geometry and van der Waals and chirality error. Confidence in this approach is based on the similarity of the refined structures for the solution conformation of the 12-mer GT duplex. The G and T bases pair through two imino-carbonyl hydrogen bonds, and stacking is maintained between the G X T wobble pair and adjacent Watson-Crick G X C pairs. The experimental distance information is restricted to base and sugar protons, and hence structural features such as base pair overlap, glycosidic torsion angles, and sugar pucker are well-defined by this combination of NMR and distance geometry methods. By contrast, we are unable to define the torsion angles about the bonds C3'-O3'-P-O5'-C5'-C4' in the backbone of the nucleic acid.     

Towards automatic measurement of anteversion and neck–shaft angles in human femurs using CT images

Automatic assessment of human femur morphology may provide useful clinical information with regard to hip and knee surgery, prosthesis design and management of hip instability. To this end, neck–shaft and anteversion angles are usually used. We propose a full automatic method to estimate these angles in human femurs. Multislice CT images from 18 dried bones were analysed. The algorithm fits 3D cylinders to different regions of the bone to estimate the angles. A manual segmentation and a conventional angle assessment were used for validation. We found anteversion angle as 20 ± 7° and neck–shaft angle as 130 ± 9°. Mean distances from femur surface to cylinders were 5.5 ± 0.6, 3.5 ± 0.6 and 2.4 ± 0.4 mm for condyles, diaphysis and neck regions, respectively. Automatic and conventional angles were positively correlated (r²>0.85). Manual and automatic segmentations did not differ. The method was fast and 100% reproducible. A robust in vivo segmentation algorithm should be integrated to advance towards a clinically compliant methodology.     

Sequence-dependent motions of DNA: a normal mode analysis at the base-pair level

Computer simulation of the dynamic structure of DNA can be carried out at various levels of resolution. Detailed high resolution information about the motions of DNA is typically collected for the atoms in a few turns of double helix. At low resolution, by contrast, the sequence-dependence features of DNA are usually neglected and molecules with thousands of base pairs are treated as ideal elastic rods. The present normal mode analysis of DNA in terms of six base-pair "step" parameters per chain residue addresses the dynamic structure of the double helix at intermediate resolution, i.e., the mesoscopic level of a few hundred base pairs. Sequence-dependent effects are incorporated into the calculations by taking advantage of "knowledge-based" harmonic energy functions deduced from the mean values and dispersion of the base-pair "step" parameters in high-resolution DNA crystal structures. Spatial arrangements sampled along the dominant low frequency modes have end-to-end distances comparable to those of exact polymer models which incorporate all possible chain configurations. The normal mode analysis accounts for the overall bending, i.e., persistence length, of the double helix and shows how known discrepancies in the measured twisting constants of long DNA molecules could originate in the deformability of neighboring base-pair steps. The calculations also reveal how the natural coupling of local conformational variables affects the global motions of DNA. Successful correspondence of the computed stretching modulus with experimental data requires that the DNA base pairs be inclined with respect to the direction of stretching, with chain extension effected by low energy transverse motions that preserve the strong van der Waals' attractions of neighboring base-pair planes. The calculations further show how one can "engineer" the macroscopic properties of DNA in terms of dimer deformability so that polymers which are intrinsically straight in the equilibrium state exhibit the mesoscopic bending anisotropy essential to DNA curvature and loop formation.     

Stereochemical analysis of ribosomal transpeptidation. Conformation of nascent peptide

Transpeptidation performed by the ribosome is considered as a nucleophilic Sn2 substitution reaction, passing through a tetrahedral intermediate. A stereochemically universal mechanism of the reaction is assumed to exist for all 20 amino acid residues, both in the attacked (donor) and in the attacking (acceptor) substrates. The angles of internal rotation around the bonds of the attacked carbonyl carbon and around the neighbouring bonds in the tetrahedral intermediate, as well as the stereoconfiguration of the intermediate, have been varied. All 54 combinations of the sterically allowed rotational isomers determined by the five torsional angles have been analysed by using Corey-Pauling-Koltun models and by direct calculations permitting the "extreme limits" in interatomic distances and +/- 7 degrees deviations in bond angles. Only one combination, i.e. one unique conformation of the tetrahedral intermediate, is found to be sterically compatible with all 400 possible pairs of the reacting amino acid residues and at the same time to be capable of cleaving into a planar trans-peptide group. The torsion angles phi and psi of this universally allowed intermediate and the peptide product resulting from its cleavage are similar to those in an alpha-helix. It is suggested that the ribosome generates the alpha-helical confirmation at the C-end of the nascent peptide.     

Specific and non-specific interactions in bacterial adhesion to solid substrata

Abstract Based on a literature review, a hypothesis is forwarded on the mechanism of initial bacterial adhesion to solid substrata, which accounts both for the role of specific microscopic surface components as well as for the role of non-specific macroscopic surface properties (surface free energy, zeta potential or hydrophobicity). Three distinct regions in the adhesion process are suggested in which at large and intermediate separation distances adhesion is mediated by the macroscopic surface properties as surface free energy and surface charge, respectively. At small separation distances specific short-range interactions can occur, leading to a strong and irreversible bonding, provided the water film present in between the interaction surfaces can be removed. A major role of hydrophobic groups, supposed to be associated with bacterial surface appendages is suggested to be its dehydrating capacity, enabling the removal of the vicinal water film yielding small areas of direct contact between protruberant parts of the cell surface and the substratum.     

The passive elastic moment at the hip

The passive elastic moment at the hip was measured in normal male subjects in situ. The influence of two joint muscles crossing the hip was evaluated by performing hip moment measurements over a continuous range of hip angles and at prescribed knee angles. The experimentally acquired data was fitted by exponential functions which separately modeled the moment contribution of tissues deformed by hip flexion and extension. The results of the investigation are discussed with regard to the possible role of the passive joint moments as an energy storage and release mechanism during human walking.     

Importance of maintaining the basic stress pathway above the acetabular dome during acetabular reconstruction

The basic stress pathway above the acetabular dome is important for the maintenance of implant stability in press-fit acetabular reconstruction of total hip arthroplasty. However, information on the basic stress pathway and its impact factors remains unclear. The objective of this study was to investigate the effects of the orientations and positions of the acetabular component on the basic stress pathway. The basic stress pathway above the acetabular dome was defined as two parts: 3D basic trabecular bone stress distribution and quantified basic cortical bone stress level, using two subject-specific finite element normal hip models. The effects were then analysed by generating 32 reconstructed acetabular cases with different cup abduction and anteversion angles within a range of 35–50° and 10–25°, respectively, and 12 cases with different hip centre heights within a range of 0–15 mm above the acetabular dome. The 3D trabecular stress distribution decreased remarkably in all cases, while the 80% of the basic cortical bone stress level was maintained in cases when the acetabular component was positioned at 10° or 15° anteversion and 40° or 45° abduction angles. The basic stress pathway above the acetabular dome was disturbed when the superior displacement of the hip centre exceeded 5 mm above the anatomical hip centre. Positioning the acetabular component correctly contributes to maintain the stress balance between the acetabular cup and the bone during acetabular reconstruction, thus helping restore the normal hip biomechanics and preserve the stability of the implants.     

Age-related and obstacle height-related differences in movements while stepping over obstacles

Background

This study aims to examine age-related and obstacle height-related differences in movements while stepping over obstacles.

Methods

The participants included 16 elderly and nine young women. Obstacles that were either 5 or 20 cm high were positioned at the center of a 4-m walking path. The participants were instructed to walk along the path as quickly as possible. The participants’ movements were analyzed using a three-dimensional motion analysis system that recorded their movements as they walked and stepped over the obstacles.

Results and conclusions

Seven joint angles and the distances between the ground and six markers were examined in the initial contact and swing instants of the leading and trailing limbs. In the initial contact instant, the elderly women prepared for stepping with a lower toe height than the young women when stepping over the 20-cm obstacle. Trunk rotation was greater in the young women than in the elderly women. In the swing instant, the elderly women showed greater ankle dorsiflexion and hip adduction angles for the leading limb when stepping over the 20-cm obstacle. They moved the trailing limb with increased ankle dorsiflexion, knee flexion, hip flexion, and foot inversion to ensure that they did not touch the obstacle as they stepped over it. These movement patterns are characteristic of elderly individuals who cannot easily lift their lower limbs because of decreased lower-limb strength.     

Calculation of the minimum energy conformation of biomolecules using a global optimization technique I. Methodology and application to a model molecular fragment (Normal pentane)

The conformational energy of a molecule is minimized with respect to interatomic distances using Bremermann's method of unconstrained global optimization (1970). The optimal set of distances is then used for calculating the preferred conformation of the molecule. A simultaneous optimization of all the dihedral angles is achieved. The classical potential function is used in this study. An illustration of the method is given by applying it to normal pentane, which is a commonly occurring fragment of biomolecules. Results show that, for the standard geometry (bond lengths and bond angles), the all-trans) conformation is the preferred one. However, fluctuations of the geometry within the limits of the vibrational spectra can lead to preferred conformations that are not necessarily all-trans.     

Statistical measures on residue-level protein structural properties

The atomic-level structural properties of proteins, such as bond lengths, bond angles, and torsion angles, have been well studied and understood based on either chemistry knowledge or statistical analysis. Similar properties on the residue-level, such as the distances between two residues and the angles formed by short sequences of residues, can be equally important for structural analysis and modeling, but these have not been examined and documented on a similar scale. While these properties are difficult to measure experimentally, they can be statistically estimated in meaningful ways based on their distributions in known proteins structures. Residue-level structural properties including various types of residue distances and angles are estimated statistically. A software package is built to provide direct access to the statistical data for the properties including some important correlations not previously investigated. The distributions of residue distances and angles may vary with varying sequences, but in most cases, are concentrated in some high probability ranges, corresponding to their frequent occurrences in either α-helices or β-sheets. Strong correlations among neighboring residue angles, similar to those between neighboring torsion angles at the atomic-level, are revealed based on their statistical measures. Residue-level statistical potentials can be defined using the statistical distributions and correlations of the residue distances and angles. Ramachandran-like plots for strongly correlated residue angles are plotted and analyzed. Their applications to structural evaluation and refinement are demonstrated. With the increase in both number and quality of known protein structures, many structural properties can be derived from sets of protein structures by statistical analysis and data mining, and these can even be used as a supplement to the experimental data for structure determinations. Indeed, the statistical measures on various types of residue distances and angles provide more systematic and quantitative assessments on these properties, which can otherwise be estimated only individually and qualitatively. Their distributions and correlations in known protein structures show their importance for providing insights into how proteins may fold naturally to various residue-level structures.     

A model of semitendinosus muscle sarcomere length, knee and hip joint interaction in the frog hindlimb

The interaction between the semitendinosus muscle and both hip and knee joint angles was examined in the frog (Rana pipiens) hindlimb. Sarcomere length was measured by laser diffraction in passive muscle during hip and knee rotation. A model was then developed to predict semitendinosus sarcomere length as a function of both hip and knee flexion angle. Based on published frog muscle fiber length-tension [Gordon, A. M. et al., J. Physiol. 184, 170-192 (1966)] and force-velocity [Edman, K. A. P., J. Physiol. 291, 143-159 (1979)] properties, and published joint angles during hopping [Calow, L. J. and Alexander, R. McN., J. Zool. (Lond.) 171, 293-321 (1973)], muscle sarcomere length, force and hip and knee torque during a hop were predicted. The semitendinosus muscle generally operated on the descending limb of the length-tension curve at normal joint angle combinations. The model predicted that, during a single coordinated movement, a period of sarcomere shortening (concentric) was followed by a period of sarcomere lengthening (eccentric). Based on calculated torque profiles at the hip and knee joints, this study suggested that the semitendinosus muscle probably functions more as a hip extensor than a knee flexor. In addition, based on the nature of the shortening-lengthening cycle, the semitendinosus may act to mechanically link the force of knee extension to hip extension.     

Solution structure of a cis-opened (10R)-N6-deoxyadenosine adduct of (9S,10R)-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in a DNA duplex

The solution structure of an 11-mer DNA duplex, d(CGGTCA*CGAGG) x d(CCTCGTGACCG), containing a 10R adduct at dA* that corresponds to the cis addition of the N(6)-amino group of dA(6) to (+)-(9S,10R)-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene was studied by 2D NMR methods. The NOESY cross-peak patterns indicate that the hydrocarbon is intercalated on the 5'-side of the modified base. This observation is the same as that observed for other oligonucleotides containing (10R)-dA adducts but opposite to that observed for the corresponding (10S)-dA adducts which are intercalated on the 3'-side of the modified base. The hydrocarbon is intercalated from the major groove without significant disruption of either the anti glycosidic torsion angle of the modified residue or the base pairing of the modified residue with the complementary residue on the opposite strand. The ensemble of 10 structures determined exhibits relatively small variations (6-15 degrees) in the characteristic hydrocarbon-base dihedral angles (alpha' and beta') as well as the glycosidic torsion angle chi. These angles are similar to those in a previously determined cis-opened benzo[a]pyrene diol epoxide-(10R)-dA adduct structure. Comparison of the present structure with the cis-opened diol epoxide adduct suggests that the absence of the 7- and 8-hydroxyl groups results in more efficient stacking of the aromatic moiety with the flanking base pairs and deeper insertion of the hydrocarbon into the helix. Relative to normal B-DNA, the duplex containing the present tetrahydroepoxide adduct is unwound at the lesion site, whereas the diol epoxide adduct structure is more tightly wound than normal B-DNA. Buckling of the adducted base pair as well as the C(5)-G(18) base pair that lies immediately above the hydrocarbon is much less severe in the present adducted structure than its cis-opened diol epoxide counterpart.     

Refinement of the solution structure of the DNA hexamer 5'd(GCATGC)2: combined use of nuclear magnetic resonance and restrained molecular dynamics

The solution structure of the self-complementary DNA hexamer 5'd(GCATGC)2 comprising the specific target site for the restriction endonuclease Sph 1 is investigated by using nuclear magnetic resonance spectroscopy and restrained molecular dynamics. All the nonexchangeable proton resonances are assigned sequentially, and from time-dependent nuclear Overhauser enhancement measurements a set of 158 approximate interproton distances are determined. These distances are used as the basis of a structure refinement using restrained molecular dynamics in which the interproton distances are incorporated into the total energy function of the system in the form of an effective potential term. Two restrained molecular dynamics simulations are carried out, starting from classical B- and A-DNA [atomic root mean square (rms) difference 3.3 A]. In both cases convergence is achieved to essentially identical structures satisfying the experimental restraints and having a root mean square difference of only 0.3 A between them, which is within the rms fluctuations of the atoms about their average positions. These results suggest that the restrained molecular dynamics structures represent reasonable approximations of the solution structure. The converged structures are of the B type and exhibit clear sequence-dependent variations of helical parameters, some of which follow Calladine's rules and can be attributed to the relief of interstrand purine-purine clash at adjacent base pairs. In addition, the converged restrained dynamics structures appear bent with a radius of curvature of approximately 20 A. This bending appears to be due almost entirely to the large positive base roll angles, particularly at the Pyr-Pur steps. Further, the global and local helix axes are not coincident, and the global helix axis represents a superhelical axis which the bent DNA, when extended into an "infinite" helix by repeated translation and rotation, wraps around.