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Biomedical subjects

P Schuck

Publications and source records attributed to P Schuck.

At least 19 recordsLinked to original sources
PubMed2001

Oligomeric structure of virion-associated and soluble forms of the simian immunodeficiency virus envelope protein in the prefusion activated conformation.

The envelope proteins (env) of simian immunodeficiency virus (SIV) and HIV type 1 assemble to form noncovalently associated oligomers in the endoplasmic reticulum. After cleavage in a Golgi compartment, oligomeric env complexes are transported to the surface of infected cells, where incorporation into budding virions can occur. Difficulties in obtaining adequate quantities of virions retaining env, as well as the unstable nature and hydrophobicity of the oligomer, may account for the absence of previous biophysical studies to determine the oligomeric valency of membrane-associated env. The aim of this study was to evaluate the oligomeric state of SIV env before membrane-fusion activation. Virion-associated env, obtained by crosslinking and detergent extraction, and non-crosslinked secreted env ectodomain (recombinant gp140) were purified by lentil-lectin chromatography and gel filtration as single predominant species. Sedimentation equilibrium-derived mass values for both forms of SIV env were close to those predicted for trimeric assemblies. Determination of the mass of individual molecules by scanning transmission electron microscopy confirmed that SIV virion-associated env and gp140 formed largely homogeneous populations of trimers. Furthermore, a triangular or tri-lobed morphology was clearly visualized in a subset of the trimers.

Biopolymers↗
PubMed2001

Alpha cluster condensation in 12C and 16O.

A new alpha-cluster wave function is proposed which is of the alpha-particle condensate type. Applications to 12C and 16O show that states of low density close to the 3 and 4 alpha-particle thresholds in both nuclei are possibly of this kind. It is conjectured that all self-conjugate 4n nuclei may show similar features.

Journal Article↗
PubMed2001

Class of exactly solvable pairing models.

We present three classes of exactly solvable models for fermion and boson systems, based on the pairing interaction. These models are solvable in any dimension. As an example we show the first results for fermions interacting with repulsive pairing forces in a two-dimensional square lattice. In spite of the repulsive pairing force the exact results show attractive pair correlations.

Journal Article↗
PubMed2001

Adaptation of a surface plasmon resonance biosensor with microfluidics for use with small sample volumes and long contact times.

The efficient delivery of sample to surface-immobilized sites is a key element in biosensing. For a surface plasmon resonance (SPR) biosensor, this has been addressed by constant flow through a microfluidic system with a sample injection loop (Sjölander, S.; Urbaniczky, C. Anal. Chem. 1991, 63, 2338-2345). The present study describes an alternative mode of sample delivery without constant unidirectional flow. It was implemented on a commercial Biacore X SPR biosensor equipped with a microfluidic cartridge, but with the fluidic handling performed by an externally computer-controlled syringe pump. We demonstrate that sample volumes as low as 2 microL can be reproducibly positioned to cover the sensor surfaces, manipulated in a serial fashion, efficiently mixed by applying an oscillatory flow pattern, and fully recovered. Compared to the traditional continuous unidirectional flow configuration, we found very similar kinetic responses at high analyte concentrations and slightly slower responses at low concentrations, most likely due to depletion of analyte from the small sample volumes due to surface binding. With the antibody-antigen systems tested, binding parameters were obtained that are generally within 10% of those from conventional experiments. In the new configuration, biosensor experiments can be conducted without the usual constraints in the surface contact time that are correlated with sample volume and mass transport rate. This can translate to improved detection limits for slow reactions and can facilitate kinetic and thermodynamic binding studies.

Biosensing Techniques↗
PubMed2001

[A hospital suited version of the German SF-36 and its psychometric comparison with the original questionnaire].

OBJECTIVES: The application of the Short-Form 36 Health Survey (SF-36) in hospitals is sometimes problematic, because some formulations like "at work" or "at home" in some items are not adequate in a hospital context. To account for this problem, we deleted 23 words like "at work" and "at home" from the SF-36 and generated thus a slightly modified version, the SF-36 m. The present paper deals primarily with two questions: is the SF-36 m well applicable in hospitals and is it still equivalent with the original version? METHODS: The SF-36 m was applied in more than 300 patients in two different hospitals. In one hospital a randomised design was used to assess the equivalence of SF-36 and SF-36 m before treatment. RESULTS: The reported high percentages of missing values in four critical scales were no more observed, when the SF-36 m was applied at the end of hospital stays. Only minor differences between SF-36 and SF-36 m with effect sizes < 0.20 were observed, although the scale "physical role function" and especially item 4d showed unexpectedly significantly higher values in the SF-36 m (p < 0.10). Additionally, higher and significant differences were observed between the applications of the questionnaires at home and in the hospital. CONCLUSIONS: Opposed to the original SF-36 the SF-36 m is well applicable at the end of hospital stays. SF-36 and SF-36 m show only minor differences in the comparison of their equivalence. The application of SF-36 and SF-36 m in hospitals seems to yield more favourable scores of health related quality of life than the application at home.

Chronic Disease↗
PubMed2001

Non-ideality by sedimentation velocity of halophilic malate dehydrogenase in complex solvents.

We have investigated the potential of sedimentation velocity analytical ultracentrifugation for the measurement of the second virial coefficients of proteins, with the goal of developing a method that allows efficient screening of different solvent conditions. This may be useful for the study of protein crystallization. Macromolecular concentration distributions were modeled using the Lamm equation with the approximation of linear concentration dependencies of the diffusion constant, D = D(o) (1 + k(D)c), and the reciprocal sedimentation coefficient s = s(o)/(1 + k(s)c). We have studied model distributions for their information content with respect to the particle and its non-ideal behavior, developed a strategy for their analysis by direct boundary modeling, and applied it to data from sedimentation velocity experiments on halophilic malate dehydrogenase in complex aqueous solvents containing sodium chloride and 2-methyl-2,4-pentanediol, including conditions near phase separation. Using global modeling for three sets of data obtained at three different protein concentrations, very good estimates for k(s) and s degrees and also for D degrees and the buoyant molar mass were obtained. It was also possible to obtain good estimates for k(D) and the second virial coefficients. Modeling of sedimentation velocity profiles with the non-ideal Lamm equation appears as a good technique to investigate weak inter-particle interactions in complex solvents and also to extrapolate the ideal behavior of the particle.

Diffusion↗
PubMed2001

Crystal structure of a superantigen bound to the high-affinity, zinc-dependent site on MHC class II.

MHC class II molecules possess two binding sites for bacterial superantigens (SAGs): a low-affinity site on the alpha chain and a high-affinity, zinc-dependent site on the beta chain. Only the former has been defined crystallographically. We report the structure of streptococcal pyrogenic exotoxin C (SPE-C) complexed with HLA-DR2a (DRA*0101, DRB5*0101) bearing a self-peptide from myelin basic protein (MBP). SPE-C binds the beta chain through a zinc bridge that links the SAG and class II molecules. Surprisingly, SPE-C also makes extensive contacts with the MBP peptide, such that peptide accounts for one third of the surface area of the MHC molecule buried in the complex, similar to TCR-peptide/MHC complexes. Thus, SPE-C may optimize T cell responses by mimicking the peptide dependence of conventional antigen presentation and recognition.

Animals↗
PubMed2000

Rotavirus nonstructural protein NSP2 self-assembles into octamers that undergo ligand-induced conformational changes.

The nonstructural protein NSP2 is a component of the rotavirus replication machinery and binds single-stranded RNA cooperatively, with high affinity, and independent of sequence. Recently, NSP2 has been shown to form multimers and to possess an NTPase activity, but its precise function remains unclear. In the present study, we have characterized the solution structure of recombinant NSP2 by velocity and equilibrium ultracentrifugation, dynamic light scattering, and circular dichroism spectroscopy. We found that NSP2 exists as an octamer, which is functional in the binding of RNA and ADP. In the presence of magnesium, a partial dissociation of the octamer into smaller oligomers was observed. This was reversed by binding of ADP and RNA. We observed an increased sedimentation rate in the presence of ADP and a nonhydrolyzable ATP analogue, which suggests a change toward a significantly more compact octameric conformation. The secondary structure of NSP2 showed a high fraction of beta-sheet, with small changes induced by magnesium that were reversed in the presence of RNA. That NSP2 can exist in different conformations lends support to the previously proposed hypothesis (Taraporewala, Z., Chen, D., and Patton, J. T. (1999) J. Virol. 73, 9934-9943) of its function as a molecular motor involved in the packaging of viral mRNA.

Adenosine Diphosphate↗
PubMed2000

Self-association of human apolipoprotein E3 and E4 in the presence and absence of phospholipid.

Human apolipoprotein E (apoE) exists as three main isoforms, differing by single amino acid substitutions, with the apoE4 isoform strongly linked to the incidence of late onset Alzheimer's disease. We have expressed and purified apoE3 and apoE4 from Escherichia coli and compared their hydrodynamic properties by gel permeation liquid chromatography, capillary electrophoresis, circular dichroism, and sedimentation methods. Sedimentation velocity experiments, employing a new method for determining the size distribution of polydisperse macromolecules in solution (Schuck, P. (2000) Biophys. J. 78, 1606-1619), provide direct evidence for the heterogeneous solution structures of apoE3 and apoE4. In a lipid-free environment, apoE3 and apoE4 exist as a slow equilibrium mixture of monomer, tetramer, octamer, and a small proportion of higher oligomers. Both sedimentation velocity and equilibrium experiments indicate that apoE4 has a greater propensity to self-associate. We also demonstrate that apoE3 and apoE4 oligomers dissociate significantly in the presence of dihexanoylphosphatidylcholine micelles (20 mm) and to a lesser extent at submicellar concentrations (4 mm). The alpha-helical content for both isoforms was almost identical (50%) in the presence and absence of dihexanoylphosphatidylcholine. These results reveal that apoE oligomers undergo phospholipid-induced dissociation to folded monomers, suggesting the monomeric form prevails on the lipoprotein surface in vivo.

Apolipoprotein E3↗
PubMed2000

Determination of the sedimentation coefficient distribution by least-squares boundary modeling.

A new method is presented for the calculation of apparent sedimentation coefficient distributions g*(s) for the size-distribution analysis of polymers in sedimentation velocity experiments. Direct linear least-squares boundary modeling by a superposition of sedimentation profiles of ideal nondiffusing particles is employed. It can be combined with algebraic noise decomposition techniques for the application to interference optical ultracentrifuge data at low loading concentrations with significant systematic noise components. Because of the use of direct boundary modeling, residuals are available for assessment of the quality of the fits and the consistency of the g*(s) distribution with the experimental data. The method can be combined with regularization techniques based on F statistics, such as used in the program CONTIN, or alternatively, the increment of s values can be adjusted empirically. The method is simple, has advantageous statistical properties, and reveals precise sedimentation coefficients. The new least-squares ls-g*(s) exhibits a very high robustness and resolution if data acquired over a large time interval are analyzed. This can result in a high resolution for large particles, and for samples with a high degree of heterogeneity. Because the method does not require a high frequency of scans, it can also be easily used in experiments with the absorbance optical scanning system. Published 2000 John Wiley & Sons, Inc.

Immunoglobulin G↗
PubMed2000

Analysis of transport experiments using pseudo-absorbance data.

The measurement of the concentration distribution of a macromolecule across a solution column by absorption optics usually requires optical transmission profiles of both the sample solution and the buffer, measured under identical conditions, to calculate the absorbance as the logarithm of the ratio of reference to sample intensity. For transport experiments, however, where the changes in the local macromolecule concentration with time are measured, a reference buffer intensity is not necessarily required. We demonstrate that the logarithm of the light transmitted through the sample solution, referred to as pseudo-absorbance, can suffice to determine macromolecular transport parameters of interest, with little loss of precision. Local changes in illumination of the sample column or in the detection efficiency of the transmitted light, as well as temporal fluctuations of the light source intensity can be well-described by consideration of time-invariant and radial-invariant signal components in the pseudo-absorbance data, using the systematic noise decomposition techniques developed recently (Schuck, P., and Demeler, B. (1999) Biophys. J. 76, 2288-2296). The practical use of the method is demonstrated with double-sector and single-sector sedimentation velocity experiments, and with analytical electrophoresis experiments. It is shown that pseudo-absorbance analysis can increase the capacity of a sedimentation velocity experiment in ultracentrifugation, and, in general, can considerably simplify the requirements of optical design.

Animals↗
PubMed2000

Calcium-sensitive interaction between calmodulin and modified forms of rat brain neurogranin/RC3.

Neurogranin (NG) binding of calmodulin (CaM) at its IQ domain is sensitive to Ca(2+) concentration and to modifications by protein kinase C (PKC) and oxidants. The PKC phosphorylation site of NG is within the IQ domain whereas the four oxidant-sensitive Cys residues are outside this region. These Cys residues were oxidized forming two pairs of intramolecular disulfides, and could also be glutathiolated by S-nitrosoglutathione resulting in the incorporation of four glutathiones per NG. Circular dichroism (CD) showed that modification of NG by phosphorylation, oxidation forming intramolecular disulfides, or glutathiolation did not affect the alpha-helical content of this protein. Mutation of the four Cys residues [Cys(-)-NG] to Gly and Ser did not affect the alpha-helical content either. Interaction of CaM with the reduced (red)-, glutathiolated (GS)-, or Cys(-)-NG in the Ca(2+)-free solution resulted in an increase in the alpha-helicity determined by their CD spectra, but relatively little change was seen with the oxidized NG (ox-NG) or phosphorylated NG (PO(4)-NG). The binding affinities between the various modified forms of NG and CaM were determined by CD spectrometry and sedimentation equilibrium: their affinities were Cys(-)-NG > red-NG, GS-NG > ox-NG > PO(4)-NG. Unlike Cys(-)-, red-, and GS-NG, neither ox- nor PO(4)-NG bound to a CaM-affinity column. Thus, both oxidation of NG to form intramolecular disulfides and phosphorylation of NG by PKC are effective in modulating the intracellular level of CaM. These results indicate that modification of NG to form intramolecular disulfides outside the IQ domain provides an alternative mechanism for regulation of its binding affinity to CaM.

Animals↗
PubMed2000

Crystal structure of an NK cell immunoglobulin-like receptor in complex with its class I MHC ligand.

Target cell lysis is regulated by natural killer (NK) cell receptors that recognize class I MHC molecules. Here we report the crystal structure of the human immunoglobulin-like NK cell receptor KIR2DL2 in complex with its class I ligand HLA-Cw3 and peptide. KIR binds in a nearly orthogonal orientation across the alpha1 and alpha2 helices of Cw3 and directly contacts positions 7 and 8 of the peptide. No significant conformational changes in KIR occur on complex formation. The receptor footprint on HLA overlaps with but is distinct from that of the T-cell receptor. Charge complementarity dominates the KIR/HLA interface and mutations that disrupt interface salt bridges substantially diminish binding. Most contacts in the complex are between KIR and conserved HLA-C residues, but a hydrogen bond between Lys 44 of KIR2DL2 and Asn 80 of Cw3 confers the allotype specificity. KIR contact requires position 8 of the peptide to be a residue smaller than valine. A second KIR/HLA interface produced an ordered receptor-ligand aggregation in the crystal which may resemble receptor clustering during immune synapse formation.

Amino Acid Sequence↗
PubMed2000

Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling.

A new method for the size-distribution analysis of polymers by sedimentation velocity analytical ultracentrifugation is described. It exploits the ability of Lamm equation modeling to discriminate between the spreading of the sedimentation boundary arising from sample heterogeneity and from diffusion. Finite element solutions of the Lamm equation for a large number of discrete noninteracting species are combined with maximum entropy regularization to represent a continuous size-distribution. As in the program CONTIN, the parameter governing the regularization constraint is adjusted by variance analysis to a predefined confidence level. Estimates of the partial specific volume and the frictional ratio of the macromolecules are used to calculate the diffusion coefficients, resulting in relatively high-resolution sedimentation coefficient distributions c(s) or molar mass distributions c(M). It can be applied to interference optical data that exhibit systematic noise components, and it does not require solution or solvent plateaus to be established. More details on the size-distribution can be obtained than from van Holde-Weischet analysis. The sensitivity to the values of the regularization parameter and to the shape parameters is explored with the help of simulated sedimentation data of discrete and continuous model size distributions, and by applications to experimental data of continuous and discrete protein mixtures.

Confidence Intervals↗
PubMed2000

The human immunodeficiency virus type 1 gp120 V2 domain mediates gp41-independent intersubunit contacts.

The envelope protein of human immunodeficiency virus type 1 HIV-1 undergoes proteolytic cleavage in the Golgi complex to produce subunits designated gp120 and gp41, which remain noncovalently associated. While gp41 has a well-characterized oligomeric structure, the maintenance of gp41-independent gp120 intersubunit contacts remains a contentious issue. Using recombinant vaccinia virus to achieve high-level expression of gp120 in mammalian cells combined with gel filtration analysis, we were able to isolate a discrete oligomeric form of gp120. Oligomerization of gp120 occurred intracellularly between 30 and 120 min after synthesis. Analysis by sedimentation equilibrium unequivocally identified the oligomeric species as a dimer. In order to identify the domains involved in the intersubunit contact, we expressed a series of gp120 proteins lacking various domains and assessed the effects of mutation on oligomeric structure. Deletion of the V1 or V3 loops had little effect on the relative amounts of monomer and dimer in comparison to wild-type gp120. In contrast, deletion of either all or part of the V2 loop drastically reduced dimer formation, indicating that this domain is required for intersubunit contact formation. Consistent with this, the V2 loop of the dimer was less accessible than that of the monomer to a specific monoclonal antibody. Previous studies have shown that while the V2 loop is not an absolute requirement for viral entry, the absence of this domain reduces viral resistance to neutralization by monoclonal antibodies or sera. We propose that the quaternary structure of gp120 may contribute to resistance to neutralization by limiting the exposure of conserved epitopes.

Cell Line↗
PubMed1999

Salt links dominate affinity of antibody HyHEL-5 for lysozyme through enthalpic contributions.

The binding of murine monoclonal antibody HyHEL-5 to lysozyme has been the subject of extensive crystallographic, computational, and experimental investigations. The complex of HyHEL-5 with hen egg lysozyme (HEL) features salt bridges between Fab heavy chain residue Glu(50), and Arg(45) and Arg(68) of HEL. This interaction has been predicted to play a dominant role in the association on the basis of molecular electrostatics calculations. The association of aspartic acid and glutamine mutants at position 50(H) of the cloned HyHEL-5 Fab with HEL and bobwhite quail lysozyme (BQL), an avian variant bearing an Arg(68) --> Lys substitution in the epitope, was characterized by isothermal titration calorimetry and sedimentation equilibrium. Affinities for HEL were reduced by 400-fold (E50(H)D) and 40,000-fold (E50(H)Q) (DeltaDeltaG degrees estimated at 4.0 and 6.4 kcal mol(-1), respectively). The same mutations reduce affinity for BQL by only 7- and 55-fold, respectively, indicating a reduced specificity for HEL. The loss of affinity upon mutation is in each case primarily due to an unfavorable change in the enthalpy of the interaction; the entropic contribution is virtually unchanged. An enthalpy-entropy compensation exists for each interaction; DeltaH degrees decreases, while DeltaS degrees increases with temperature. The DeltaCp for each mutant interaction is less negative than the wild-type. Mutant-cycle analysis suggests the mutations present in the HyHEL-5 Fab mutants are linked to those present in the BQL with coupling energies between 3 and 4 kcal mol(-1).

Animals↗
PubMed1999

Sedimentation equilibrium analysis of interference optical data by systematic noise decomposition.

An analytical method is described for removal of systematic signal offsets from interference optical data of sedimentation equilibrium gradients. It is demonstrated that the time-invariant signal contributions can be extracted from hydrodynamic modeling of interference profiles acquired during the approach to sedimentation equilibrium. This method is based on a technique for the explicit algebraic calculation of time-invariant noise components from sedimentation data, recently described for the direct modeling of sedimentation velocity experiments (P. Schuck and B. Demeler, Biophys. J. 76, 2288-2296, 1999). The calculated systematic signal offset is very well defined by the experimental data, stable over time, and its calculation is robust and to a large extent independent of the hydrodynamic model. The calculated time-invariant signal can be used to reduce the systematic errors in the measured sedimentation equilibrium profiles by more than an order of magnitude. It is shown that the resulting net equilibrium fringe profiles after subtraction of the time-invariant noise component allow equilibrium analyses consistent with those obtained from absorbance profiles. However, due to a higher dynamic range and the higher number of data points, the parameters derived from the net interference analysis can exhibit significantly improved precision. The presented study demonstrates the feasibility and potential of this analytical method for full exploitation of the remarkable precision of the interference optical data acquisition system, allowing sedimentation equilibrium experiments at loading concentrations below 0.05 mg/ml.

Histocompatibility Antigens Class I↗