Possible three-dimensional backbone folding around antibody combining site of immunoglobulin MOPC167

Using a recently developed method of predicting possible three dimensional foldings of immunoglobulin backbones around antibody combining sites, we have attempted to construct the structure of immunoglobulin MOPC167 which could bind phosphorylcholine. A small pocket was present in the middle of the predicted structure similar to that of another phosphorylcholine binding immunoglobulin, McPC603, the tertiary structure of which has been determined by X-ray diffraction studies. Although detailed atomic co-ordinates of McPC603 were not available, a rough comparison between these structures strongly suggested that the foldings of complementarity determining regions were alike only for those with identical lengths and similar amino acid sequences. The predicted structure of MOPC167 as compared with the previously predicted structure of MOPC315, a 2,4-dinitrophenol binding immunoglobulin, further suggested that the light chain of one immunoglobulin could indeed combine with the heavy chain of another immunoglobulin, but might not result in a reasonable site for binding antigens.     

A novel method for mapping antibody combining sites by 1H nuclear magnetic resonance spectroscopy

A series of paramagnetic probes for Dnp binding antibodies has been developed by cupling a metal chelating group onto the immunodominant Dnp-group. The approach is illustrated for protein 315. The haptens used in the present study are of the form N-Dnp-2-aminoethyl (phosphate)_n and the paramagnetic ion chosen in Mn(II). The use of proton high resolution nuclear magnetic resonances (n.m.r.) difference spectroscopy allows quantitative measurements of the perturbation caused by the Mn(II) on several of the protein resonances. The sum of the distances between the metal and the ring centres of the two histidine residues, His 97_L and His 102_H, in the third hypervariable regions of the light (L) and heary (H) chain is found to be 20 ± 1 Å. This is in good agreement with the value of 19.4 a between the two histidines calculated from the coordinates of the predicted model of padlan et. al.⁴     

A novel method for sampling alpha-helical protein backbones

We present a novel technique of sampling the configurations of helical proteins. Assuming knowledge of native secondary structure, we employ assembly rules gathered from a database of existing structures to enumerate the geometrically possible three-dimensional arrangements of the constituent helices. We produce a library of possible folds for 25 helical protein cores. In each case, our method finds significant numbers of conformations close to the native structure. In addition, we assign coordinates to all atoms for four of the 25 proteins and show that this has a small effect on the number of near-native conformations. In the context of database driven exhaustive enumeration our method performs extremely well, yielding significant percentages of conformations (between 0.02% and 82%) within 6 A of the native structure. The method's speed and efficiency make it a valuable tool for predicting protein structure.     

A method for the characterization of foldings in protein ribbon models

The ribbon model of chain macromolecules is a useful tool for analyzing some of the targe-scale shape features of these complex systems. Up to now, the ribbon model has been used mostly to produce graphical displays, which are usually analyzed by visual inspection. In this work we suggest a computational method for characterizing automatically, in a concise and algebraic fashion, some of the important shape features of these ribbon models. The procedure is based on a graph-theoretical and knot-theoretical characterization of three well-defined projections of a space curve associated with the ribbon. The labeled graphs can be characterized by the handedness of the crossovers in the ribbon that are the vertices of the graph. The method can be used to provide a fully algebraic representation of the changes occurring when a molecule, such as a protein, undergoes conformational rearrangements (folding), as well as to provide a shape comparison for a pair of related molecular ribbons. This algebraic representation is well suited for easy storage, retrieval, and computer manipulation of the information on the ribbon's shape. Illustrative examples of the method are provided.     

Protein-lipid interactions: correlation of a predictive algorithm for lipid-binding sites with three-dimensional structural data

Background  

Over the past decade our laboratory has focused on understanding how soluble cytoskeleton-associated proteins interact with membranes and other lipid aggregates. Many protein domains mediating specific cell membrane interactions appear by fluorescence microscopy and other precision techniques to be partially inserted into the lipid bilayer. It is unclear whether these protein-lipid-interactions are dependent on shared protein motifs or unique regional physiochemistry, or are due to more global characteristics of the protein.     

Analysis of correlated motion in antibody combining sites from molecular dynamics simulations

The crystal structures of the NC6.8-antisweet taste ligand complex and the uncomplexed antibody structures display significant differences in the conformations of residues in the combining site. A molecular dynamics method was employed to understand the flexibility and correlated motion of key combining site residues in the uncomplexed antibody. The simulations reveal that residues that show conformational differences between the complex and uncomplexed structures display strong dynamical correlations. Extensive analysis of the dynamics trajectory using time correlation methods is presented.     

A method for determining tissue sulfate

A method for the determination of the inorganic sulfate present in rat liver homogenates has been developed. In order to determine sulfate, a protein-free extract is required. The classical protein precipitation methods of preparing protein-free extracts gave 2.5–40% recovery of added ³⁵SO₄^2?. Separation of the protein by ultrafiltration gave only 29% recovery when 0.15 m KCl was the homogenizing medium. A homogenization medium containing 0.154 m NH₄OH and 20 g EDTA per liter gave 102 ± 11% recovery of added ³⁵SO₄^2? when the protein was separated by ultrafiltration.     

A graphical method for determining inhibition constants

A new simple graphical method is described for the determination of inhibition type and inhibition constants of an enzyme reaction without any replot. The method consists of plotting experimental data as (V–v)/v versus the inhibitor concentration at two or more concentrations of substrate, where V and v represent the maximal velocity and the velocity in the absence and presence of inhibitor with given concentrations of the substrate, respectively. Competitive inhibition gives straight lines that converge on the abscissa at a point where [I]?=??K_i. Uncompetitive inhibition gives parallel lines with the slope of 1/K’_i. For mixed type inhibition, the intersection in the plot is given by [I]?=??K_i and (V–v)/v?=??K_i/K’_i in the third quadrant, and in the special case where K_i?=?K’_i (noncompetitive inhibition) the intersections occur at the point where [I]?=??K_i and (V–v)/v?=??1. The present method, the “quotient velocity plot,” provides a simple way of determining the inhibition constants of all types of inhibitors.     

A method for determining phospholipases in microorganisms

A method is suggested for the determination of bacteria phospholipases in dense nutrient medium. The medium contains the egg-yolk solution, buffer pH 9.2, meat extract, calcium chloride and toluidine blue. The enzyme activity is estimated by the diameter value of the medium clarification zone around the bacterial mass inoculation by injection into an agar plate. It is possible to study 6-8 strains on one Petri dish. This method is a simple one and thus it can be used in the bacteriological practice when determining phospholipases of bacteria, especially in strains of those species where this enzyme is a pathogenicity factor.