Search

To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used.

Protein-protein interactions identified through high-throughput proteomics efforts continue to advance our understanding of the protein interactome. In addition to highly specific protein-protein interactions, it is becoming increasingly more common for yeast two-hybrid, pull-down assays, and other proteomics techniques to identify multiple protein ligands that bind to the same target protein. A resulting challenge is to accurately characterize the assembly of these multiprotein complexes and the competition among multiple protein ligands for a given target. The Association of Biomolecular Resource Facilities-Molecular Interactions Research Group recently conducted a benchmark study to assess participants' ability to correctly describe the interactions between two protein ligands and their target protein using primarily biosensor technologies, such as surface plasmon resonance. Participants were provided with microgram quantities of three proteins (A, B, and C) and asked to determine if a ternary A-B-C complex can form or if protein-B and protein-C bind competitively to protein-A. This article will summarize the experimental approaches taken by participants to characterize the molecular interactions, the interpretation of the data, and the results obtained using different biosensor instruments. [ABSTRACT FROM AUTHOR]

In this study, the sperm protein profile was compared between fertile and infertile men using 2-dimensional gel electrophoresis, liquid chromatography mass spectrometer analysis, and matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry. Four unique proteins, semenogelin II precursor, prolactin-induced protein, clusterin isoform 1, and prostate-specific antigen isoform 1 preproprotein, were predominantly present in the semen of healthy men; however, semenogelin II precursor and clusterin isoform 1 were not seen in the semen of infertile men, suggesting unique differences in the spermatozoa protein profiles of fertile and infertile men. [ABSTRACT FROM AUTHOR]

Soluble oligomers of Aβ42 peptide are believed to play a major role in the pathogenesis of Alzheimer disease (AD). It was recently found that at least some of the neurotoxic effects of these oligomers may be mediated by specific binding to the prion protein, PrPC, on the cell surface (Laurén, J., Gimbel, D. A., Nygaard, H. B., Gilbert, J. W., and Strittmatter, S. M. (2009) Nature 457, 1128-1132). Here we characterized the interaction between synthetic Aβ42 oligomers and the recombinant human prion protein (PrP) using two biophysical techniques: site-directed spin labeling and surface plasmon resonance. Our data indicate that this binding is highly specific for a particular conformation adopted by the peptide in soluble oligomeric species. The binding appears to be essentially identical for the Met129 and Val129 polymorphic forms of human PrP, suggesting that the role of PrP codon 129 polymorphism as a risk factor in AD is due to factors unrelated to the interaction with Aβ oligomers. It was also found that in addition to the previously identified ∼95-110 segment, the second region of critical importance for the interaction with Aβ42 oligomers is a cluster of basic residues at the extreme N terminus of PrP (residues 23-27). The deletion of any of these segments results in a major loss of the binding function, indicating that these two regions likely act in concert to provide a high affinity binding site for Aβ42 oligomers. This insight may help explain the interplay between the postulated protective and pathogenic roles of PrP in AD and may contribute to the development of novel therapeutic strategies as well. [ABSTRACT FROM AUTHOR]

The leukocyte integrin αMβ2 (Mac-1) is a multiligand receptor that mediates a range of adhesive reactions of leukocytes during the inflammatory response. This integrin binds the coagulation protein fibrinogen providlng a key link between thrombosis and inflammation.However, the mechanism by which αMβ2 binds fibrino-gen remains unknown. Previous studies indicated that a model in which two fibrinogen γC domain sequences, P1 (γ190-202) and P2 (γ377-395), serve as the αMβ2 binding sites cannot fully account for recognition of fibrinogen by integrin. Here, using surface plasmon resonance, we examined the interaction of the ligand binding αMI-domain of αMβ2 with the D fragment of fibrinogen and showed that this ligand is capable of associating with several αMI-domain molecules. To localize the alternatire αMI-domain binding sites, we screened peptide libraries covering the complete sequences of the γC and βC domains, comprising the majority of the D fragment structure, for αMI-domain binding. In addition to the P2 and P1 peptides, the αMI-domain bound to many other sequences in the γC and βC scans. Similar to P1 and P2, synthetic peptides derived from γC and βC were efficient inhibitors of αMβ2-mediated cell adhesion and were able to directly support adhesion suggesting that they contain identical recognition information. Analyses of recognition specificity using substitutional peptide libraries demonstrated that the αMI-domain binding depends on basic and hydrophobic residues. These findings establish a new model of αMβ2 binding in which the αMI-domain interacts with multiple sites in fibrinogen and has the potential to recognize numerous sequences. This paradigm may have implications for mechanisms of promiscuity in ligand binding exhibited by integrin αMβ2. [ABSTRACT FROM AUTHOR]