Transformation of aqueous protein attenuated total reflectance infra-red absorbance spectroscopy to transmission.

Infrared (IR) spectroscopy is increasingly being used to probe the secondary structure of proteins, especially for high-concentration samples and biopharmaceuticals in complex formulation vehicles. However, the small path lengths required for aqueous protein transmission experiments, due to high water absorbance in the amide I region of the spectrum, means that the path length is not accurately known, so only the shape of the band is ever considered. This throws away a dimension of information. Attenuated total reflectance (ATR) IR spectroscopy is much easier to implement than transmission IR spectroscopy and, for a given instrument and sample, gives reproducible spectra. However, the ATR-absorbance spectrum varies with sample concentration and instrument configuration, and its wavenumber dependence differs significantly from that observed in transmission spectroscopy. In this paper, we determine, for the first time, how to transform water and aqueous protein ATR spectra into the corresponding transmission spectra with appropriate spectral shapes and intensities. The approach is illustrated by application to water, concanavalin A, haemoglobin and lysozyme. The transformation is only as good as the available water refractive index data. A hybrid of literature data provides the best results. The transformation also allows the angle of incidence of an ATR crystal to be determined. This opens the way to using both spectral shape and spectra intensity for protein structure fitting.

The MPB83 antigen from Mycobacterium bovis contains O-linked mannose and (1-->3)-mannobiose moieties.

Mycobacterium tuberculosis and Mycobacterium bovis, the causative agents of human and bovine tuberculosis, have been reported to express a range of surface and secreted glycoproteins, although only one of these has been subjected to detailed structural analysis. We describe the use of a genetic system, in conjunction with lectin binding, to characterize the points of attachment of carbohydrate moieties to the polypeptide backbone of a second mycobacterial glycoprotein, antigen MPB83 from M. bovis. Biochemical and structural analysis of the native MPB83 protein and derived peptides demonstrated the presence of 3 mannose units attached to two threonine residues. Mannose residues were joined by a (1 --> 3) linkage, in contrast to the (1 --> 2) linkage previously observed in antigen MPT32 from M. tuberculosis and the (1 --> 2) and (1 --> 6) linkages in other mycobacterial glycolipids and polysaccharides. The identification of glycosylated antigens within the M. tuberculosis complex raises the possibility that the carbohydrate moiety of these glycoproteins might be involved in pathogenesis, either by interaction with mannose receptors on host cells, or as targets or modulators of the cell-mediated immune response. Given such a possibility characterization of mycobacterial glycoproteins is a step toward understanding their functional role and elucidating the mechanisms of mycobacterial glycosylation.

Identification of major outer surface proteins of Streptococcus agalactiae.

To identify the major outer surface proteins of Streptococcus agalactiae (group B streptococcus), a proteomic analysis was undertaken. An extract of the outer surface proteins was separated by two-dimensional electrophoresis. The visualized spots were identified through a combination of peptide sequencing and reverse genetic methodologies. Of the 30 major spots identified as S. agalactiae specific, 27 have been identified. Six of these proteins, previously unidentified in S. agalactiae, were sequenced and cloned. These were ornithine carbamoyltransferase, phosphoglycerate kinase, nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase, purine nucleoside phosphorylase, enolase, and glucose-6-phosphate isomerase. Using a gram-positive expression system, we have overexpressed two of these proteins in an in vitro system. These recombinant, purified proteins were used to raise antisera. The identification of these proteins as residing on the outer surface was confirmed by the ability of the antisera to react against whole, live bacteria. Further, in a neonatal-animal model system, we demonstrate that some of these sera are protective against lethal doses of bacteria. These studies demonstrate the successful application of proteomics as a technique for identifying vaccine candidates.