Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), in combination with proteolytic protection assays, has been used to identify the functional epitope on human immunodeficiency virus envelope glycoprotein gp41 for the broadly neutralizing anti-gp41 human monoclonal antibody 2F5. In this protection assay-based procedure, a soluble gp140 protein with a stabilizing intermolecular disulfide bond between the gp120 and gp41 subunits (SOS gp140) was affinity bound to immobilized 2F5 under physiological conditions. A combination of proteolytic enzymatic cleavages was then performed to remove unprotected residues. Residues of SOS gp140 protected by their binding to 2F5 were then identified based on their molecular weights as determined by direct MALDI-MS of the immobilized antibody beads. The epitope, NEQELLELDKWASLWN, determined by this MALDI-MS protection assay approach consists of 16 amino acid residues near the C terminus of gp41. It is significantly longer than the ELDKWA core epitope previously determined for 2F5 by peptide enzyme-linked immunosorbent assay. This new knowledge of the structure of the 2F5 epitope may facilitate the design of vaccine antigens intended to induce antibodies with the breadth and potency of action of the 2F5 monoclonal antibody.

Spin polarized transient EPR spectra taken at X-band (9 GHz) and K-band (24 GHz) of membrane fragments of Chlorobium tepidum and Heliobacillus mobilis are presented along with the spectra of two fractions obtained in the purification of reaction centers (RC) from C. tepidum. The lifetime of P+. is determined by measuring the decay of the EPR signals following relaxation of the initial spin polarization. All samples except one of the RC fractions show evidence of light induced charge separation and formation of chlorophyll triplet states. The lifetime of P+. is found to be biexponential with components of 1.5 ms and 30 ms for C. tepidum and 1.0 and 4.5 ms for Hc. mobilis at 100 K. In both cases, the rates are assigned to recombination from F-X. The spin polarized radical pair spectra for both species are similar and those from Hc. mobilis at room temperature and 100 K are identical. In all cases, an emission/absorption polarization pattern with a net absorption is observed. A slight narrowing of the spectra and a larger absorptive net polarization is found at K-band. No out-of-phase echo modulation is observed. Taken together, the recombination kinetics, the frequency dependence of the spin polarization and the absence of an out-of-phase echo signal lead to the assignment of the spectra to the contribution from P+. to the state P+.F-X. The origin of the net polarization and its frequency dependence are discussed in terms of singlet-triplet mixing in the precursor. It is shown that the field-dependent polarization expected to develop during the 600-700 ps lifetime of P+.A-.0 is in qualitative agreement with the observed spectra. The identity that the acceptor preceding FX and the conflicting evidence from EPR, optical methods and chemical analyses of the samples are discussed.

Mass spectrometry has evolved as a technique suitable for the characterization of peptides and proteins beyond their linear sequence. The advantages of mass spectrometric sample analysis are high sensitivity, high mass accuracy, rapid analysis time and low sample consumption. In epitope mapping, the molecular structure of an antigen (the epitope or antigenic determinant) that interacts with the paratope (recognition surface) of the antibody is identified. To obtain information on linear, conformational and/or discontinuous epitopes, various approaches have been developed in conjunction with mass spectrometry. These methods include limited proteolysis and epitope footprinting, epitope excision and epitope extraction for linear epitopes and probing the surface accessibility of residues by differential chemical modifications of specific amino acid side chains or by differential hydrogen/deuterium exchange of the protein backbone amides for conformational and discontinuous epitopes. Epitope mapping by mass spectrometry is applicable in basic biochemical research and, with increasing robustness, should soon find its implementation in routine clinical diagnosis.

Simple procedures for the anaerobic preparation of photoactive and stable P840 reaction centers from Chlorobium tepidum and Chlorobium limicola in good yield are presented and quantitated. The subunit composition was tested by cosedimentation in sucrose density gradients. For C. limicola, it minimally comprises four subunits: the P840 reaction center protein PscA, the BChla antenna protein FMO, the FeS protein PscB with centers A and B, and a positively charged 17-kDa protein denoted PscD. The preparation from Chlorobium tepidum additionally contained PscC, a cytochrome c-551. The BChla absorption peak of the purified complexes was at 810 nm, with a shoulder at 835 nm. The ratio of the shoulder to the peak was 0.25, which corresponds to 1 reaction center per 70 BChla molecules if a uniform extinction coefficient of BChla is assumed. However, bleaching at 610 nm in continuous light corresponded up to 1 photoactive reaction center per 50 BChla molecules. Therefore, either the extinction coefficient of BChla in the reaction center is overestimated or the one for photobleaching is underestimated. In any case, the major portion of the reaction center was photoactive in the preparations. A P840 reaction center subcomplex, lacking PscD and deficient in FMO and PscB, but retaining the cytochrome c subunit, was obtained as a side product. It was photoinactive and had an absorption peak at 814 nm and a 835/814 absorbance ratio of 0.42. FMO and PscB show the tendency to form a complementary subcomplex. FMO and PscD are apparently required to stabilize the photoactive reaction center, while the cytochrome c subunit is not.