Fine definition of the epitope on the gp41 glycoprotein of human immunodeficiency virus type 1 for the neutralizing monoclonal antibody 2F5.

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.

Electron transfer in reaction center core complexes from the green sulfur bacteria Prosthecochloris aestuarii and Chlorobium tepidum.

Electron transfer in reaction center core (RCC) complexes from the green sulfur bacteria Prosthecochloris aestuarii and Chlorobium tepidum was studied by measuring flash-induced absorbance changes. The first preparation contained approximately three iron-sulfur centers, indicating that the three putative electron acceptors F(X), F(A), and F(B) were present; the Chl. tepidum complex contained on the average only one. In the RCC complex of Ptc. aestuarii at 277 K essentially all of the oxidized primary donor (P840(+)) created by a flash was rereduced in several seconds by N-methylphenazonium methosulfate. In RCC complexes of Chl. tepidum two decay components, one of 0.7 ms and a smaller one of about 2 s, with identical absorbance difference spectra were observed. The fast component might be due to a back reaction of P840(+) with a reduced electron acceptor, in agreement with the notion that the terminal electron acceptors, F(A) and F(B), were lost in most of the Chl. tepidum complexes. In both complexes the terminal electron acceptor (F(A) or F(B)) could be reduced by dithionite, yielding a back reaction of 170 ms with P840(+). At 10 K in the RCC complexes of both species P840(+) was rereduced in 40 ms, presumably by a back reaction with F(X)(-). In addition, a 350 micros component occurred that can be ascribed to decay of the triplet of P840, formed in part of the complexes. For P840(+) rereduction a pronounced temperature dependence was observed, indicating that electron transfer is blocked after F(X) at temperatures below 200 K.

Composition and optical properties of reaction centre core complexes from the green sulfur bacteria Prosthecochloris aestuarii and Chlorobium tepidum.

Photosynthetically active reaction centre core (RCC) complexes were isolated from two species of green sulfur bacteria, Prosthecochloris (Ptc.) aestuarii strain 2K and Chlorobium (Chl.) tepidum, using the same isolation procedure. Both complexes contained the main reaction centre protein PscA and the iron-sulfur protein PscB, but were devoid of Fenna-Matthews-Olson (FMO) protein. The Chl. tepidum RCC preparation contained in addition PscC (cytochrome c). In order to allow accurate determination of the pigment content of the RCC complexes, the extinction coefficients of bacteriochlorophyll (BChl) a in several solvents were redetermined with high precision. They varied between 54.8 mM(-1) cm(-1) for methanol and 97.0 mM(-1) cm(-1) for diethylether in the Q(Y) maximum. Both preparations appeared to contain 16 BChls a of which two are probably the 13(2)-epimers, 4 chlorophylls (Chls) a 670 and 2 carotenoids per RCC. The latter were of at least two different types. Quinones were virtually absent. The absorption spectra were similar for the two species, but not identical. Eight bands were present at 6 K in the BChl a Q(Y) region, with positions varying from 777 to 837 nm. The linear dichroism spectra showed that the orientation of the BChl a Q(Y) transitions is roughly parallel to the membrane plane; most nearly parallel were transitions at 800 and 806 nm. For both species, the circular dichroism spectra were dominated by a strong band at 807-809 nm, indicating strong interactions between at least some of the BChls. The absorption, CD and LD spectra of the four Chls a 670 were virtually identical for both RCC complexes, indicating that their binding sites are highly conserved and that they are an essential part of the RCC complexes, possibly as components of the electron transfer chain. Low temperature absorption spectroscopy indicated that typical FMO-RCC complexes of Ptc. aestuarii and Chl. tepidum contain two FMO trimers per reaction centre.

Transient electron paramagnetic resonance spectroscopy on green-sulfur bacteria and heliobacteria at two microwave frequencies.

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.

Stable photobleaching of P840 in Chlorobium reaction center preparations: presence of the 42-kDa bacteriochlorophyll a protein and a 17-kDa polypeptide.

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.