Escherichia coli peptide binding protein OppA has a preference for positively charged peptides.

The Escherichia coli peptide binding protein OppA is an essential component of the oligopeptide transporter Opp. Based on studies on its orthologue from Salmonella typhimurium, it has been proposed that OppA binds peptides between two and five amino acids long, with no apparent sequence selectivity. Here, we studied peptide binding to E. coli OppA directly and show that the protein has an unexpected preference for basic peptides. OppA was expressed in the periplasm, where it bound to available peptides. The protein was purified in complex with tightly bound peptides. The crystal structure (up to 2.0 Å) of OppA liganded with the peptides indicated that the protein has a preference for peptides containing a lysine. Mass spectrometry analysis of the bound peptides showed that peptides between two and five amino acids long bind to the protein and indeed hinted at a preference for positively charged peptides. The preference of OppA for peptides with basic residues, in particular lysines, was corroborated by binding studies with peptides of defined sequence using isothermal titration calorimetry and intrinsic protein fluorescence titration. The protein bound tripeptides and tetrapeptides containing positively charged residues with high affinity, whereas related peptides without lysines/arginines were bound with low affinity. A structure of OppA in an open conformation in the absence of ligands was also determined to 2.0 Å, revealing that the initial binding site displays a negative surface charge, consistent with the observed preference for positively charged peptides. Taken together, E. coli OppA appears to have a preference for basic peptides.

Probing the self-assembly and the accompanying structural changes of hydrophobin SC3 on a hydrophobic surface by mass spectrometry.

The fungal class I hydrophobin SC3 self-assembles into an amphipathic membrane at hydrophilic-hydrophobic interfaces such as the water-air and water-Teflon interface. During self-assembly, the water-soluble state of SC3 proceeds via the intermediate alpha-helical state to the stable end form called the beta-sheet state. Self-assembly of the hydrophobin at the Teflon surface is arrested in the alpha-helical state. The beta-sheet state can be induced at elevated temperature in the presence of detergent. The structural changes of SC3 were monitored by various mass spectrometry techniques. We show that the so-called second loop of SC3 (C39-S72) has a high affinity for Teflon. Binding of this part of SC3 to Teflon was accompanied by the formation of alpha-helical structure and resulted in low solvent accessibility. The solvent-protected region of the second loop extended upon conversion to the beta-sheet state. In contrast, the C-terminal part of SC3 became more exposed to the solvent. The results indicate that the second loop of class I hydrophobins plays a pivotal role in self-assembly at the hydrophilic-hydrophobic interface. Of interest, this loop is much smaller in case of class II hydrophobins, which may explain the differences in their assembly.

A bacteriochlorophyll a antenna complex from purple bacteria absorbing at 963 nm.

A recently isolated species of the photosynthetic purple sulfur bacteria, provisionally called strain 970, was investigated with respect to its antenna function by means of various spectroscopic techniques, including fluorescence and pump-probe absorption difference spectroscopy. The bacterium contains bacteriochlorophyll a and an as yet unidentified carotenoid, perhaps 3,4,3',4'-tetrahydrospirilloxanthin. It has a single antenna complex of the LH1 type, with a Q(y) absorption band situated at the unusually long wavelength of 963 nm at room temperature and 990 nm at 6 K. In contrast to many other species, the reaction center showed two well-separated absorption bands of bacteriopheophytin at 6 K, located at 747 and 762 nm. The primary electron donor showed a bleaching band centered at 925 nm upon photooxidation. Thus, the energy gap between LH1 and the primary electron donor is quite large in this strain: 425 cm(-1). Nevertheless, trapping occurred with a time constant of 65 +/- 5 ps, similar to the rates observed in other purple bacteria. As in other species, no back-transfer from the reaction center to the antenna was observed. Our results show that strain 970 is a unique subject for the study of antenna and reaction center function and organization.

Energy transfer and charge separation in the purple non-sulfur bacterium Roseospirillum parvum.

The antenna reaction centre system of the recently described purple non-sulfur bacterium Roseospirillum parvum strain 930I was studied with various spectroscopic techniques. The bacterium contains bacteriochlorophyll (BChl) a, 20% of which was esterified with tetrahydrogeranylgeraniol. In the near-infrared, the antenna showed absorption bands at 805 and 909 nm (929 nm at 6 K). Fluorescence bands were located at 925 and 954 nm, at 300 and 6 K, respectively. Fluorescence excitation spectra and time resolved picosecond absorbance difference spectroscopy showed a nearly 100% efficient energy transfer from BChl 805 to BChl 909, with a time constant of only 2.6 ps. This and other evidence indicate that both types of BChl belong to a single LH1 complex. Flash induced difference spectra show that the primary electron donor absorbs at 886 nm, i.e. at 285 cm(-1) higher energy than the long wavelength antenna band. Nevertheless, the time constant for trapping in the reaction centre was the same as for almost all other purple bacteria: 55+/-5 ps. The shape as well as the amplitude of the absorbance difference spectrum of the excited antenna indicated exciton interaction and delocalisation of the excited state over the BChl 909 ring, whereas BChl 805 appeared to have a monomeric nature.

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.

Kinetics of absorbance and anisotropy upon excited state relaxation in the reaction center core complex of a green sulfur bacterium.

Properties of the excited states in reaction center core (RCC) complexes of the green sulfur bacterium Prosthecochloris aestuarii were studied by means of femtosecond time-resolved isotropic and anisotropic absorption difference spectroscopy at 275 K. Selective excitation of the different transitions of the complex resulted in the rapid establishment of a thermal equilibrium. At about 1 ps after excitation, the energy was located at the lowest energy transition, BChl a 835. Time constants varying between 0.26 and 0.46 ps were observed for the energy transfer steps leading to this equilibrium. These transfer steps were also reflected in changes in polarization. Our measurements indicate that downhill energy transfer towards excited BChl a 835 occurs via the energetically higher spectral forms BChl a 809 and BChl a 820. Low values of the anisotropy of about 0.07 were found in the 'two-color' measurements at 820 and 835 nm upon excitation at 800 nm, whereas the 'one-color' kinetics showed much higher anisotropies. Charge separation occurred with a time constant varying between 20 and 30 ps.

Excited states and trapping in reaction center complexes of the green sulfur bacterium Prosthecochloris aestuarii.

The excited states of bacteriochlorophyll (BChl) a were studied by pump-probe transient absorption spectroscopy in reaction center core (RCC), Fenna-Matthews-Olson (FMO) and FMO-RCC complexes of the green sulfur bacterium Prosthecochloris aestuarii. Excitation at 790 or 835 nm resulted in rapid equilibration of the energy between the BChl a molecules of the RCC complex: within 1 ps, most of the excitations had relaxed to the lowest energy level (835 nm), as a result of strong interactions between the BChls. Excitation of chlorophyll a 670 resulted in energy transfer to BChl a with a time constant of 1.2 ps, followed by thermal equilibration. Independent of the wavelength of excitation, the decay at 835 nm could be fitted with a time constant of about 25 ps, comparable to the 30 ps measured earlier with membrane fragments, which is ascribed to trapping in the reaction centers. Similar results were obtained with the FMO-RCC complex upon excitation at 835 or 670 nm, but the results upon 790 nm excitation were quite different. Again an equilibrium was rapidly reached, but now most of the excitations remained within the FMO complex, with a maximum bleaching at 813 nm, the same as observed in the isolated FMO. Even after 100 ps there was no bleaching at 835 nm and no evidence for charge separation. We conclude that there is no equilibration of the energy between the FMO and the RCC complex and that the efficiency of energy transfer from FMO to the reaction center core is low.

Isolation and properties of photochemically active reaction center complexes from the green sulfur bacterium Prosthecochloris aestuarii.

A new and rapid procedure was developed for the isolation of the reaction center core (RCC)-complex from the green sulfur bacterium Prosthecochloris aestuarii. Reaction center preparations containing the Fenna Matthews Olson (FMO) protein were also obtained. The procedure involved incubation of broken cells with the detergents Triton X-100 and SB12, sucrose gradient centrifugation and hydroxyapatite chromatography. Three different pigment protein complexes were obtained: one containing (about) three FMO trimers per RCC, one with one FMO per RCC and one consisting of RCC only. The last one contained polypeptides with apparent molecular masses of 64 kDa (pscA) and 35 kDa (pscB, the FA/FB, FeS subunit), but no cytochrome. Bacteriochlorophyll a and the chlorophyll a isomer functioning as primary electron acceptor were present at a ratio of 4.8:1. The complexes were also characterized spectroscopically and in terms of photochemical activity, at room temperature as well as at cryogenic temperatures. Illumination caused oxidation of the primary donor P840, with the highest activity in the RCC complex (DeltaA840/A810 = 0.06). At room temperature in the RCC complex essentially all of the P840+ produced in a flash was re-reduced slowly in the dark (several seconds). At low temperatures (150-10 K) a triplet was formed in a fraction of the reaction centers, presumably by a reversal of the charge separation, whereas in others P840+ formed in the light was re-reduced in 40-50 ms.