Light-induced conformational changes in the photosynthetic reaction center from Rhodobacter sphaeroides

The photosynthetic reaction center (RC) from purple photosynthetic bacteria is a membrane-bound protein-pigment complex that serves as an excellent model for studying biological energy conversion. This energy conversion takes place by electron transfer reactions, which occur within the protein and are often coupled to conformational changes. In order to study these conformational changes, recovery of the oxidized bacteriochlorophyll dimer, from the RC of the purple photosynthetic bacterium Rhodobacter sphaeroides , to its original state was measured by light-minus-dark optical difference spectroscopy. Laser flash excitation generated an electron transfer that takes place across the membrane; creating the primary charge-separated state with a lifetime of 100 ms. Prolonged illumination induced subsequent conformational rearrangements in the RC protein complex which result in lifetimes of the same charge-separated state that are significantly different from that measured after flash excitation. The structural details of the conformational rearrangements on the molecular level will be discussed. The conformational changes were sensitive to duration and wavelength of illumination, pH, temperature, hydrophobic environment (liposome or detergent), head-group charge of the detergent, and presence of a bound metal ion. By systematically varying these parameters, we were able to extend the lifetime of the charge-separated state up to 21 mins. Based on these results, our goal is to utilize the bacterial RC protein complex as a biocapacitor, since the positive and negative charges are separated by a hydrophobic core of the protein with a low dielectric constant. This biocapacitor can be discharged rapidly by inducing pH jump

Molecular assignment of light-induced structural changes using site-directed mutant reaction centers

The photosynthetic reaction center from purple photosynthetic bacteria is a membrane-bound protein-pigment complex that serves as an excellent model for studying biological energy conversion. This energy conversion takes place by electron transfer reactions, which occur within the protein and are often coupled to conformational changes that influence the lifetime of the charge-separated state. In order to identify these light-induced conformational changes, near the bacteriochlorophyll dimer, wild type and 11 different mutants of reaction centers from Rhodobacter sphaeroides were studied. Upon 1 min illumination the recovery of the charge-separated states, characterized by steady-state and transient optical spectroscopy, was nearly an order of magnitude slower in one group of mutants (including the wild type) than in mutants carrying the Leu to His mutation at the L131 position. The slower recovery, unlike in the mutants carrying His at the L131 position, was accompanied by a substantial decrease of the electrochromic absorption changes associated with the QY bands of the nearby bacteriochlorophyll monomers, plus a large proton release at pH 6, and a decrease up to 79 mV of the oxidation potential of the dimer during the illumination. The results in the mutants carrying His at the L131 position are modeled as arising from the loss of a proton conducting pathway from the dimer to the solvent, which inhibits the formation of the long-lived charge-separated state. On the other hand, combination of the light-induced conformational changes and lipid binding near accessory bacteriochorophyll pigment under optimized conditions resulted in unprecedented 5 orders of magnitude increase in lifetime of the charge-separated state, which sheds light on a new potential application of the reaction center in energy storage as a light-driven biocapacitor. Moreover, these conformational changes near the dimer can also be blocked by Mn2+ binding. The metal ion binding induced a significant ~ 100 mV increase in the oxidation potential of the dimer and inhibition of formation of the long-lived charge-separated state similar to mutants carrying Leu to His mutation at L131 position. The elevation of oxidation potential of the dimer upon Mn2+ binding can make reaction center protein gain some specific functional features of much more complicated photosystem II

The influence of truncating the carboxy-terminal amino acid residues of streptococcal enolase on its ability to interact with canine plasminogen

The native octameric structure of streptococcal enolase from Streptococcus pyogenes increasingly dissociates as amino acid residues are removed one by one from the carboxy-terminus. These truncations gradually convert native octameric enolase into monomers and oligomers. In this work, we investigated how these truncations influence the interaction between Streptococcal enolase and canine plasminogen. We used dual polarization interferometry (DPI), localized surface plasmon resonance (LSPR), and sedimentation velocity analytical ultracentrifugation (AUC) to study the interaction. The DPI was our first technique, was performed on all the truncations and used one exclusive kind of chip. The LSRP was used to show that the DPI results were not dependent on the type of chip used. The AUC was required to show that our surface results were not the result of selecting a minority population in any given sample; the majority of the protein was responsible for the binding phenomenon we observed. By comparing results from these techniques we identified one detail that is essential for streptococcal enolase to bind plasminogen: In our hands the individual monomers bind plasminogen; dimers, trimers, tetramers may or may not bind, the fully intact, native, octamer does not bind plasminogen. We also evaluated the contribution to the equilibrium constant made by surface binding as well as in solution. On a surface, the association coefficient is about twice that in solution. The difference is probably not significant. Finally, the fully octameric form of the protein that does not contain a hexa-his N-terminal peptide does not bind to a silicon oxynitride surface, does not bind to an Au-nanoparticle surface, does not bind to a surface coated with Ni-NTA nor does it bind to a surface coated with DPgn. The likelihood is great that the enolase species on the surface of Streptococcus pyogenes is an x-mer of the native octamer

The Interaction of Streptococcal Enolase with Canine Plasminogen: The Role of Surfaces in Complex Formation

The enolase from Streptococcus pyogenes (Str enolase F137L/E363G) is a homo-octamer shaped like a donut. Plasminogen (Pgn) is a monomeric protein composed of seven discrete separated domains organized into a lock washer. The enolase is known to bind Pgn. In past work we searched for conditions in which the two proteins would bind to one another. The two native proteins in solution would not bind under any of the tried conditions. We found that if the structures were perturbed binding would occur. We stated that only the non-native Str enolase or Pgn would interact such that we could detect binding. We report here the results of a series of dual polarization interferometry (DPI) experiments coupled with atomic force microscopy (AFM), isothermal titration calorimetry (ITC), dynamic light scattering (DLS), and fluorescence. We show that the critical condition for forming stable complexes of the two native proteins involves Str enolase binding to a surface. Surfaces that attract Str enolase are a sufficient condition for binding Pgn. Under certain conditions, Pgn adsorbed to a surface will bind Str enolase

Low potential manganese ions as efficient electron donors in native anoxygenic bacteria

Systematic control over molecular driving forces is essential for understanding the natural electron transfer processes as well as for improving the efficiency of the artificial mimics of energy converting enzymes. Oxygen producing photosynthesis uniquely employs manganese ions as rapid electron donors. Introducing this attribute to anoxygenic photosynthesis may identify evolutionary intermediates and provide insights to the energetics of biological water oxidation. This work presents effective environmental methods that substantially and simultaneously tune the redox potentials of manganese ions and the cofactors of a photosynthetic enzyme from native anoxygenic bacteria without the necessity of genetic modification or synthesis. A spontaneous coordination with bis-tris propane lowered the redox potential of the manganese (II) to manganese (III) transition to an unusually low value (~400 mV) at pH 9.4 and allowed its binding to the bacterial reaction center. Binding to a novel buried binding site elevated the redox potential of the primary electron donor, a dimer of bacteriochlorophylls, by up to 92 mV also at pH 9.4 and facilitated the electron transfer that is able to compete with the wasteful charge recombination. These events impaired the function of the natural electron donor and made BTP-coordinated manganese a viable model for an evolutionary alternative

Data for Truncation of Str enolase and its binding to DPgn

The native octameric structure of streptococcal enolase from Streptococcus pyogenes increasingly dissociates as amino acid residues are removed one by one from the carboxy-terminus. These truncations gradually convert native octameric enolase into monomers and oligomers. In this work, we investigated how these truncations influence the interaction between Streptococcal enolase and canine plasminogen. We used dual polarization interferometry (DPI), localized surface plasmon resonance (LSPR), and sedimentation velocity analytical ultracentrifugation (AUC) to study the interaction. The DPI was our first technique, was performed on all the truncations and used one exclusive kind of chip. The LSRP was used to show that the DPI results were not dependent on the type of chip used. The AUC was required to show that our surface results were not the result of selecting a minority population in any given sample; the majority of the protein was responsible for the binding of phenomenon we observed. By comparing results from these techniques we identified one detail that is essential for streptococcal enolase to bind plasminogen: In our hands the individual monomers bind plasminogen; dimers, trimers, tetramers may or may not bind, the fully intact, native, octamer does not bind plasminogen. We also evaluated the contribution to the equilibrium constant made by surface binding as well as in solution. On a surface, the association coefficient is about twice that in solution. The difference is probably not significant. Finally, the fully octameric form of the protein that does not contain a hexahis N-terminal peptide does not bind to a silicon oxynitride surface, does not bind to a Au-nanoparticle surface, does not bind to a surface coated with Ni-NTA nor does it bind to a surface coated with DPgn. The likelihood is great that the enolase species on the surface of Streptococcus pyogenes is an x-mer of the native octamer

Fluorescence of (125 µM DOPG + 1.25 µM DOPE) vesicles labeled with either rhodamine or NBD in separate vesicles.

<p>The excitation wavelength was 460(NBD, maximum absorption) while the emission wavelengths were 535 nm (NBD) and 592 (rhodamine). Fusion resulting in FRET from NBD to rhodamine is indicated by a diminution of NBD fluorescence (middle curve) and an increase in rhodamine fluorescence (lowest curve). The top curve of A is the ratio of rhodamine fluorescence to NBD. At zero time the solution was brought to 1.12 µM Str enolase. In B, 1 mM CaCl2 was added to the sample. Str enolase clearly brings about a slow fusion of about 5% of the vesicles (relative to Ca).</p

Crystal structures of Str enolase and human plasminogen.

<p>Left figures: Str enolase shown in two views. The homo-octamer is depicted in two colours. The purple atoms represent the sites where Pgn is thought to bind. Right figure: The individual domains of Pgn are colour coded. Residues 1-78 (N-Terminal Peptide), orange. Kringle 1 (79–163), red. Kringle 2 (164–249), green. Kringle 3 (250–345), yellow. Kringle 4 (346–439), blue. Kringle 5 (440–541), magenta. Preproteolytic domain (542–791), white. The figure depicts the closed form whereas it is probable that the open form (for which there is currently no structure) is that which binds to Str enolase. The two proteins are not drawn to the same scale. The dimensions of the Str enolase donut are approximately 15 nm wide and 5 nm thick. The Pgn, in contrast, is not symmetrical and consists of domains attached to, and sticking out from, a continuous string. Its largest dimensions are 10 nm×8.5 nm×5 nm. The dimensions of the two proteins will be important in determining the orientation of the two during the dual polarization interferometry experiments.</p

Isothermal titration calorimetry of Str enolase binding to DOPG vesicles.

<p>Titration of 0.00083 µM DOPG vesicles (86 µM DOPG) with 10 µM (octamer) Str enolase. The total final concentration of DOPG is 73.7 µM. The total final concentration of Str enolase bound is 0.24 µM.</p

DPI of Pgn interactions with Str enolase.

<p>The silicon oxynitride chip was injected once with 200 µL 4.4 µM Pgn and the injection stopped halfway. It was then injected with another 4.4 µM Pgn which indicated that the chip was saturated. This was followed by two injections of 5.3 µM Str enolase. The two lower curves indicate the calculated average masses of the proteins bound to the chip while the two upper curves indicate the average heights of the adsorbed layers.</p