Cyanovirin-N (CV-N) is a 101 amino acid cyanobacterial lectin with potent antiviral activity against HIV, mediated by high-affinity binding to branched N-linked oligomannosides on the viral surface envelope protein gp120. The protein contains two carbohydrate-binding domains, A and B, each of which binds short oligomannosides independently in vitro. The interaction to gp120 could involve either a single domain or both domains simultaneously; it is not clear which mode would elicit the antiviral activity. The model is complicated by the formation of a domain-swapped dimer form, in which part of each domain is exchanged between two monomers, which contains four functional carbohydrate-binding domains. To clarify whether multivalent interactions with gp120 are necessary for the antiviral activity, we engineered a novel mutant, P51G-m4-CVN, in which the binding site on domain A has been knocked out; in addition, a [P51G] mutation prevents the formation of domain-swapped dimers under physiological conditions. Here, we present the crystal structures at 1.8 A of the free and of the dimannose-bound forms of P51G-m4-CVN, revealing a monomeric structure in which only domain B is bound to dimannose. P51G-m4-CVN binds gp120 with an affinity almost 2 orders of magnitude lower than wt CV-N and is completely inactive against HIV. The tight binding to gp120 is recovered in the domain-swapped version of P51G-m4-CVN, prepared under extreme conditions. Our findings show that the presence of at least two oligomannoside-binding sites, either by the presence of intact domains A and B or by formation of domain-swapped dimers, is essential for activity.

Cyanovirin (CV-N) is a small lectin with potent HIV neutralization activity, which could be exploited for a mucosal defense against HIV infection. The wild-type (wt) protein binds with high affinity to mannose-rich oligosaccharides on the surface of gp120 through two quasi-symmetric sites, located in domains A and B. We recently reported on a mutant of CV-N that contained a single functional mannose-binding site, domain B, showing that multivalent binding to oligomannosides is necessary for antiviral activity. The structure of the complex with dimannose determined at 1.8 Å resolution revealed a different conformation of the binding site than previously observed in the NMR structure of wt CV-N. Here, we present the 1.35 Å resolution structure of the complex, which traps three different binding conformations of the site and provides experimental support for a locking and gating mechanism in the nanoscale time regime observed by molecular dynamics simulations.

The mutation, HL(M182), in the Rhodobacter sphaeroides reaction center (RC) results in the replacement of the monomer bacteriochlorophyll (BChl) on the inactive side (B side) of the RC with a bacteriopheophytin (BPheo; the new cofactor is referred to as φB). In φB-containing RCs, the first excited state of the primary donor (P*) decays with an accelerated time constant of 2.6 ± 0.1 ps at room temperature as compared to 3.1 ± 0.2 ps in wild-type (WT) RCs. At low temperatures, P* decay is essentially the same in the HL(M182) mutant and WT RCs:  1.4 ± 0.1 ps at 77 K and 1.1 ± 0.1 ps at 9 K. The yield of the charge-separated P+φB - state decreases from 35% at room temperature to 12% at 77 and 9 K. The decreased P+φB - yield is apparently due to the fact that the rate of the charge separation along the A side of the RC at low temperature increases, while the rate along the B side remains essentially unchanged. From measurements of the long-lived fluorescence decay at room temperature, the standard free energy of the P+φB - state is estimated to be about 0.16 ± 0.04 eV below P*. Given a difference between the midpoint potentials of BChl and BPheo of 0.26 ± 0.03 V, the standard free energy of the P+BB - state in WT RC is estimated to be 0.1 ± 0.07 eV above P*.

The mutation HL(M182) in the Rb. sphaeroides reaction center (RC) results in the replacement of the monomer bacteriochlorophyll on the inactive side (B side) of the reaction center with a bacteriopheophytin (φB). In φB containing reaction centers, excitation of the initial electron donor, the special pair P, results in about 35% electron transfer along the normally inactive B side. However, the electron is transferred only to the exchanged cofactor φB. Several additional mutations in close proximity to bacteriopheophytins φB or HB have been created with the goal of altering the energetics of charge-separated states P+φB - and P+HB -. Aspartic acid residues were introduced to replace methionine L174 or valine M175 in the vicinity of the φB cofactor in order to raise the free energy of state P+φB -. Threonine M133 was mutated to the aspartic acid to add a hydrogen bond to the HB cofactor and lower the free energy of state P+HB -. The mutations in the environment surrounding the φB pigment resulted in a decrease in the quantum yield of P+φB - as well as a decrease in the recombination lifetime of this state. The mutation of valine M175 to aspartic acid showed the largest effect. The yield of state P+φB - decreased to about 25% and its recombination lifetime shortened from 200 to 125 ps. This additional mutation also resulted in the loss of the carotenoid molecule from the reaction centers. None of the three additional mutations altered the free energies sufficiently to result in observable electron transfer to HB. However, these measurements have allowed a more accurate assignment of the B-side charge-separated states' energetics than was previously possible.

Experimental evidence is presented showing that excitons in circular antenna complexes from photosynthetic bacteria are dynamically self trapped in about 200 fs by coupling to nuclear vibrations. The induced deformation covers ∼20% of the complex circumference at low temperature. This self trapping, the first of its kind observed in biological systems, results in a broad fluorescence spectrum and considerably improves energy resonance between heterogeneous antenna complexes. Exciton self trapping may thus be a part of nature's strategy, increasing the speed and efficiency of energy transfer in photosynthesis.

A new approach to creating fluorescent signaling aptamers using fluorescent nucleotide analogues is presented. The fluorescence quantum yield of nucleotide analogues such as 2-aminopurine strongly depends on base stacking interactions when incorporated into double or single stranded DNA. This property is used to generate a binding-specific fluorescence signal. Aptamers for human alpha-thrombin, immunoglobulin E, and platelet-derived growth factor B were modified with fluorescent nucleotide analogues in positions that undergo conformational changes. The resulting signaling aptamers show a specific, binding-induced increase in the fluorescence signal of up to 30-fold. Conformation-changing positions in these aptamers were identified by screening a set of modified aptamer sequences that each included a fluorescent nucleotide analogue at a different position. The positions for these modifications were estimated by modeling the aptamer secondary structure. It is likely that this approach to producing fluorescent signaling aptamers is of general use for protein-binding aptamers because of their "induced fit" binding mechanism.

The histidine (H) ligand of the bacteriochlorophyll monomer molecule on the B-side of the photosynthetic reaction center (RC) from Rhodobacter (Rb.) sphaeroides was replaced with a glutamic acid residue (E) (mutant HE(M182)). The photochemical properties of this mutant are markedly different from those of wild-type RCs. The excited state of the initial electron donor (P*) decays with a lifetime of 2.8 ± 0.1 ps, which is about 10% faster than in wild-type RCs. The faster decay of the excited state is due to an additional electron-transfer pathway in the mutant from P to the monomer bacteriochlorophyll on the B-side (BB) of the RC, forming the state P+BB -. The initial yield of the B-side electron transfer is estimated at about 35%, whereas the remaining 65% of P* leads to electron transfer along the A-side pigments forming the charge-separated state P+HA -. The P+BB - state formed during initial charge separation decays with a lifetime of 45 ps. Of the 35% P+BB - initially formed, 10% decays to form P+HA - via back electron transfer to P* and subsequent A-side charge separation. The other 25% of the state P+BB - recombines to the ground state. There is no observable further electron transfer from P+BB - to the B-side bacteriopheophytin molecule, HB. Apparently, P+HB - is at least as high in free energy as is P+BB - in this mutant, preventing further B-side electron transfer. From analysis of the long-lived fluorescence kinetics and transient absorbance data, the standard free energy of the state P+BB - in the HE(M182) mutant is estimated to be 70 meV below P*. Thus, the standard free energy of the state P+HB -, which should be similar in the mutant and the wild-type RCs, is apparently less than or equal to 70 meV below P*.

Time-resolved fluorescence of chromatophores isolated from strains of Rhodobacter sphaeroides containing light harvesting complex I (LHI) and reaction center (RC) (no light harvesting complex II) was measured at several temperatures between 295 K and 10 K. Measurements were performed to investigate energy trapping from LHI to the RC in RC mutants that have a P/P(+) midpoint potential either above or below wild-type (WT). Six different strains were investigated: WT + LHI, four mutants with altered RC P/P(+) midpoint potentials, and an LHI-only strain. In the mutants with the highest P/P(+) midpoint potentials, the electron transfer rate decreases significantly, and at low temperatures it is possible to directly observe energy transfer from LHI to the RC by detecting the fluorescence kinetics from both complexes. In all mutants, fluorescence kinetics are multiexponential. To explain this, RC + LHI fluorescence kinetics were analyzed using target analysis in which specific kinetic models were compared. The kinetics at all temperatures can be well described with a model which accounts for the energy transfer between LHI and the RC and also includes the relaxation of the charge separated state P(+)H(A)(-), created in the RC as a result of the primary charge separation.