Design of an artificial phage-display library based on a new scaffold improved for average stability of the randomized proteins.

Scaffold-based protein libraries are designed to be both diverse and rich in functional/folded proteins. However, introducing an extended diversity while preserving stability of the initial scaffold remains a challenge. Here we developed an original approach to select the ensemble of folded proteins from an initial library. The thermostable CheY protein from Thermotoga maritima was chosen as scaffold. Four loops of CheY were diversified to create a new binding surface. The subset of the library giving rise to folded proteins was first selected using a natural protein partner of the template scaffold. Then, a gene shuffling approach based on a single restriction enzyme was used to recombine DNA sequences encoding these filtrated variants. Taken together, the filtration strategy and the shuffling of the filtrated sequences were shown to enrich the library in folded and stable sequences while maintaining a large diversity in the final library (Lib-Cheytins 2.1). Binders of the Oplophorus luciferase Kaz domain were then selected by phage display from the final library, showing affinities in the µM range. One of the best variants induced a loss of 92% of luminescent activity, suggesting that this Cheytin preferentially binds to the Kaz active site.

Use of differential display to identify novel Sesbania rostrata genes enhanced by Azorhizobium caulinodans infection.

Upon infection of the tropical legume Sesbania rostrata with Azorhizobium caulinodans ORS571, nodules are formed on the roots as well as on the stems. Stem nodules appear at multiple predetermined sites consisting of dormant root primordia, which are positioned in vertical rows along the stem of the plant. We used the differential display method to isolate and characterize three cDNA clones (differential display; didi-2, didi-13, and didi-20), corresponding to genes whose expression is enhanced in the dormant root primordia after inoculation. Database searches revealed that the deduced (partial) didi-2 gene product shares significant similarity with hydroxyproline-rich cell wall proteins. The (partial) didi-13 and didi-20 products are similar to chitinases and chalcone reductases, respectively. Transcripts corresponding to the cDNA clones didi-2 and didi-13 were first detectable 1 day after inoculation. In contrast, didi-20 transcripts were found at low levels in uninfected root primordia and were enhanced significantly around 3 days after inoculation. In addition, a cDNA was isolated (didi-42) that corresponds to the previously identified leghemoglobin gene Srlb6. These studies show that differential display is a useful method for the isolation of infection-related genes.

A native electrostatic environment near Q(B) is not sufficient to ensure rapid proton delivery in photosynthetic reaction centers.

Flash-induced absorption spectroscopy has been used to characterize Rhodobacter capsulatus reaction centers mutated in the secondary quinone acceptor site (Q(B). We compared the wild-type, the L212Glu-L213Asp --> Ala-Ala photosynthetically incompetent double mutant (DM), and two photocompetent revertants, the DM+L217Arg --> Cys and the DM+M5Asn- --> Asp strains. The electrostatic environment for Q(B)- is different in the two revertant strains. Only the L217Arg --> Cys mutation nearly restores the native electrostatic environment of Q(B)-. However, the level of recovery of the reaction center function, measured by the rates of second electron transfer and cytochrome c turnover, is quite incomplete in both strains. This shows that a wild-type-like electrostatic environment of Q(B)- cannot ensure on its own, rapid and efficient proton transfer to Q(B)-.

In bacterial reaction centers, a key residue suppresses mutational blockage of two different proton transfer steps.

In reaction centers of Rhodobacter (Rb.) capsulatus, the M43Asn --> Asp substitution is capable of restoring rapid rates for delivery of the second proton to QB in a mutant that lacks L212Glu. Flash-induced absorbance spectroscopy was used to show a nearly native rate for transfer of the second proton to QB (approximately 700 s-1) in the L212Gln+M43Asp double-mutant reaction center; this rate was shown to decrease more than 1000-fold in the photoincompetent L212Glu --> Gln mutant [Miksovska, J., Kálmán, L., Maróti, P., Schiffer, M., Sebban, P., and Hanson, D.K. (1997) Biochemistry 36, 12216-12226]. In Rb. sphaeroides, the equivalent M44Asn --> Asp mutation was reported to restore the rate of transfer of the first proton to a wild-type level when it is added to the L213Asp --> Asn photoincompetent mutant [Rongey, S.H., Paddock, M.L., Feher, G., and Okamura, M.Y. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 1325-1329]. It is remarkable that the same second-site mutation can compensate for both of these mutations which severely impair reaction center function by blocking two different proton-transfer reactions. We suggest that residue M43Asp is situated in a key position which can link pathways for delivery of both the first and second protons (involving structured water molecules) to QB.

Mutations in the environment of the primary quinone facilitate proton delivery to the secondary quinone in bacterial photosynthetic reaction centers.

In Rhodobacter capsulatus, we constructed a quadruple mutant that reversed a structural asymmetry that contributes to the functional asymmetry of the two quinone sites. In the photosynthetically incompetent quadruple mutant RQ, two acidic residues near QB, L212Glu and L213Asp, have been mutated to Ala; conversely, in the QA pocket, the symmetry-related residues M246Ala and M247Ala have been mutated to Glu and Asp. We have selected photocompetent phenotypic revertants (designated RQrev3 and RQrev4) that carry compensatory mutations in both the QA and QB pockets. Near QA, the M246Ala --> Glu mutation remains in both revertants, but M247Asp is replaced by Tyr in RQrev3 and by Ala in RQrev4. The engineered L212Ala and L213Ala substitutions remain in the QB site of both revertants but are accompanied by an additional electrostatic-type mutation. To probe the respective influences of the mutations occurring near the QA and QB sites on electron and proton transfer, we have constructed two additional types of strains. First, "half" revertants were constructed that couple the QB site of the revertants with a wild-type QA site. Second, the QA sites of the two revertants were linked with the L212Glu-L213Asp --> Ala-Ala mutations of the QB site. We have studied the electron and proton-transfer kinetics on the first and second flashes in reaction centers from these strains by flash-induced absorption spectroscopy. Our data demonstrate that substantial improvements of the proton-transfer capabilities occur in the strains carrying the M246Ala --> Glu + M247Ala --> Tyr mutations near QA. Interestingly, this is not observed when only the M246Ala --> Glu mutation is present in the QA pocket. We suggest that the M247Ala --> Tyr mutation in the QA pocket, or possibly the coupled M246Ala --> Glu + M247Ala --> Tyr mutations, accelerates the uptake and delivery of protons to the QB anions. The M247Tyr substitution may enable additional pathways for proton transfer that are located near QA.