D. Youvan - Academia.edu (original) (raw)

Papers by D. Youvan

Research paper thumbnail of Characterization of a symmetrized mutant RC with 42 residues from the QA site replacing residues in the Q(B) site

Photosynthesis research, 2000

The electron transfer reactions involving Q(A) and Q(B) were investigated in Rb. capsulatus RCs w... more The electron transfer reactions involving Q(A) and Q(B) were investigated in Rb. capsulatus RCs where the Q(B) site was mutated to contain 42 residues from the Q(A) site. The RCs have M220-M261 in the Q(A) site substituted for L193-L227 in the Q(B) site plus the M subunit second-site mutations, M144MI and M145AS, which had been found to restore the ability of the bacteria to grow photosynthetically. These mutants lack L210D, L212E, L213D, and L223S which have been previously shown to affect the electron transfer from Q(A) (-) to Q(B). Despite the large change in the Q(B) pocket, secondary quinone function still can be reconstituted. The UQ(4) dissociation constant for the Q(B) site in the mutant is only three times as large as in the wild type RCs. The rate of charge recombination (P(+)Q(A)Q(B) (-) --> PQ(A)Q(B)) (k (BP)) is reduced from 8.9 s(-1) in wild type RCs to 0.05s(-1) in the mutant, This indicates that Q(A)Q(B) (-) is stabilized relative to Q(A) (-)Q(B) by at least 60 me...

Research paper thumbnail of Green fluorescent protein: untapped potential in immunotechnology

Immunotechnology : an international journal of immunological engineering, 1995

Many invertebrates produce bioluminescence using green-fluorescent proteins (GFPs) as energy-tran... more Many invertebrates produce bioluminescence using green-fluorescent proteins (GFPs) as energy-transfer acceptors. GFPs fluoresce in vivo upon receiving energy from either a luciferase-oxyluciferin excited-state complex or a Ca(2+)-activated photoprotein depending upon the organism. These highly fluorescent proteins are unique due to the chemical nature of their chromophore, which is comprised of modified amino acid residues within the polypeptide chain. Recently GFP was sequenced and cloned. GFP, GFP mutants or related proteins with altered spectra will have widespread use as a markers of gene expression and as a protein tags in cell culture and in multicellular organisms. Many of the uses of fluorescent-labeled proteins or antibodies in immunotechnology will be improved by the use of GFP. Many new applications were discussed at a recent international symposium [1].

Research paper thumbnail of Partial symmetrization of the photosynthetic reaction center

Science, 1990

The bacterial photosynthetic reaction center (RC) is a pigmented intrinsic membrane protein that ... more The bacterial photosynthetic reaction center (RC) is a pigmented intrinsic membrane protein that performs the primary charge separation event of photosynthesis, thereby converting light to chemical energy. The RC pigments are bound primarily by two homologous peptides, the L and M subunits, each containing five transmembrane helices. These alpha helices and pigments are arranged in an approximate C2 symmetry and form two possible electron transfer pathways. Only one of these pathways is actually used. In an attempt to identify nonhomologous residues that are responsible for functional differences between the two branches, homologous helical regions that interact extensively with the pigments were genetically symmetrized (that is, exchanged). For example, replacement of the fourth transmembrane helix (D helix) in the M subunit with the homologous helix from the L subunit yields photosynthetically inactive RCs lacking a critical photoactive pigment. Photosynthetic revertants have been isolated in which single amino acid substitutions (intragenic suppressors) compensate for this partial symmetrization.

Research paper thumbnail of Light-harvesting II (B800-B850 complex) structural genes from Rhodopseudomonas capsulata

Proceedings of the National Academy of Sciences, 1985

The light-harvesting II (LHII) structural genes coding for the (B800-B850 complex) beta- and alph... more The light-harvesting II (LHII) structural genes coding for the (B800-B850 complex) beta- and alpha-polypeptides have been cloned and the nucleotide and deduced polypeptide sequences have been determined. This completes the sequencing of all seven structural genes coding for the structural polypeptides of the photosynthetic apparatus that bind the pigments and cofactors participating in the primary light reactions of photosynthesis. Unlike the structural genes coding for the reaction center L, M, and H subunits and the light-harvesting I (LHI) (B870 complex) structural polypeptides, the LHII structural genes are not within the 46-kilobase photosynthetic gene cluster carried by the R-prime plasmid pRPS404. Identical organization of the beta and alpha structural genes for both LHI and LHII and sequence homologies between the two beta-polypeptides and between the two alpha-polypeptides suggests that both complexes arose by gene duplication from a single ancestral light-harvesting complex and that the putative bacteriochlorophyll binding sequence Ala-X-X-X-His has been absolutely conserved.

Research paper thumbnail of Chromosomal deletion and plasmid complementation of the photosynthetic reaction center and light-harvesting genes from Rhodopseudomonas capsulata

Gene, 1985

Using in vitro interposon mutagenesis, Rhodopseudomonas capsulata strains have been constructed w... more Using in vitro interposon mutagenesis, Rhodopseudomonas capsulata strains have been constructed wherein all or part of the reaction center (RC), light-harvesting I (LHI), and light-harvesting II (LHII) structural genes have been deleted. In one series of strains, the 2778-bp ApaI fragment bearing more than 90% of the rxcA operon (promoter and structural genes coding for LHI beta, LHI alpha, RC-L and RC-M) has been deleted from the chromosome. When the rxcA operon is deleted, resultant strains possess only LHII and are photosynthetically defective. The rxcA deletion in an LHII- background results in a strain lacking all LH antennae and RC subunits. As expected this strain has no near-infrared absorption characteristic of LH or RC bacteriochlorophyll. The rxcA deletion may be complemented by a pBR322 derivative carrying the entire rxcA operon (pU21). In a second series of deletion mutants, the 2500-bp BstEII-StuI fragment, including the beta and alpha structural genes coding for LHII has been deleted from the chromosome. In the wild-type background, functional RC and LHI are synthesized. LHII may be restored in the deletion strain by conjugal transfer of the plasmid pU2 which carries the LHII operon.

Research paper thumbnail of Structure of psoralen-crosslinked ribosomal RNA from Drosophila melanogaster

Proceedings of the National Academy of Sciences, 1978

Ribosomal RNA from Drosophila melanogaster photoreacted with hydroxymethyltrioxsalen has been exa... more Ribosomal RNA from Drosophila melanogaster photoreacted with hydroxymethyltrioxsalen has been examined by electron microscopy. Reproducible patterns of hairpins were found in both the 26S and 18S RNA. The frequency of these hairpins and the amount of incorporated drug were dependent upon the conditions under which the crosslinking was performed. A prominent central hairpin occurs in the 26S RNA and the break that interrupts the continuity of the RNA chain is located within it. In addition to several small hairpins, the crosslinked 18S RNA contains a large open loop.

Research paper thumbnail of Characterization of a symmetrized mutant RC with 42 residues from the QA site replacing residues in the Q(B) site

Photosynthesis research, 2000

The electron transfer reactions involving Q(A) and Q(B) were investigated in Rb. capsulatus RCs w... more The electron transfer reactions involving Q(A) and Q(B) were investigated in Rb. capsulatus RCs where the Q(B) site was mutated to contain 42 residues from the Q(A) site. The RCs have M220-M261 in the Q(A) site substituted for L193-L227 in the Q(B) site plus the M subunit second-site mutations, M144MI and M145AS, which had been found to restore the ability of the bacteria to grow photosynthetically. These mutants lack L210D, L212E, L213D, and L223S which have been previously shown to affect the electron transfer from Q(A) (-) to Q(B). Despite the large change in the Q(B) pocket, secondary quinone function still can be reconstituted. The UQ(4) dissociation constant for the Q(B) site in the mutant is only three times as large as in the wild type RCs. The rate of charge recombination (P(+)Q(A)Q(B) (-) --> PQ(A)Q(B)) (k (BP)) is reduced from 8.9 s(-1) in wild type RCs to 0.05s(-1) in the mutant, This indicates that Q(A)Q(B) (-) is stabilized relative to Q(A) (-)Q(B) by at least 60 me...

Research paper thumbnail of Green fluorescent protein: untapped potential in immunotechnology

Immunotechnology : an international journal of immunological engineering, 1995

Many invertebrates produce bioluminescence using green-fluorescent proteins (GFPs) as energy-tran... more Many invertebrates produce bioluminescence using green-fluorescent proteins (GFPs) as energy-transfer acceptors. GFPs fluoresce in vivo upon receiving energy from either a luciferase-oxyluciferin excited-state complex or a Ca(2+)-activated photoprotein depending upon the organism. These highly fluorescent proteins are unique due to the chemical nature of their chromophore, which is comprised of modified amino acid residues within the polypeptide chain. Recently GFP was sequenced and cloned. GFP, GFP mutants or related proteins with altered spectra will have widespread use as a markers of gene expression and as a protein tags in cell culture and in multicellular organisms. Many of the uses of fluorescent-labeled proteins or antibodies in immunotechnology will be improved by the use of GFP. Many new applications were discussed at a recent international symposium [1].

Research paper thumbnail of Partial symmetrization of the photosynthetic reaction center

Science, 1990

The bacterial photosynthetic reaction center (RC) is a pigmented intrinsic membrane protein that ... more The bacterial photosynthetic reaction center (RC) is a pigmented intrinsic membrane protein that performs the primary charge separation event of photosynthesis, thereby converting light to chemical energy. The RC pigments are bound primarily by two homologous peptides, the L and M subunits, each containing five transmembrane helices. These alpha helices and pigments are arranged in an approximate C2 symmetry and form two possible electron transfer pathways. Only one of these pathways is actually used. In an attempt to identify nonhomologous residues that are responsible for functional differences between the two branches, homologous helical regions that interact extensively with the pigments were genetically symmetrized (that is, exchanged). For example, replacement of the fourth transmembrane helix (D helix) in the M subunit with the homologous helix from the L subunit yields photosynthetically inactive RCs lacking a critical photoactive pigment. Photosynthetic revertants have been isolated in which single amino acid substitutions (intragenic suppressors) compensate for this partial symmetrization.

Research paper thumbnail of Light-harvesting II (B800-B850 complex) structural genes from Rhodopseudomonas capsulata

Proceedings of the National Academy of Sciences, 1985

The light-harvesting II (LHII) structural genes coding for the (B800-B850 complex) beta- and alph... more The light-harvesting II (LHII) structural genes coding for the (B800-B850 complex) beta- and alpha-polypeptides have been cloned and the nucleotide and deduced polypeptide sequences have been determined. This completes the sequencing of all seven structural genes coding for the structural polypeptides of the photosynthetic apparatus that bind the pigments and cofactors participating in the primary light reactions of photosynthesis. Unlike the structural genes coding for the reaction center L, M, and H subunits and the light-harvesting I (LHI) (B870 complex) structural polypeptides, the LHII structural genes are not within the 46-kilobase photosynthetic gene cluster carried by the R-prime plasmid pRPS404. Identical organization of the beta and alpha structural genes for both LHI and LHII and sequence homologies between the two beta-polypeptides and between the two alpha-polypeptides suggests that both complexes arose by gene duplication from a single ancestral light-harvesting complex and that the putative bacteriochlorophyll binding sequence Ala-X-X-X-His has been absolutely conserved.

Research paper thumbnail of Chromosomal deletion and plasmid complementation of the photosynthetic reaction center and light-harvesting genes from Rhodopseudomonas capsulata

Gene, 1985

Using in vitro interposon mutagenesis, Rhodopseudomonas capsulata strains have been constructed w... more Using in vitro interposon mutagenesis, Rhodopseudomonas capsulata strains have been constructed wherein all or part of the reaction center (RC), light-harvesting I (LHI), and light-harvesting II (LHII) structural genes have been deleted. In one series of strains, the 2778-bp ApaI fragment bearing more than 90% of the rxcA operon (promoter and structural genes coding for LHI beta, LHI alpha, RC-L and RC-M) has been deleted from the chromosome. When the rxcA operon is deleted, resultant strains possess only LHII and are photosynthetically defective. The rxcA deletion in an LHII- background results in a strain lacking all LH antennae and RC subunits. As expected this strain has no near-infrared absorption characteristic of LH or RC bacteriochlorophyll. The rxcA deletion may be complemented by a pBR322 derivative carrying the entire rxcA operon (pU21). In a second series of deletion mutants, the 2500-bp BstEII-StuI fragment, including the beta and alpha structural genes coding for LHII has been deleted from the chromosome. In the wild-type background, functional RC and LHI are synthesized. LHII may be restored in the deletion strain by conjugal transfer of the plasmid pU2 which carries the LHII operon.

Research paper thumbnail of Structure of psoralen-crosslinked ribosomal RNA from Drosophila melanogaster

Proceedings of the National Academy of Sciences, 1978

Ribosomal RNA from Drosophila melanogaster photoreacted with hydroxymethyltrioxsalen has been exa... more Ribosomal RNA from Drosophila melanogaster photoreacted with hydroxymethyltrioxsalen has been examined by electron microscopy. Reproducible patterns of hairpins were found in both the 26S and 18S RNA. The frequency of these hairpins and the amount of incorporated drug were dependent upon the conditions under which the crosslinking was performed. A prominent central hairpin occurs in the 26S RNA and the break that interrupts the continuity of the RNA chain is located within it. In addition to several small hairpins, the crosslinked 18S RNA contains a large open loop.