The Year 1948-1949 in Prelog Laboratory at E.T.H., Zürich: a Reminiscence

After completing my work for a Ph. D. degree at the University of Liverpool in 1948, I wanted very much to spend a period of time in a laboratory in a German-speaking region of Europe. I thought of going to Zürich at the Eidgenössische Technische Hochschule known for its great tradition of excellence in organic chemistry. Professor Leopold Ružička was the Director at that time, but I wanted to work, in particular, with Professor Vladimir Prelog, who had joined the ETH from Zagreb some years earlier and whose work I had admired

Light-stable rhodopsin. II. An opsin mutant (TRP-265→PHE) and a retinal analog with a nonisomerizable 11-cis configuration form a photostable chromophore

In order to prepare a completely light-stable rhodopsin, we have synthesized an analog, II, of 11-cis retinal in which isomerization at the C11-C12 cis-double bond is blocked by formation of a cyclohexene ring from the C10 to C13-methyl. We used this analog to generate a rhodopsin-like pigment from opsin expressed in COS-1 cells and opsin from rod outer segments (Bhattacharya, S., Ridge, K.D., Knox, B.E., and Khorana, H. G. (1992) J. Biol. Chem. 267, 6763-6769). The pigment (lambda max, 512 nm) formed from opsin and analog II (rhodospin-II) showed ground state properties very similar to those of rhodopsin, but was not entirely stable to light. In the present work, 12 opsin mutants (Ala-117→Phe, Glu-122→Gln(Ala, Asp), Trp-126→Phe(Leu, Ala), Trp-265→Ala(Tyr, Phe), Tyr-268→Phe, and Ala-292→Asp), where the mutations were presumed to be in the retinal binding pocket, were reconstituted with analog II. While all mutants formed rhodopsin-like pigments with II, blue-shifted (12-30 nm) chromophores were obtained with Ala-117→Phe, Glu-122→Gln(Ala), Trp-126→Leu(Ala), and Trp-265→Ala(Tyr, Phe) opsins. The extent of chromophore formation was markedly reduced in the mutants Ala-117→Phe and Trp-126→Ala. Upon illumination, the reconstituted pigments showed varying degrees of light sensitivity; the mutants Trp-126→Phe(Leu) showed light sensitivity similar to wild-type. Continuous illumination of the mutants Glu-122→Asp, Trp-265→Ala, Tyr-268→Phe, and Ala-292→Asp resulted in hydrolysis of the retinyl Schiff base. Markedly reduced light sensitivity was observed with the mutant Trp-265→Tyr, while the mutant Trp-265→Phe was light-insensitive. Consistent with this result, the mutant Trp-265→Phe showed no detectable light-dependent activation of transducin or phosphorylation by rhodopsin kinase

The Roles of Transmembrane Domain Helix-III during Rhodopsin Photoactivation

Background: Rhodopsin, the prototypic member of G protein-coupled receptors (GPCRs), undergoes isomerization of 11- cis-retinal to all-trans-retinal upon photoactivation. Although the basic mechanism by which rhodopsin is activated is well understood, the roles of whole transmembrane (TM) helix-III during rhodopsin photoactivation in detail are not completely clear. Principal Findings: We herein use single-cysteine mutagenesis technique to investigate conformational changes in TM helices of rhodopsin upon photoactivation. Specifically, we study changes in accessibility and reactivity of cysteine residues introduced into the TM helix-III of rhodopsin. Twenty-eight single-cysteine mutants of rhodopsin (P107C-R135C) were prepared after substitution of all natural cysteine residues (C140/C167/C185/C222/C264/C316) by alanine. The cysteine mutants were expressed in COS-1 cells and rhodopsin was purified after regeneration with 11-cis-retinal. Cysteine accessibility in these mutants was monitored by reaction with 4, 49-dithiodipyridine (4-PDS) in the dark and after illumination. Most of the mutants except for T108C, G109C, E113C, I133C, and R135C showed no reaction in the dark. Wide variation in reactivity was observed among cysteines at different positions in the sequence 108–135 after photoactivation. In particular, cysteines at position 115, 119, 121, 129, 131, 132, and 135, facing 11-cis-retinal, reacted with 4-PDS faster than neighboring amino acids. The different reaction rates of mutants with 4-PDS after photoactivation suggest that the amino acids in different positions in helix-III are exposed to aqueous environment to varying degrees. Significance: Accessibility data indicate that an aqueous/hydrophobic boundary in helix-III is near G109 and I133. The lack of reactivity in the dark and the accessibility of cysteine after photoactivation indicate an increase of water/4-PDS accessibility for certain cysteine-mutants at Helix-III during formation of Meta II. We conclude that photoactivation resulted in water-accessible at the chromophore-facing residues of Helix-III.National Institutes of Health (U.S.) (grant GM28289)National Eye Institute (Grant Grant EY11716)National Science Foundation (U.S.) (grant EIA-0225609

Total synthesis of a gene

The method developed for the total synthesis of a given DNA containing biologically specific sequences consists of the following. The DNA in the double-stranded form is carefully divided into short single-stranded segments with suitable overlaps in the complementary strands. All the segments are chemically synthesized starting with protected nucleosides and mono-nucleotides. The 5′-OH ends of the appropriate oligonucleotides are then phosphorylated with the use of [γ-32P]ATP and polynucleotide kinase. A few to several neighboring oligonucleotides are then allowed to form bihelical complexes in aqueous solution, and the latter are joined end to end by polynucleotide ligase to form covalently linked duplexes. Subsequent head-to-tail joining of the short duplexes leads to the total DNA. The methods are described for the construction of a biologically functional suppressor transfer RNA gene. The total work involved (i) the synthesis of a 126-nucleotide-long bihelical DNA corresponding to a known precursor to the tyrosine suppressor transfer RNA, (ii) the sequencing of the promoter region and the distal region adjoining the C-C-A end, which contained a signal for the processing of the RNA transcript, (iii) total synthesis of the 207 base-pair-long DNA, which included the control elements, as well as the Eco R1 restriction endonu-clease specific sequences at the two ends, and (iv) full characterization by transcription in vitro and amber suppressor activity in vivo of the synthetic gene

A new photo-crosslinking reagent for the study of protein-protein interactions

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