The protein HC chromophore is linked to the cysteine residue at position 34 of the polypeptide chain by a reduction-resistant bond and causes the charge heterogeneity of protein HC.

Protein HC, an extremely charge-heterogeneous lipocalin, carries a yellow-brown fluorescent chromophore of unknown structure covalently bound at an unidentified site of its polypeptide chain. Two chromophore-carrying peptides with 60% of the chromophore material (defined as material absorbing light at 330 nm) were isolated from pepsin-digested native human protein HC by reversed-phase high performance liquid chromatography. Sequence analysis of these peptides indicated that the chromophore was bound to the cysteine residue at position 34 of the protein HC polypeptide chain. Sequence analysis of a native chromophore-tripeptide complex, isolated from pronase digests of the pepsin-produced peptides, identified the sequence Thr33-X34-Pro35, corroborating the position of the chromophore linkage. Quantitative amino acid analysis of the hydrolyzed, performic acid-oxidized, chromophore-tripeptide complex demonstrated approximately equal amounts of threonine, cysteic acid, and proline in the complex. Reduction and carboxymethylation of the native chromophore-tripeptide complex did not remove the chromophore from the peptide. The absorption spectrum of the chromophore-tripeptide complex was similar to that of native protein HC, implying that all of the heterogeneity of protein HC resides in its chromophore.     

Binding, tuning and mechanical function of the 4-hydroxy-cinnamic acid chromophore in photoactive yellow protein.

The bacterial photoreceptor protein photoactive yellow protein (PYP) covalently binds the chromophore 4-hydroxy coumaric acid, tuning (spectral) characteristics of this cofactor. Here, we study this binding and tuning using a combination of pointmutations and chromophore analogs. In all photosensor proteins studied to date the covalent linkage of the chromophore to the apoprotein is dispensable for light-induced catalytic activation. We analyzed the functional importance of the covalent linkage using an isosteric chromophore-protein variant in which the cysteine is replaced by a glycine residue and the chromophore by thiomethyl-p-coumaric acid (TMpCA). The model compound TMpCA is shown to weakly complex with the C69G protein. This non-covalent binding results in considerable tuning of both the pKa and the color of the chromophore. The photoactivity of this system, however, was strongly impaired, making PYP the first known photosensor protein in which the covalent linkage of the chromophore is of paramount importance for the functional activity of the protein in vitro. We also studied the influence of chromophore analogs on the color and photocycle of PYP, not only in WT, but especially in the E46Q mutant, to test if effects from both chromophore and protein modifications are additive. When the E46Q protein binds the sinapinic acid chromophore, the color of the protein is effectively changed from yellow to orange. The altered charge distribution in this protein also results in a changed pKa value for chromophore protonation, and a strongly impaired photocycle. Both findings extend our knowledge of the photochemistry of PYP for signal generation.     

The protein-chromophore bond in B phycoerythrin from Porphyridium cruentum. Radiosulfur labeling experiments

Red algae of the species Porphyridium cruentum were grown in a minimum sulfate medium containing 35SO42-. 35S-labeled phycoerythrin was extracted. B Phycoerythrin, b phycoerythrin and R phycocyanin could be separated from other proteins by using a carrier-free electrophoresis on columns. The final ratio A545/A280 of B phycoerythrin thus obtained was greater than or equal to 5. 35S-labeled B phycoerythrin was digested proteolytically with trypsin and pepsin. The resulting 35S-containing bilipeptides were separated by isoelectric focusing. Zones of enhanced chromophore concentration always showed an enhanced radioactivity. Peptide fractions with a low molar ratio sulfur/chromophore (1.1-1.8) were purified to remove sucrose and the carrier ampholyte. A modified, optimized Edman degradation followed. A butylacetate-soluble, red Edman product was obtained that contained most of the chromophore and the bulk of the radioactivity. This product was purified by two-dimensional thin-layer chromatography. The main spot of the chromatogram was subjected to acidic hydrolysis. The major part of the radioactivity in the hydrolysate cochromatographed with cysteine. That proves cysteine to be the binding amino acid in all cases investigated.     

A new method for the selective isolation of cysteine-containing peptides. Specific labelling of the thiol group with a hydrophobic chromophore.

A new method for the selective isolation of cysteine-containing peptides was designed. The method is based on the specific labelling of thiol groups with a hydrophobic chromophore followed by enzymic fragmentation of the labelled protein and reversed-phase high-pressure liquid-chromatographic separation of the peptide mixture. This new method has several distinct advantages: (1) the hydrophobic-chromophore-labelled cysteine-containing peptides are easily separated from non-cysteine-containing peptides by reversed-phase high-pressure liquid chromatography; (2) only cysteine-containing peptides are detected in the visible region with sensitivity at the low picomole level; this high sensitivity allows isolation of nanogram amounts of pure cysteine-containing peptide; (3) during sequence determination of the chromophore-labelled cysteine-containing peptides, the cysteine residues are released as coloured anilinothiazolinone derivatives and can be detected directly in the picomole range; (4) with proteins bearing several disulphide groups, each disulphide group may undergo a different degree of reduction, and therefore the recovery of individual cysteine-containing peptides may be used to deduce the disulphide linkages present in the native protein. Two thiol-specific reagents, 4-dimethylaminoazobenzene-4'-iodoacetamide and 4-dimethylaminoazobenzene-4'-N-maleimide, were synthesized and characterized. The method was successfully used to isolate five cysteine-containing peptides from a completely reduced monoclonal-antibody kappa-light chain raised against the azobenzenearsonate determinant and six cysteine-containing peptides from a kappa-light chain raised against streptococcal group A polysaccharide. The principle of this method is applicable to the isolation of any peptide containing amino acid residues that can be specifically labelled with a hydrophobic chromophore.     

The biliverdin chromophore binds covalently to a conserved cysteine residue in the N-terminus of Agrobacterium phytochrome Agp1

Phytochromes are widely distributed biliprotein photoreceptors. Typically, the chromophore becomes covalently linked to the protein during an autocatalytic lyase reaction. Plant and cyanobacterial phytochromes incorporate bilins with a ring A ethylidene side chain, whereas other bacterial phytochromes utilize biliverdin as chromophore, which has a vinyl ring A side chain. For Agrobacterium phytochrome Agp1, site-directed mutagenesis provided evidence that biliverdin is bound to cysteine 20. This cysteine is highly conserved within bacterial homologues, but its role as attachment site has as yet not been proven. We therefore performed mass spectrometry studies on proteolytic holopeptide fragments. For that purpose, an Agp1 expression vector was re-engineered to produce a protein with an N-terminal affinity tag. Following proteolysis, the chromophore co-purified with a ca. 5 kDa fragment during affinity chromatography, showing that the attachment site is located close to the N-terminus. Mass spectrometry analyses performed with the purified chromopeptide confirmed the role of the cysteine 20 as biliverdin attachment site. We also analyzed the role of the highly conserved histidine 250 by site-directed mutagenesis. The homologous amino acid plays an important but yet undefined role in plant phytochromes and has been proposed as chromophore attachment site of Deinococcus phytochrome. We found that in Agp1, this amino acid is dispensable for covalent attachment, but required for tight chromophore-protein interaction.     

Environment around the chromophore in pharaonis phoborhodopsin: mutation analysis of the retinal binding site.

Phoborhodopsin (pR or sensory rhodopsin II, sRII) and pharaonis phoborhodopsin (ppR or pharaonis sRII, psRII) have a unique absorption maximum (lambda(max)) compared with three other archaeal rhodopsins: lambda(max) of pR and ppR is approx. 500 nm and of others (e.g. bacteriorhodopsin, bR) is 560-590 nm. To determine the residue contributing to the opsin shift from ppR to bR, we constructed various ppR mutants, in which a single residue was substituted for a residue corresponding to that of bR. The residues mutated were those which differ from that of bR and locate within 5 A from the conjugated polyene chain of the chromophore or any methyl group of the polyene chain. The shifts of lambda(max) of all mutants were small, however. We constructed a mutant in which all residues which differ from those of bR in the retinal binding site were simultaneously substituted for those of bR, but the shift was only from 499 to 509 nm. Next, we constructed a mutant in which 10 residues located within 5 A from the polyene as described above were simultaneously substituted. Only 44% of the opsin shift (lambda(max) of 524 nm) from ppR to bR was obtained even when all amino acids around the chromophore were replaced by the same residues as bR. We therefore conclude that the structural factor is more important in accounting for the difference of lambda(max) between ppR and bR rather than amino acid substitutions. The possible structural factors are discussed.     

The suicide inactivation of ox liver short-chain acyl-CoA dehydrogenase by propionyl-CoA. Formation of an FAD adduct.

R-Phycoerythrin contains two covalently bound bilin prosthetic groups, phycoerythrobilin and phycourobilin. The two chromophore types were separated as their peptide-bound derivatives by subjecting tryptic digests of R-phycoerythrin to adsorption chromatography on Sephadex G-25. The structure and apoprotein linkages of the bound phycoerythrobilin were found to be identical with those previously reported for this phycobilin [Killilea, O'Carra & Murphy (1980) Biochem. J. 187, 311-320]. Phycourobilin is a tetrapyrrole, containing no oxo bridges and has the same order of side chains as IX alpha bilins. The chromophore is linked to the peptide through two and possibly three of its pyrrole rings. One linkage possibly consists of an ester bond between the hydroxy group of a serine residue and the propionic acid side chain of one of the inner rings. The second linkage is a labile thioether bond between a cysteine residue and the C2 side chain of pyrrole ring A. The third linkage is a stable thioether bond between a cysteine residue and the alpha-carbon atom of the C2 side chain of pyrrole ring D. Ring D is unsaturated and is attached to ring C through a saturated carbon bridge. Rings B and C have a conjugated system of five bonds, as found in other urobilinoid pigments. Ring A is attached to ring B via a saturated carbon bridge. Both of the alpha-positions of ring A are in the reduced state, but the ring does contain an unsaturated centre (probably a double bond between the beta-carbon and the ring nitrogen atom). The presence of this double bond and its isomerization into the bridge position between rings A and B would explain the extension of the conjugated system of phycourobilin to that of a phycoerythrobilinoid/rhodenoid pigment in acid or alkali.     

Homologous expression of a bacterial phytochrome. The cyanobacterium Fremyella diplosiphon incorporates biliverdin as a genuine, functional chromophore

Bacteriophytochromes constitute a light-sensing subgroup of sensory kinases with a chromophore-binding motif in the N-terminal half and a C-terminally located histidine kinase activity. The cyanobacterium Fremyella diplosiphon (also designated Calothrix sp.) expresses two sequentially very similar bacteriophytochromes, cyanobacterial phytochrome A (CphA) and cyanobacterial phytochrome B (CphB). Cyanobacterial phytochrome A has the canonical cysteine residue, by which covalent chromophore attachment is accomplished in the same manner as in plant phytochromes; however, its paralog cyanobacterial phytochrome B carries a leucine residue at that position. On the basis of in vitro experiments that showed, for both cyanobacterial phytochrome A and cyanobacterial phytochrome B, light-induced autophosphorylation and phosphate transfer to their cognate response regulator proteins RcpA and RcpB [Hübschmann T, Jorissen HJMM, B?rner T, G?rtner W & deMarsac NT (2001) Eur J Biochem268, 3383-3389], we aimed at the identification of a chromophore that is incorporated in vivo into cyanobacterial phytochrome B within the cyanobacterial cell. The approach was based on the introduction of a copy of cphB into the cyanobacterium via triparental conjugation. The His-tagged purified, recombinant protein (CphBcy) showed photoreversible absorption bands similar to those of plant and bacterial phytochromes, but with remarkably red-shifted maxima [lambda(max) 700 and 748 nm, red-absorbing (P(r)) and far red-absorbing (P(fr)) forms of phytochrome, respectively]. A comparison of the absorption maxima with those of the heterologously generated apoprotein, assembled with phycocyanobilin (lambda(max) 686 and 734 nm) or with biliverdin IXalpha (lambda(max) 700 and 750 +/- 2 nm), shows biliverdin IXalpha to be a genuine chromophore. The kinase activity of CphBcy and phosphotransfer to its cognate response regulator was found to be strictly P(r)-dependent. As an N-terminally located cysteine was found as an alternative covalent binding site for several bacteriophytochrome photoreceptors that bind biliverdin and lack the canonical cysteine residue (e.g. Agrobacterium tumefaciens and Deinococcus radiodurans), this corresponding residue in heterologously expressed cyanobacterial phytochrome B was mutated into a serine (C24S); however, there was no change in its spectral properties. On the other hand, the mutation of His267, which is located directly after the canonical cysteine, into alanine (H267A), caused complete loss of the capability of cyanobacterial phytochrome B to form a chromoprotein.     

Identification and characterization of a new class of bilin lyase: the cpcT gene encodes a bilin lyase responsible for attachment of phycocyanobilin to Cys-153 on the beta-subunit of phycocyanin in Synechococcus sp. PCC 7002

Synechococcus sp. PCC 7002 and all other cyanobacteria that synthesize phycocyanin have a gene, cpcT, that is paralogous to cpeT, a gene of unknown function affecting phycoerythrin synthesis in Fremyella diplosiphon. A cpcT null mutant contains 40% less phycocyanin than wild type and produces smaller phycobilisomes with red-shifted absorbance and fluorescence emission maxima. Phycocyanin from the cpcT mutant has an absorbance maximum at 634 nm compared with 626 nm for the wild type. The phycocyanin beta-subunit from the cpcT mutant has slightly smaller apparent molecular weight on SDS-PAGE. Purified phycocyanins from the cpcT mutant and wild type were cleaved with formic acid, and the products were analyzed by SDS-PAGE. No phycocyanobilin chromophore was bound to the peptide containing Cys-153 derived from the phycocyanin beta-subunit of the cpcT mutant. Recombinant CpcT was used to perform in vitro bilin addition assays with apophycocyanin (CpcA/CpcB) and phycocyanobilin. Depending on the source of phycocyanobilin, reaction products with CpcT had absorbance maxima between 597 and 603 nm as compared with 638 nm for the control reactions, in which mesobiliverdin becomes covalently bound. After trypsin digestion and reverse phase high performance liquid chromatography, the CpcT reaction product produced one major phycocyanobilin-containing peptide. This peptide had a retention time identical to that of the tryptic peptide that includes phycocyanobilin-bound, cysteine 153 of wild-type phycocyanin. The results from characterization of the cpcT mutant as well as the in vitro biochemical assays demonstrate that CpcT is a new phycocyanobilin lyase that specifically attaches phycocyanobilin to Cys-153 of the phycocyanin beta-subunit.     

Primary structure of a photoactive yellow protein from the phototrophic bacterium Ectothiorhodospira halophila,with evidence for the mass and the binding site of the chromophore.

The complete amino acid sequence of the 125-residue photoactive yellow protein (PYP) from Ectothiorhodospira halophila has been determined to be MEHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAAEGDITGRDPKEVIGKNFFKDVAP+ ++ CTDSPEFYGKFKEGVASGNLNTMFEYTFDYQMTPTKVKVHMKKALSGDSYWVFVKRV. This is the first sequence to be reported for this class of proteins. There is no obvious sequence homology to any other protein, although the crystal structure, known at 2.4 A resolution (McRee, D.E., et al., 1989, Proc. Natl. Acad. Sci. USA 86, 6533-6537), indicates a relationship to the similarly sized fatty acid binding protein (FABP), a representative of a family of eukaryotic proteins that bind hydrophobic molecules. The amino acid sequence exhibits no greater similarity between PYP and FABP than for proteins chosen at random (8%). The photoactive yellow protein contains an unidentified chromophore that is bleached by light but recovers within a second. Here we demonstrate that the chromophore is bound covalently to Cys 69 instead of Lys 111 as deduced from the crystal structure analysis. The partially exposed side chains of Tyr 76, 94, and 118, plus Trp 119 appear to be arranged in a cluster and probably become more exposed due to a conformational change of the protein resulting from light-induced chromophore bleaching. The charged residues are not uniformly distributed on the protein surface but are arranged in positive and negative clusters on opposite sides of the protein. The exact chemical nature of the chromophore remains undetermined, but we here propose a possible structure based on precise mass analysis of a chromophore-binding peptide by electrospray ionization mass spectrometry and on the fact that the chromophore can be cleaved off the apoprotein upon reduction with a thiol reagent. The molecular mass of the chromophore, including an SH group, is 147.6 Da (+/- 0.5 Da); the cysteine residue to which it is bound is at sequence position 69.     

Alternative cyclization in GFP-like proteins family. The formation and structure of the chromophore of a purple chromoprotein from Anemonia sulcata

Anemonia sulcata purple protein (asFP595) belongs to a family of green fluorescent protein (GFP)-like proteins from the Anthozoa species. Similar to GFP, asFP595 apparently forms its chromophore by modifying amino acids within its polypeptide chain. Until now, the GFP-like proteins from Anthozoa were thought to contain chromophores with the same imidazolidinone core as GFP. Mass spectral analysis of a chromophore-containing tryptic pentapeptide from asFP595 demonstrates that chromophore formation in asFP595 is stoichiometrically the same as that in GFP: one H(2)O and two H(+) are released while a Schiff base and dehydrotyrosine are formed. However, structural studies of this asFP595 chromopeptide show that in contrast to GFP, the other peptide bond nitrogen and carbonyl carbon are required for chromophore cyclization, a reaction that yields the six-membered heterocycle 2-(4-hydroxybenzylidene)-6-hydroxy-2,5-dihydropyrazine. Spectrophotometric titration reveals three pH-dependent forms of the asFP595 chromopeptide: yellow (absorption maximum = 430 nm) at pH 3.0; red (absorption maximum = 535 nm) at pH 8.0; and colorless (absorption maximum = 380 nm) at pH 14.0. The pK(a) values for these spectral transitions (6.8 and 10.9) are consistent with the ionization of the phenolic group of dehydrotyrosine and deprotonation of the amidinium cation in the chromophore heterocycle, respectively. The amidinium group in asFP595 accounts for the unique absorption spectrum of the protein, which is substantially red-shifted relative to that of GFP. When the asFP595 chromophore cyclizes, the Cys-Met bond adjacent to the chromophore hydrolyzes, splitting the chromoprotein into 8- and 20-kDa fragments. High performance liquid chromatography analysis of a tryptic digest of denatured asFP595 shows that a pentapeptide with the cleaved Cys-Met bond is the only fragment associated with the red-shifted absorbance. These results imply that fragmentation of asFP595 is a critical step in protein maturation.     

Investigation of C-phycoerythrin from the cyanobacterium Pseudanabaena W 1174

A cyanobacterium which produces high amounts of C-phycoerythrin was classified as a new Pseudanabaena strain. This strain (number W 1173 of our collection) has been cultivated for 6 years without changing its properties. It resembles Pseudanabaena catenata (strain B 1464-1) morphologically but differs in the pigmentation. Contrary to strain B 1464-1, no chromatic adaptation was observed with strain W 1173. It was found that phycoerythrins from both strains differ in the following properties: isoelectric points, number of bilin chromophores, and immunochemical properties. Besides native C-phycoerythrin (PEI, _max = 558 nm), a degradation product (PEII, _max = 544 nm and 562nm) has been found in crude extracts from strain W 1173. Criteria for integrity of C-phycoerythrin were discussed which are essential if this biliprotein is used as taxonomic character.Abbreviations PE C-Phycoerythrin - SDS sodiumdodecylsulfate Dedicated to Professor Dr. O. Kandler on the occasion of his 60th birthday     

Coupling of hydrogen bonding to chromophore conformation and function in photoactive yellow protein

To understand in atomic detail how a chromophore and a protein interact to sense light and send a biological signal, we are characterizing photoactive yellow protein (PYP), a water-soluble, 14 kDa blue-light receptor which undergoes a photocycle upon illumination. The active site residues glutamic acid 46, arginine 52, tyrosine 42, and threonine 50 form a hydrogen bond network with the anionic p-hydroxycinnamoyl cysteine 69 chromophore in the PYP ground state, suggesting an essential role for these residues for the maintenance of the chromophore's negative charge, the photocycle kinetics, the signaling mechanism, and the protein stability. Here, we describe the role of T50 and Y42 by use of site-specific mutants. T50 and Y42 are involved in fine-tuning the chromophore's absorption maximum. The high-resolution X-ray structures show that the hydrogen-bonding interactions between the protein and the chromophore are weakened in the mutants, leading to increased electron density on the chromophore's aromatic ring and consequently to a red shift of its absorption maximum from 446 nm to 457 and 458 nm in the mutants T50V and Y42F, respectively. Both mutants have slightly perturbed photocycle kinetics and, similar to the R52A mutant, are bleached more rapidly and recover more slowly than the wild type. The effect of pH on the kinetics is similar to wild-type PYP, suggesting that T50 and Y42 are not directly involved in any protonation or deprotonation events that control the speed of the light cycle. The unfolding energies, 26.8 and 25.1 kJ/mol for T50V and Y42F, respectively, are decreased when compared to that of the wild type (29.7 kJ/mol). In the mutant Y42F, the reduced protein stability gives rise to a second PYP population with an altered chromophore conformation as shown by UV/visible and FT Raman spectroscopy. The second chromophore conformation gives rise to a shoulder at 391 nm in the UV/visible absorption spectrum and indicates that the hydrogen bond between Y42 and the chromophore is crucial for the stabilization of the native chromophore and protein conformation. The two conformations in the Y42F mutant can be interconverted by chaotropic and kosmotropic agents, respectively, according to the Hofmeister series. The FT Raman spectra and the acid titration curves suggest that the 391 nm form of the chromophore is not fully protonated. The fluorescence quantum yield of the mutant Y42F is 1.8% and is increased by an order of magnitude when compared to the wild type.     

Accessibility of the iodopsin chromophore.

Iodopsin can replace its chromophore (11-cis retinal) by added 9-cis retinal, resulting in the formation of isoiodopsin. NaBH4 bleaches iodopsin in the dark. In a relatively low concentration of digitonin, the scotopsin (the protein moiety of chicken rhodopsin) removes 11-cis retinal from iodopsin in the dark. These facts suggest that the linkage of the chromophore to opsin in the iodopsin molecule (presumably a Schiff-base linkage) is accessible to these reagents, which is different from the situation in rhodopsin.     

Nano-patterned layers of a grafted coumarinic chromophore.

We report on the grafting of coumarin chromophores on flat silicon surfaces and in regions of nanometric dimensions drawn on silicon surfaces. The coumarin derivative was grafted by using the quaternization of a tertiary amine group of the chromophore with a ((chloromethyl)phenylethyl)-dimethylchlorosilane (CMPDCS) grafted on silicon. Complete characterization of the grafted layer was performed as a function of reaction time by X-ray photoelectron spectroscopy, X-ray reflectometry, atomic force microscopy, fluorescence spectroscopy and laser-scanning confocal microscopy. The results indicate that about one chromophore molecule is grafted every second CMPDCS molecule, resulting in a surface density of coumarin of slightly more than one coumarin per nm2. A broadening of the distribution of the fluorescence lifetimes was observed, suggesting that the grafted molecules experience a larger distribution of environments in the grafted layer than in solution. Since this reaction is fully compatible with silicon processing technology, the grafting could also be performed in nano-regions of size as small as 250 nm defined by combining electron-beam lithography with silanization. In such nano-sized regions the distribution of fluorescence lifetimes was narrower, suggesting a possible influence of the confinement on the organization of the molecules.     

Photochromic biliproteins from the cyanobacterium Anabaena sp. PCC 7120: lyase activities, chromophore exchange, and photochromism in phytochrome AphA

Photochromic biliproteins can be switched by light between two states, initiated by Z/E photoisomerization of the linear tetrapyrrole chromophore. The cyanobacterium Anabaena sp. PCC 7120 contains three genes coding for such biliproteins, two coding for phytochromes (aphA/B) and one for the alpha subunit of phycoerythrocyanin (pecA). (a) aphA was overexpressed in Escherichia coli with N-terminal His and S tags, and the protein was reconstituted by an optimized protocol with phycocyanobilin (PCB), to yield the photochromic chromoprotein, PCB-AphA, carrying the PCB chromophore. (b) AphA chromophorylation is autocatalytic such as in other phytochromes. (c) AphA chromophorylation is also possible by chromophore transfer from the PCB-carrying biliprotein, phycocyanin (CPC). The autocatalytic transfer is very slow, and it is enhanced more than 100-fold by catalysis of PCB:CpcA lyase and alpha-CPC as donor. (d) Through deletion mutations of aphA, a short sequence IQPHGV [amino acids (aa) 26-31] was found essential for the lyase activity of AphA, indicating an interaction of the N terminus with the chromophore-binding domain around cysteine 259. (e) A motif of at least 23 aa, starting with this sequence and located approximately 250 aa N terminal of the chromophore-binding cysteine, is proposed to relate to the lyase function in plant and most prokaryotic phytochromes. (f) Long-range interactions in AphA are further supported by blue-shifted absorptions (相似文献   

Direct photoaffinity labeling of cysteine 211 or a nearby amino acid residue of beta-tubulin by guanosine 5'-diphosphate bound in the exchangeable site

Tubulin with [8-14C]GDP bound in the exchangeable site was exposed to ultraviolet light, and radiolabel was cross-linked to two peptide regions of the beta-subunit. Following enrichment for peptides cross-linked to guanosine by boronate chromatography, we confirmed that the cysteine 12 residue was the major site of cross-linking. However, significant radiolabel was also incorporated into a peptide containing amino acid residues 206 through 224. Although every amino acid in this peptide except cysteine 211 was identified by sequential Edman degradation, implying that this was the amino acid residue cross-linked to guanosine, radiolabel at C-8 was usually lost during peptide processing (probably during chromatography at pH 10). Consequently, the radiolabeled amino acid could not be unambiguously identified.     

Photochemical and chemical studies on the chromophore of bacteriorhodopsin.

The chromophore (purple complex) of bacteriorhodopsin is reduced by sodium borohydride upon illumination to RPhv with a three-peaked absorption band at 360 nm. Treatment of this reduction product with ultraviolet light or acid yields a modified product from which retro-retinyllysine can be obtained by alkaline hydrolysis. No reduction of the 412 nm complex was found. Under specific conditions the purple complex equilibrates with a photochemically active 460 nm form that can be reduced by borohydride in the dark. This reduction product RP460 behaves idential to RPHV. Reconstitution of the purple complex from chromophore-free membrane (apomembrane) and retinal occurs via intermediates. The first (lambdamax 400nm) shows a three-peaked absorption band and is reduced to RP400 without a change of the three-peaked absorption (lambdamax 360 nm). The same product is obtained from apomembrane and retinol. Detergents shift the absorption band to 330 nm in all cases. From the experiments described no participation of retro-retinal structures during the photochemical cycle can be concluded but stereospecific interaction of the retinal moiety with the protein resulting in a specific retinal conformation os omdocated by the spectral changes observed.     

Circular dichroism of corticotropin, fragment 1-24, and model compounds. An assessment of the contributions of the peptide chromophore and armoatic residues.

The far-ultraviolet circular dichroic spectrum of the 39-residue peptide hormone porcine corticotropin and the biologically active fragment corticotropin 1–24 is negative from 250 nm to 195 nm in water, but in 6M guanidinium chloride a positive band appears at about 225 nm. The temperature and guanidinium chloride dependence of this spectral transition indicates the absence of any stable ordered secondary structure in corticotropin and the spectrum is seen to be in only partial agreement with results using the model peptide chromophore, Ala-Ala-Ala. Using oligopeptides containing aromatic amino acid residues sandwiched between glycyl residues, it is shown that the shape and intensity of the corticotropin 225 nm positive band which appears in 6M guanidinium chloride is in agreement with the far-ultraviolet transitions of the aromatic chromophores in the hormone. Curve resolution of the near-ultraviolet circular dichroic spectrum of corticotropin and comparison of the rotational strengths of the phenylalanyl and tyrosyl bands reveals no evidence for increased rotational freedom in 6M guanidinium hydrochloride. Spectral changes are observed, however, in the transitions arising from the single tryptophan. This study suggests that corticotropin in aqueous solution may serve as a better model for the circular dichroic spectrum of the aperiodic regions in globular proteins than either synthetic homopolypeptides or reference proteins for which spectral and X-ray diffraction data are available.     

Differential effects of mutations in the chromophore pocket of recombinant phytochrome on chromoprotein assembly and Pr-to-Pfr photoconversion.

Site-directed mutagenesis was performed with the chromophore-bearing N-terminal domain of oat phytochrome A apoprotein (amino acid residues 1-595). Except for Trp366, which was replaced by Phe (W366F), all the residues exchanged are in close proximity to the chromophore-binding Cys321 (i.e. P318A, P318K, H319L, S320K, H322L and the double mutant L323R/Q324D). The mutants were characterized by their absorption maxima, and the kinetics of chromophore-binding and the Pr-->Pfr conversion. The strongest effect of mutation on the chromoprotein assembly, leading to an almost complete loss of the chromophore binding capability, was found for the exchanges of His322 by Leu (H322L) and Pro318 by Lys (P318K), whereas a corresponding alanine mutant (P318A) showed wild-type behavior. The second histidine (H319) is also involved in chromophore fixation, as indicated by a slower assembly rate upon mutation (H319L). For the other mutants, an assembly process very similar to that of the wild-type protein was found. The light-induced Pr-->Pfr conversion kinetics is altered in the mutations H319L and S320K and in the double mutant L323R/Q324D, all of which exhibited a significantly faster I700 decay and accelerated Pfr formation. P318 is also involved in the Pr-->Pfr conversion, the millisecond steps (formation of Pfr) being significantly slower for P318A. Lacking sufficient amounts of W366F, assembly kinetics could not be determined in this case, while the fully assembled mutant underwent the Pr-->Pfr conversion with kinetics similar to wild-type protein.