Molecular dynamics simulation of dark-adapted rhodopsin and free opsin

Molecular dynamics simulation was carried out for the rhodopsin protein to investigate its conformational changes in respect to inclusion of 11-cis retinal chromophore. Molecular dynamics calculations were performed within the time frame 3000 ps. Totally, 3 X 10(6) configurations ofrhodopsin and free opsin were analyzed and compared. It has been shown that the 11-cis retinal rearrangement (adaptation) in opsin strongly affects the surrounding amino acid residues of protein binding pocket and the protein cytoplasmic region. The extracellular part, however, shows comparatively little changes. On basis of the simulation results obtained we propose a molecular mechanism for the rhodopsin protein function as a G-protein-coupled receptor in the state of darkness. We discuss the role of the retinal chromophore as a ligand-antagonist stabilizing the inactive conformation of rhodopsin.     

The amino acid sequence of Clostridium pasteurianum iron protein, a component of nitrogenase. I. Tryptic peptides

A total of 25 tryptic peptides was isolated from the S-beta-carboxymethyl derivative of Clostridium pasteurianum iron protein (N2). In order to obtain the various peptides in pure state, a combination of gel permeation, cation and anion exchange column chromatographic methods, as well as various ascending paper chromatographic methods were adopted. Sequence studies of the tryptic peptides were carried out mainly by a modified manual Edman degradation procedure and also by automated analysis, carboxypeptidase digestion, and by hydrazinolysis. Thus, 242 residues (88.6%) out of a total of 273 amino acid residues were sequenced in the present study. The sum of the amino acid residues in the tryptic peptides isolated from iron protein (N2) accounted for the 273 amino acid residues present in the iron protein.     

The microenvironment around the chromophores and its changes due to the association states in C-phycocyanin isolated from the cyanobacterium Mastigocladus laminosus

The chromophore-protein interaction in C-phycocyanin was investigated as a function of the association state of the protein. Changes in the microenvironment around the chromophores were monitored by the following three indices: (1) the accessibility of a small molecule to the chromophore; (2) the fluorescence from aromatic amino acid residues; and (3) the effect of configurational changes of the chromophore on the conformation of the polypeptide chain. In the C-phycocyanin trimer, all the chromophores are shielded from the aqueous phase, probably by contact between subunits, and by a loop structure which surrounds the chromophores, even though that loop structure is not shown by X-ray analysis (Schirmer, T., Bode, W., Huber, R., Sidler, W. and Zuber, H. (1985) J. Mol. Biol. 184, 257–277). The polypeptide folding depends on the electronic structure of the chromophores; the oxidized chromophore of the α subunit inhibits the formation of the trimer and the reduced state of the chromophore of the α subunit allows the formation of trimers, in which the chromophores have the same electronic structure as in the monomers. The fluorescence from the aromatic amino acid residues showed that the conformational changes were induced by the reduced chromophore. These results indicate that the chromophore structure and the protein conformation affect each other. A definite configuration of the chromophore and also a definite conformation of the polypeptide are necessary for the intact energy transfer within C-phycocyanin.     

Photochemical properties of Escherichia coli DNA photolyase: selective photodecomposition of the second chromophore

Escherichia coli DNA photolyase contains a stable flavin radical and a second chromophore (SC) of unknown structure. The effects of flash (both conventional and laser) excitation of either the radical alone or both the radical and the second chromophore have been investigated by variation of the excitation wavelengths. Radical excitation leads to an electron abstraction by the lowest excited doublet state of the radical from an amino acid residue, probably a cysteine or tyrosine. On a longer time scale, a back-reaction occurs that can be prevented by the presence of certain electron donors, e.g., thiols, NADH, or tyrosine, but not pyrimidine dimers. Excitation of the second chromophore leads to electronic energy transfer from second chromophore excited states to the ground-state flavin radical doublet state, thus increasing the population of the lowest excited doublet state. Repetitive excitation of the enzyme with white light leads to photodecomposition of the second chromophore but not of the flavin adenine dinucleotide cofactor. Enzyme with photodecomposed SC retains full activity.     

Primary structure of a high potential iron-sulfur protein from the purple non-sulfur photosynthetic bacterium Rhodopseudomonas gelatinosa.

The third amino acid sequence of a high potential iron-sulfur protein, that of the non-sulfur purple photosynthetic bacterium Rhodopseudomonas gelatinosa, has been determined. It consists of a single polypeptide chain of 74 amino acid residues, which is slightly smaller than the high potential iron-sulfur proteins from the sulfur purple bacteria Chromatium vinosum (85 residues) and Thiocapsa pfennigii (81 residues). The sequence of the gelatinosa protein is similar to the C. vinosum and T. pfennigii proteins with 38% and 37% identically matching residues, although six gaps are proposed for the comparison (the C. vinosum and T. pfennigii proteins have 44% identically matching residues out of 73 positions compared with only one 4-residue gap). Only 17 redisues, including the 4 cystein residues essential for binding the four-iron-sulfur chromophore, are invariant in the three known sequences. A discussion of the role of conserved residues in maintenance of the three-dimensional structure and in electron transport is presented.     

The complete amino-acid sequence of C-phycoerythrin from the cyanobacterium Fremyella diplosiphon

The amino-acid sequences of both subunits of C-phycoerythrin from the cyanobacterium Fremyella diplosiphon have been determined. The alpha-subunit contains 164 amino acid residues, two phycoerythrobilin (PEB) chromophores and has a molecular mass of 18,368 Da (protein: 17,192 Da + 2 PEB, one PEB accounting for 588 Da). The beta-subunit consists of 184 residues, three PEB chromophores and has a molecular mass of 20,931 Da (protein: 19,168 Da and 3 PEB: 1,764 Da). The five PEB chromophores (open chain tetrapyrroles) are covalently bound to six cysteine residues (one of them doubly bound to two cysteine residues). On the alpha-subunit, the first chromophore was found at position 84, homologous to the chromophore binding site of the other biliproteins APC, PC and PEC. The second chromophore, unique for the alpha-subunit of PE, is inserted together with a pentapeptide at position 143 a. On the beta-subunit, a doubly bound chromophore is attached to cysteine residues 50 and 61, similar to the rhodophytan phycoerythrins (B-PE and R-PE). The second and third chromophores were found at positions 84 and 155, homologous to the other biliproteins. A unique peptide insertion of 14 amino acid residues (without chromophore) was found at position 141 a-o in the beta-subunit and probably is located in the three-dimensional model near the additional chromophores of the C-PE alpha- and beta-subunits. Both additional chromophores of the C-PE alpha- and beta-subunit may be located at the periphery of the C-PE-trimer. The amino-acid sequence homology between C-PE alpha- and beta-subunit is 26% and to the alpha- and beta-subunits of C-PC from Mastigocladus laminosus 49% and 48%, respectively.     

FTIR difference spectroscopy of bacteriorhodopsin: Toward a molecular model

Bacteriorhodopsin (bR) is a light-driven proton pump whose function includes two key membrane-based processes, active transport and energy transduction. Despite extensive research on bR and other membrane proteins, these processes are not fully understood on the molecular level. In the past ten years, the introduction of Fourier transform infrared (FTIR) difference spectroscopy along with related techniques including time-resolved FTIR difference spectroscopy, polarized FTIR, and attenuated total reflection FTIR has provided a new approach for studying these processes. A key step has been the utilization of site-directed mutagenesis to assign bands in the FTIR difference spectrum to the vibrations of individual amino acid residues. On this basis, detailed information has been obtained about structural changes involving the retinylidene chromophore and protein during the bR photocycle. This includes a determination of the protonation state of the four membrane-embedded Asp residues, identification of specific structurally active amino acid residues, and the detection of protein secondary structural changes. This information is being used to develop an increasingly detailed picture of the bR proton pump mechanism.     

Proton NMR of aequorin. Structural changes concomitant with calcium-independent light emission

Aequorin, a Ca(II)-sensitive bioluminescent protein from jellyfish, emits light at 469 nm from an excited state of a substituted pyrazine (oxyluciferin) which results from the oxidation of a chromophore molecule that is noncovalently bound to the protein. The chromophore is oxidized when Ca(II) or other activating metal ions are bound by aequorin. In the absence of Ca(II), spontaneous emission of light, referred to as Ca(II)-independent light emission, occurs at a rate less than 10(-6) of that for Ca(II)-induced emission. Proton nuclear magnetic resonance (NMR), circular dichroism (CD), and fluorescence were used to study structural changes of aequorin accompanying Ca(II)-independent light emission. Time course studies by 1H NMR and CD demonstrate that as a result of Ca(II)-independent light emission, aequorin progressively changes from a rigid, fully active form showing little segmental mobility to a practically unfolded, discharged (i.e., inactive) form in which a number of amino acid residues are significantly mobile. This slow discharged protein (SDP) is distinct in nature and conformation from aequorin which has been discharged by Ca(II), i.e., the blue fluorescent protein. The rate of Ca(II)-independent discharge of aequorin is substantially reduced in the presence of excess Mg(II); the time constant for inactivation at 5 degrees C is 30 days with no Mg(II) present and 70 days with Mg(II) present. The NMR spectra are nearly identical at a given stage of inactivation whether or not Mg(II) is present. Oxyluciferin remains bound to SDP. If it is removed, however, by column chromatography, the resulting apo-SDP partially refolds, and the segmental mobility acquired in the formation of SDP is significantly attenuated particularly for some of the aromatic amino acid residues.     

Chromophore protonation state controls photoswitching of the fluoroprotein asFP595

Fluorescent proteins have been widely used as genetically encodable fusion tags for biological imaging. Recently, a new class of fluorescent proteins was discovered that can be reversibly light-switched between a fluorescent and a non-fluorescent state. Such proteins can not only provide nanoscale resolution in far-field fluorescence optical microscopy much below the diffraction limit, but also hold promise for other nanotechnological applications, such as optical data storage. To systematically exploit the potential of such photoswitchable proteins and to enable rational improvements to their properties requires a detailed understanding of the molecular switching mechanism, which is currently unknown. Here, we have studied the photoswitching mechanism of the reversibly switchable fluoroprotein asFP595 at the atomic level by multiconfigurational ab initio (CASSCF) calculations and QM/MM excited state molecular dynamics simulations with explicit surface hopping. Our simulations explain measured quantum yields and excited state lifetimes, and also predict the structures of the hitherto unknown intermediates and of the irreversibly fluorescent state. Further, we find that the proton distribution in the active site of the asFP595 controls the photochemical conversion pathways of the chromophore in the protein matrix. Accordingly, changes in the protonation state of the chromophore and some proximal amino acids lead to different photochemical states, which all turn out to be essential for the photoswitching mechanism. These photochemical states are (i) a neutral chromophore, which can trans-cis photoisomerize, (ii) an anionic chromophore, which rapidly undergoes radiationless decay after excitation, and (iii) a putative fluorescent zwitterionic chromophore. The overall stability of the different protonation states is controlled by the isomeric state of the chromophore. We finally propose that radiation-induced decarboxylation of the glutamic acid Glu215 blocks the proton transfer pathways that enable the deactivation of the zwitterionic chromophore and thus leads to irreversible fluorescence. We have identified the tight coupling of trans-cis isomerization and proton transfers in photoswitchable proteins to be essential for their function and propose a detailed underlying mechanism, which provides a comprehensive picture that explains the available experimental data. The structural similarity between asFP595 and other fluoroproteins of interest for imaging suggests that this coupling is a quite general mechanism for photoswitchable proteins. These insights can guide the rational design and optimization of photoswitchable proteins.     

Alterations in photosynthetic pigments and amino acid composition of D1 protein change energy distribution in photosystem II

The marine cyanobacterium Prochlorococcus marinus accumulates divinyl chlorophylls instead of monovinyl chlorophylls to harvest light energy. As well as this difference in its chromophore composition, some amino acid residues in its photosystem II D1 protein were different from the conserved amino acid residues in other photosynthetic organisms. We examined PSII complexes isolated from mutants of Synechocystis sp. PCC 6803, in which chromophore and D1 protein were altered (Hisashi Ito and Ayumi Tanaka, 2011) to clarify the effects of chromophores/D1 protein composition on the excitation energy distribution. We prepared the mutants accumulating divinyl chlorophyll (DV mutant). The amino acid residues of V205 and G282 in the D1 protein were substituted with M205 and C282 in the DV mutant to mimic Prochlorococcus D1 protein (DV-V205M/G282C mutant). Isolated PSII complexes were analyzed by time-resolved fluorescence spectroscopy. Energy transfer in CP47 was interrupted in PSII containing divinyl chlorophylls. The V205M/G282C mutation did not recover the energy transfer pathway in CP47, instead, the mutation allowed the excitation energy transfer from CP43 to CP47, which neighbors in the PSII dimer. Mutual orientation of the subcomplexes of PSII might be affected by the substitution. The changes of the energy transfer pathways would reduce energy transfer from antennae to the PSII reaction center, and allow Prochlorococcus to acquire light tolerance.     

Electronic and vibrational spectroscopy of the cytochrome c:cytochrome c oxidase complexes from bovine and Paracoccus denitrificans.

The 1:1 complex between horse heart cytochrome c and bovine cytochrome c oxidase, and between yeast cytochrome c and Paracoccus denitrificans cytochrome c oxidase have been studied by a combination of second derivative absorption, circular dichroism (CD), and resonance Raman spectroscopy. The second derivative absorption and CD spectra reveal changes in the electronic transitions of cytochrome a upon complex formation. These results could reflect changes in ground state heme structure or changes in the protein environment surrounding the chromophore that affect either the ground or excited electronic states. The resonance Raman spectrum, on the other hand, reflects the heme structure in the ground electronic state only and shows no significant difference between cytochrome a vibrations in the complex or free enzyme. The only major difference between the Raman spectra of the free enzyme and complex is a broadening of the cytochrome a3 formyl band of the complex that is relieved upon complex dissociation at high ionic strength. These data suggest that the differences observed in the second derivative and CD spectra are the result of changes in the protein environment around cytochrome a that affect the electronic excited state. By analogy to other protein-chromophore systems, we suggest that the energy of the Soret pi* state of cytochrome a may be affected by (1) changes in the local dielectric, possibly brought about by movement of a charged amino acid side chain in proximity to the heme group, or (2) pi-pi interactions between the heme and aromatic amino acid residues.     

Tryptophan fluorescence monitors structural changes accompanying signalling state formation in the photocycle of photoactive yellow protein.

Photoactive yellow protein, a small, water-soluble blue-light absorbing photoreceptor protein from Ectothiorhodospira(Halorhodospira)[space]halophila has a structure with two hydrophobic cores, of which the main one houses its light-sensitive chromophore (p-coumaric acid), separated by a central [small beta]-sheet. This photoreceptor protein contains a single tryptophan residue (W119) that is situated at the interface between the central beta-sheet and its N-terminal cap. The fluorescence properties of W119 in the dark state pG (lambda(max)= 328 nm; Phi(fl)= 0.01; nearly pH-independent) are typical for a buried tryptophan in a hydrophobic environment with significant quenching by nearby amino acid residues. Signalling state formation leads to pH-dependent fluorescence changes: At pH values <6.5 the fluorescence emission increases, with a minor blue shift of the emission maximum. Above this pH, the emission maximum of the tryptophan shifts considerably to the red, whereas its total intensity decreases. These results further support the contention that signalling state formation in PYP leads to significant changes in the structure of this protein, even at sites that are at a considerable distance from the chromophore. The nature of these changes in pB, however, depend upon the pH imposed upon the protein: At slightly alkaline pH, which presumably is closest to the pH to which this protein is exposed in vivo, these changes lead to an exposure of the part of the central beta-sheet harbouring W119. At slightly acidic pH the polarity of the environment of W119 is hardly affected by the formation of the signalling state but the quenching of its fluorescence emission, possibly by nearby amino acids, is reduced. On the other hand, its accessibility for quenching by small molecules in the solution is enhanced at acidic and alkaline pH in the signalling state (pB) compared to the dark state (pG). This latter observation points towards a more flexible structure of the N-terminal cap, having a looser interaction with the central beta-sheet in pB.     

Distribution of amino acids in functional sites of proteins with high melting temperature

The stability of proteins in its native state has an important implication on its function and evolution. The functional site analysis may lead to better understanding of how these amino acid distributions influence the melting temperature of proteins. It has been reported that increasing the fraction of hydrophobic contacts in a protein tends to raise melting temperature; increasing the fraction of repulsive charge contacts decrease the melting temperature and consistent with a destabilizing effect. The role of amino acid distribution as hydrophobic, charged and polar residues in proteins and mainly in its functional sites has been studied. Due to limited data availability, redundancy check and controlled environment parameters, the study was carried out with ten single chain-wild proteins having melting temperature above 80°C at pH 7. The analysis depicts that, the entire protein, hydrophobic residues are more frequent in single chain proteins and charged residues are more frequent in multi-chains proteins. In functional sites of these proteins, hydrophobic and charged residues are equally frequent in single chain proteins and charged residues are very high in multi-chains proteins. But, the polar residue distribution remains same for both single chain and multi-chain proteins and its functional sites.     

Spectrophotometric estimation of protein concentration in the presence of tryptophan modified by 2-hydroxy-5-nitrobenzyl bromide

A spectrophotometric method makes it possible to determine the concentration of a protein after covalent modification of tryptophan residues by 2-hydroxy-5-nitrobenzyl bromide. Molar absorption coefficients for the 2-hydroxy-5-nitrobenzyl chromophore, reported here in the pH range from 4.0 to 10.9, can be used to correct the protein absorbance values at 280 nm, which then provides the basis for calculating protein concentration in the usual way. The method was tested with alpha-lactalbumin, beta-lactoglobulin, pepsin, and soybean trypsin inhibitor; spectrophotometrically estimated concentrations of these proteins agreed closely with values obtained by amino acid analysis.     

Molecular determinants of spectral properties and signal transduction in the visual pigments.

Site-directed mutagenesis of the visual pigment rhodopsin has provided a wealth of information regarding amino acid residues responsible for the determination of the spectral properties of the chromophore, the amino acids involved in activation and inactivation of the protein, and the effect of amino acid substitutions found in patients with retinitis pigmentosa. In addition, cell culture systems have now been established for expression of the three human color vision pigments, opening the way for a similar attack on the structure and function of these important proteins.     

Synthesis and properties of the red chromophore of the green-to-red photoconvertible fluorescent protein Kaede and its analogs

Green fluorescent protein (GFP) and homologous proteins possess a unique pathway of chromophore formation based on autocatalytic modification of their own amino acid residues. Green-to-red photoconvertible fluorescent protein Kaede carries His-Tyr-Gly chromophore-forming triad. Here, we describe synthesis of Kaede red chromophore (2-[(1E)-2-(5-imidazolyl)ethenyl]-4-(p-hydroxybenzylidene)-5-imidazolone) and its analogs that can be potentially formed by natural amino acid residues. Chromophores corresponding to the following tripeptides were obtained: His-Tyr-Gly, Trp-Tyr-Gly, Phe-Trp-Gly, Tyr-Trp-Gly, Asn-Tyr-Gly, Phe-Tyr-Gly, and Tyr-Tyr-Gly. In basic conditions they fluoresced red with relatively high quantum yield (up to 0.017 for Trp-derived compounds). The most red-shifted absorption peak at 595 nm was found for the chromophore Trp-Tyr-Gly in basic DMSO. Surprisingly, in basic DMF non-aromatic Asn-derived chromophore Asn-Tyr-Gly demonstrated the most red-shifted emission maximum at 642 nm. Thus, Asn residue may be a promising substituent, which can potentially diversify posttranslational chemistry in GFP-like proteins.     

Analysis of cloned cDNA and genomic sequences for phytochrome: complete amino acid sequences for two gene products expressed in etiolated Avena.

Cloned cDNA and genomic sequences have been analyzed to deduce the amino acid sequence of phytochrome from etiolated Avena. Restriction endonuclease site polymorphism between clones indicates that at least four phytochrome genes are expressed in this tissue. Sequence analysis of two complete and one partial coding region shows approximately 98% homology at both the nucleotide and amino acid levels, with the majority of amino acid changes being conservative. High sequence homology is also found in the 5'-untranslated region but significant divergence occurs in the 3'-untranslated region. The phytochrome polypeptides are 1128 amino acid residues long corresponding to a molecular mass of 125 kdaltons. The known protein sequence at the chromophore attachment site occurs only once in the polypeptide, establishing that phytochrome has a single chromophore per monomer covalently linked to Cys-321. Computer analyses of the amino acid sequences have provided predictions regarding a number of structural features of the phytochrome molecule.     

Selective photochemical modification by trichloroethanol of tryptophan residues in proteins with a high tyrosine-to-tryptophan ratio.

We present an improved procedure for the selective modification of tryptophan residues in proteins. A simple, low-cost set-up allows rapid tryptophan photoreaction upon ultraviolet irradiation in the presence of 2,2,2-trichloroethanol. This photochemical reaction is carried out under native conditions, occurs only in the excited state of tryptophan, and yields a single, as yet unidentified, photoproduct. Except for tyrosine, no reaction with other amino acid side chains are known. Stringent photoselection of tryptophan, ensuring that tyrosine residues are not affected, is achieved in situ without the need for an elaborate system of optical filters or lenses. Illumination with a medium-wave uv lamp of samples placed in disposable, dual pathlength, polystyrene fluorescence cuvettes allows treatment of small sample volumes (greater than or equal to 100 microliters) of various optical density. Chromophore accessibility in oligomeric assemblies or protein-nucleic acid complexes can be assessed by this reaction since the integrity of these structures is preserved. Moreover, this technique can be used to evaluate the involvement of tryptophan residues in catalytic or ligand binding processes.     

Oligomeric protein structure networks: insights into protein-protein interactions

Background  

Protein-protein association is essential for a variety of cellular processes and hence a large number of investigations are being carried out to understand the principles of protein-protein interactions. In this study, oligomeric protein structures are viewed from a network perspective to obtain new insights into protein association. Structure graphs of proteins have been constructed from a non-redundant set of protein oligomer crystal structures by considering amino acid residues as nodes and the edges are based on the strength of the non-covalent interactions between the residues. The analysis of such networks has been carried out in terms of amino acid clusters and hubs (highly connected residues) with special emphasis to protein interfaces.     

The binding of amino acids to the herbicide 2,4-dichlorophenoxy acetic acid

Summary. The interaction of amino acids with the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was studied by charge-transfer chromatography carried out on diatomaceous layers covered with different amount of 2,4-D and the effect of salts on the strength of interaction was elucidated. It was established that Arg, His, Lys, Orn, Phe and Trp binds to 2,4-D, the binding process is of saturation character. Principal component analysis proved that the concentration of 2,4-D exerts the highest impact on the interaction and the effect of salts is of secondary importance. The results suggest that these amino acid residues may account for the binding of 2,4-D to proteins and can play a considerable role in the detoxification processes by forming conjugates with 2,4-D. Received April 10, 1998, Accepted September 15, 1998