Biomedical subjects

J M Guss

Publications and source records attributed to J M Guss.

At least 19 recordsLinked to original sources

PubMed1998

Structure and mechanism of a proline-specific aminopeptidase from Escherichia coli.

The structure of the proline-specific aminopeptidase (EC 3.4.11.9) from Escherichia coli has been solved and refined for crystals of the native enzyme at a 2.0-A resolution, for a dipeptide-inhibited complex at 2.3-A resolution, and for a low-pH inactive form at 2.7-A resolution. The protein crystallizes as a tetramer, more correctly a dimer of dimers, at both high and low pH, consistent with observations from analytical ultracentrifuge studies that show that the protein is a tetramer under physiological conditions. The monomer folds into two domains. The active site, in the larger C-terminal domain, contains a dinuclear manganese center in which a bridging water molecule or hydroxide ion appears poised to act as the nucleophile in the attack on the scissile peptide bond of Xaa-Pro. The metal-binding residues are located in a single subunit, but the residues surrounding the active site are contributed by three subunits. The fold of the protein resembles that of creatine amidinohydrolase (creatinase, not a metalloenzyme). The C-terminal catalytic domain is also similar to the single-domain enzyme methionine aminopeptidase that has a dinuclear cobalt center.

Aminopeptidases↗

PubMed1998

The crystal structure of bikunin from the inter-alpha-inhibitor complex: a serine protease inhibitor with two Kunitz domains.

Bikunin is a serine protease inhibitor found in the blood serum and urine of humans and other animals. Its sequence shows internal repetition, suggesting that it contains two domains that resemble bovine pancreatic trypsin inhibitor (BPTI). A fragment of bikunin has been crystallised, its structure solved and subsequently refined against 2.5 A data. The two BPTI-like domains pack closely together and are related by an approximate 60 degrees rotation combined with a translation. These domains are very similar to each other and other proteins with this fold. The largest variations occur in the loops responsible for protease recognition. The loops of the first domain are unobstructed by the remaining protein. However, the loops of the second domain are close to the first domain and it is possible that protease binding may be affected or, in some cases, abolished by the presence of the first domain. Thus, cleavage of the two domains could alter the substrate specificity of domain II. Bikunin has a hydrophobic patch close to the N terminus of domain I, which is the most likely site for cell-surface receptor binding. In addition, there is a basic patch at one end of domain II that may be responsible for the inhibition of calcium oxalate crystallization in urine.

Amino Acid Sequence↗

PubMed1997

Crystal structures of the copper-containing amine oxidase from Arthrobacter globiformis in the holo and apo forms: implications for the biogenesis of topaquinone.

The crystal structures of the copper enzyme phenylethylamine oxidase from the Gram-positive bacterium Arthrobacter globiformis (AGAO) have been determined and refined for three forms of the enzyme: the holoenzyme in its active form (at 2.2 A resolution), the holoenzyme in an inactive form (at 2.8 A resolution), and the apoenzyme (at 2.2 A resolution). The holoenzyme has a topaquinone (TPQ) cofactor formed from the apoenzyme by the post-translational modification of a tyrosine residue in the presence of Cu2+. Significant differences between the three forms of AGAO are limited to the active site. The polypeptide fold is closely similar to those of the amine oxidases from Escherichia coli [Parsons, M. R., et al. (1995) Structure 3, 1171-1184] and pea seedlings [Kumar, V., et al. (1996) Structure 4, 943-955]. In the active form of holo-AGAO, the active-site Cu atom is coordinated by three His residues and two water molecules in an approximately square-pyramidal arrangement. In the inactive form, the Cu atom is coordinated by the same three His residues and by the phenolic oxygen of the TPQ, the geometry being quasi-trigonal-pyramidal. There is evidence of disorder in the crystals of both forms of holo-AGAO. As a result, only the position of the aromatic group of the TPQ cofactor, but not its orientation about the Cbeta-Cgamma bond, is determined unequivocally. In apo-AGAO, electron density consistent with an unmodified Tyr occurs at a position close to that of the TPQ in the inactive holo-AGAO. This observation has implications for the biogenesis of TPQ. Two features which have not been described previously in amine oxidase structures are a channel from the molecular surface to the active site and a solvent-filled cavity at the major interface between the two subunits of the dimer.

Amine Oxidase (Copper-Containing)↗

PubMed1997

Crystal structure of the epsilon subunit of the proton-translocating ATP synthase from Escherichia coli.

BACKGROUND: Proton-translocating ATP synthases convert the energy generated from photosynthesis or respiration into ATP. These enzymes, termed F0F1-ATPases, are structurally highly conserved. In Escherichia coli, F0F1-ATPase consists of a membrane portion, F0, made up of three different polypeptides (a, b and c) and an F1 portion comprising five different polypeptides in the stoichiometry alpha 3 beta 3 gamma delta epsilon. The minor subunits gamma, delta and epsilon are required for the coupling of proton translocation with ATP synthesis; the epsilon subunit is in close contact with the alpha, beta, gamma and c subunits. The structure of the epsilon subunit provides clues to its essential role in this complex enzyme. RESULTS: The structure of the E. coli F0F1-ATPase epsilon subunit has been solved at 2.3 A resolution by multiple isomorphous replacement. The structure, comprising residues 2-136 of the polypeptide chain and 14 water molecules, refined to an R value of 0.214 (Rfree = 0.288). The molecule has a novel fold with two domains. The N-terminal domain is a beta sandwich with two five-stranded sheets. The C-terminal domain is formed from two alpha helices arranged in an antiparallel coiled-coil. A series of alanine residues from each helix form the central contacting residues in the helical domain and can be described as an 'alanine zipper'. There is an extensive hydrophobic contact region between the two domains providing a stable interface. The individual domains of the crystal structure closely resemble the structures determined in solution by NMR spectroscopy. CONCLUSIONS: Sequence alignments of a number of epsilon subunits from diverse sources suggest that the C-terminal domain, which is absent in some species, is not essential for function. In the crystal the N-terminal domains of two epsilon subunits make a close hydrophobic interaction across a crystallographic twofold axis. This region has previously been proposed as the contact surface between the epsilon and gamma subunits in the complete F1-ATPase complex. In the crystal structure we observe what is apparently a stable interface between the two domains of the epsilon subunit, consistent with the fact that the crystal and solution structures are quite similar despite close crystal packing. This suggests that a gross conformational change in the epsilon subunit, to transmit the effect of proton translocation to the catalytic domain, is unlikely, but cannot be ruled out.

Amino Acid Sequence↗

PubMed1997

Structure of a human lysosomal sulfatase.

BACKGROUND: . Sulfatases catalyze the hydrolysis of sulfuric acid esters from a wide variety of substrates including glycosaminoglycans, glycolipids and steroids. There is sufficient common sequence similarity within the class of sulfatase enzymes to indicate that they have a common structure. Deficiencies of specific lysosomal sulfatases that are involved in the degradation of glycosamino-glycans lead to rare inherited clinical disorders termed mucopolysaccharidoses. In sufferers of multiple sulfatase deficiency, all sulfatases are inactive because an essential post-translational modification of a specific active-site cysteine residue to oxo-alanine does not occur. Studies of this disorder have contributed to location and characterization of the sulfatase active site. To understand the catalytic mechanism of sulfatases, and ultimately the determinants of their substrate specificities, we have determined the structure of N-acetylgalactosamine-4-sulfatase. RESULTS: . The crystal structure of the enzyme has been solved and refined at 2.5 resolution using data recorded at both 123K and 273K. The structure has two domains, the larger of which belongs to the alpha/beta class of proteins and contains the active site. The enzyme active site in the crystals contains several hitherto undescribed features. The active-site cysteine residue, Cys91, is found as the sulfate derivative of the aldehyde species, oxo-alanine. The sulfate is bound to a previously undetected metal ion, which we have identified as calcium. The structure of a vanadate-inhibited form of the enzyme has also been solved, and this structure shows that vanadate has replaced sulfate in the active site and that the vanadate is covalently linked to the protein. Preliminary data is presented for crystals soaked in the monosaccharide N-acetylgalactosamine, the structure of which forms a product complex of the enzyme. CONCLUSIONS: . The structure of N-acetylgalactosamine-4-sulfatase reveals that residues conserved amongst the sulfatase family are involved in stabilizing the calcium ion and the sulfate ester in the active site. This suggests an archetypal fold for the family of sulfatases. A catalytic role is proposed for the post-translationally modified highly conserved cysteine residue. Despite a lack of any previously detectable sequence similarity to any protein of known structure, the large sulfatase domain that contains the active site closely resembles that of alkaline phosphatase: the calcium ion in sulfatase superposes on one of the zinc ions in alkaline phosphatase and the sulfate ester of Cys91 superposes on the phosphate ion found in the active site of alkaline phosphatase.

Alkaline Phosphatase↗

PubMed1996

The structure of a phytocyanin, the basic blue protein from cucumber, refined at 1.8 A resolution.

The crystal structure of the cucumber basic protein (CBP), a type 1 or blue copper protein, has been refined at 1.8 A resolution. The molecule resembles other blue copper proteins in having a Greek key beta-barrel structure, except that the barrel is open on one side and is better described as a "beta-sandwich" or "beta-taco". The Cu atom has the normal blue copper NNSS' co-ordination with bond lengths Cu-N(His39) = 1.93 A, Cu-S(Cys79) = 2.16 A, Cu-N(His84) = 1.95 A, Cu-S(Met89) = 2.61 A. The Cu-S(Met) bond is the shortest so far observed in a blue copper protein. A disulphide link, (Cys52)-S-S-(Cys85), appears to play an important role in stabilising the molecular structure. It is suggested that the polypeptide fold is typical of a sub-family of blue copper proteins (phytocyanins) as well as a non-metalloprotein, ragweed allergen Ra3, with which CBP has a high degree of sequence identify. The proteins currently identifiable as phytocyanins are CBP, stellacyanin, mavicyanin, umecyanin, a cucumber peeling cupredoxin, a putative blue copper protein in pea pods, and a blue copper protein from Arabidopsis thaliana. In all except CBP and the pea-pod protein, the axial methionine ligand normally found at blue copper sites is replaced by glutamine. The structure of CBP was originally solved by the multiple wavelength anomalous scattering method, using data recorded at four wavelengths. All these data were included in the restrained least squares refinement. The final model comprises 96 amino acid residues, 122 solvent molecules and a copper atom. Several residues are modelled as having more than one conformation. The residual R is 0.141 for 41,910 observations (including Bijvoet-related observations) of 8.142 unique reflections in the resolution range 7 to 1.8 A.

Allergens↗

PubMed1996

Crystal structure of a eukaryotic (pea seedling) copper-containing amine oxidase at 2.2 A resolution.

BACKGROUND: Copper-containing amine oxidases catalyze the oxidative deamination of primary amines to aldehydes, in a reaction that requires free radicals. These enzymes are important in many biological processes, including cell differentiation and growth, would healing, detoxification and signalling. The catalytic reaction requires a redox cofactor, topa quinone (TPQ), which is derived by post-translational modification of an invariant tyrosine residue. Both the biogenesis of the TPQ cofactor and the reaction catalyzed by the enzyme require the presence of a copper atom at the active site. The crystal structure of a prokaryotic copper amine oxidase from E. coli (ECAO) has recently been reported. RESULTS: The first structure of a eukaryotic (pea seedling) amine oxidase (PSAO) has been solved and refined at 2.2 A resolution. The crystallographic phases were derived from a single phosphotungstic acid derivative. The positions of the tungsten atoms in the W12 clusters were obtained by molecular replacement using E. coli amine oxidase as a search model. The methodology avoided bias from the search model, and provides an essentially independent view of a eukaryotic amine oxidase. The PSAO molecule is a homodimer; each subunit has three domains. The active site of each subunit lies near an edge of the beta-sandwich of the largest domain, but is not accessible from the solvent. The essential active-site copper atom is coordinated by three histidine side chains and two water molecules in an approximately square-pyramidal arrangement. All the atoms of the TPQ cofactor are unambiguously defined, the shortest distance to the copper atom being approximately 6 A. CONCLUSIONS: There is considerable structural homology between PSAO and ECAO. A combination of evidence from both structures indicates that the TPQ side chain is sufficiently flexible to permit the aromatic grouf to rotate about the Cbeta-Cgamma bond, and to move between bonding and non-bonding positions with respect to the Cu atom. Conformational flexibility is also required at the surface of the molecule to allow the substrates access to the active site, which is inaccessible to solvent, as expected for an enzyme that uses radical chemistry.

Amine Oxidase (Copper-Containing)↗

PubMed1993

Formation in vivo, purification and crystallization of a complex of the gamma and epsilon subunits of the F0F1-ATPase of Escherichia coli.

A complex comprising the epsilon subunit of Escherichia coli F1-ATPase (ECF1-ATPase) and a glutathione-S-transferase gamma subunit (of ECF1-ATPase) fusion protein was formed in vivo and purified from cell extracts by binding to glutathione-agarose beads. The glutathione-S-transferase was released from the complex by digestion with thrombin and the gamma/epsilon complex purified by cation-exchange chromatography. Crystals of the complex were grown by vapour diffusion using PEG8000 as precipitant. The crystals are orthorhombic, space-group P2(1)2(1)2 with a = 161.9 A, b = 44.1 A and c = 63.4 A. The volume of the asymmetric unit is consistent with the presence of a complex of one gamma subunit and one epsilon subunit.

Chromatography, Ion Exchange↗

PubMed1993

Crystallization and preliminary crystallographic characterization of the copper-containing amine oxidase from pea seedlings.

The copper-containing amine oxidase from pea seedlings has been crystallized using lithium sulfate as precipitant at pH 5.2. The unit cell is orthorhombic, space group P2(1)2(1)2(1), with dimensions a = 89.3 A, b = 113.4 A, c = 199.0 A. The mass of the asymmetric unit is 131(+/- 13) kDa, consistent with independent evidence that the molecule has two approximately 66 kDa subunits. The crystals diffract to 2.5 A in a synchrotron X-ray beam.

Amine Oxidase (Copper-Containing)↗

PubMed1992

Accuracy and precision in protein structure analysis: restrained least-squares refinement of the structure of poplar plastocyanin at 1.33 A resolution.

The structure of the electron-transfer protein, plastocyanin (99 amino acids, one Cu atom, 10,500 Da) from poplar leaves, has been refined at 1.33 A resolution to a residual R = 0.15. The space group is orthorhombic, P2(1)2(1)2(1), a = 29.60 (1), b = 46.86 (3), c = 57.60 (3) A. The 14,303 reflections used in the refinement were obtained from a data set recorded on a four-circle diffractometer with radiation from a sealed fine-focus tube, combined with a data set measured on oscillation films exposed at the DESY synchrotron. The final model comprises 1442 (738 non-H) protein atoms, one Cu atom and 110 solvent molecules. Nine residues are described as disordered. The root-mean-square deviation from ideal bond lengths is 0.016 A and the root-mean-square difference between the positions of the C alpha atoms in this refined model and in the structure previously refined at 1.6 A resolution is 0.11 A. The effects of manual model adjustment, resolution, choice of standard values for geometrical parameters, inclusion of H atoms and inclusion of anomalous-scattering corrections on the copper-site geometry have been explored. The final values of the Cu-ligand bond lengths are: Cu--N(His37) 1.91, Cu--S(Cys84) 2.07, Cu--N(His87) 2.06, Cu--S(Met92) 2.82 A.

Amino Acid Sequence↗

PubMed1992

The expression, purification and crystallization of the epsilon subunit of the F1 portion of the ATPase of Escherichia coli.

The epsilon subunit of the F0F1-ATPase from Escherichia coli has been expressed in E. coli as a fusion protein with glutathione S-transferase from the parasitic helminth Schistosoma japonicum. The epsilon subunit released by thrombin treatment of the purified fusion protein carried two amino acid changes, A1G and M2S, and was obtained in a yield of about five milligrams per litre of cultured cells. The two amino acid changes were shown not to affect function. The protein has been crystallized in a form suitable for X-ray diffraction structure analysis. The crystals are hexagonal, space group P6(1)22 (or P6(5)22), with a = b = 94.9 A, c = 57.1 A and gamma = 120 degrees. The diffraction from small crystals extends to at least 2.9 A resolution.

Crystallization↗

PubMed1991

Three-dimensional model for stellacyanin, a "blue" copper-protein.

A three-dimensional model of the "blue" copper-glycoprotein stellacyanin from Rhus vernicifera has been derived by computer graphics, energy minimization and molecular dynamics techniques. The initial atomic co-ordinates were obtained by making substitutions and insertions in the known structure of another blue copper-protein, cucumber basic protein (CBP), which is 46% homologous with stellacyanin and has similar spectroscopic properties. An important difference between CBP and stellacyanin is that the latter lacks methionine, a residue that forms an exceptionally long bond to the copper atom in all blue copper-proteins of known structure. In the aligned amino acid sequences, stellacyanin has glutamine 97 at the position that corresponds to the copper-binding methionine 89 in CBP. The hypothesis that the copper atom in stellacyanin is co-ordinated by the side-chain functional groups of histidine 46, cysteine 87, histidine 92 and glutamine 97 leads to a model that enables the spectroscopic properties, redox potential and electron-transfer kinetics of the protein to be rationalized. The present model for stellacyanin is more plausible than an antecedent model derived from the structure of plastocyanin. This demonstrates that the output from molecular modeling calculations is strongly dependent on the input, and that sequence homology with the target molecule is an important criterion for the selection of a starting model.

Amino Acid Sequence↗

PubMed1990

Crystal structure of plastocyanin from a green alga, Enteromorpha prolifera.

The crystal structure of the Cu-containing protein plastocyanin (Mr 10,500) from the green alga Enteromorpha prolifera has been solved by molecular replacement. The structure was refined by constrained-restrained and restrained reciprocal space least-squares techniques. The refined model includes 111 solvent sites. There is evidence for alternate conformers at eight residues. The residual is 0.12 for a data set comprising 74% of all observations accessible at 1.85 A resolution. The beta-sandwich structure of the algal plastocyanin is effectively the same as that of poplar leaf (Populus nigra var. italica) plastocyanin determined at 1.6 A resolution. The sequence homology between the two proteins is 56%. Differences between the contacts in the hydrophobic core create some significant (0.5 to 1.2 A) movements of the polypeptide backbone, resulting in small differences between the orientations and separations of corresponding beta-strands. These differences are most pronounced at the end of the molecule remote from the Cu site. The largest structural differences occur in the single non-beta strand, which includes the sole turn of helix in the molecule: two of the residues in a prominent kink of the poplar plastocyanin backbone are missing from the algal plastocyanin sequence, and there is a significant change in the position of the helical segment in relation to the beta-sandwich. Several other small but significant structural differences can be correlated with intermolecular contacts in the crystals. An intramolecular carboxyl-carboxylate hydrogen bond in the algal plastocyanin may be associated with an unusually high pKa. The dimensions of the Cu site in the two plastocyanins are, within the limits of precision, identical.

Amino Acid Sequence↗

PubMed1988

Phase determination by multiple-wavelength x-ray diffraction: crystal structure of a basic "blue" copper protein from cucumbers.

A novel x-ray diffraction technique, multiple-wavelength anomalous dispersion (MAD) phasing, has been applied to the de novo determination of an unknown protein structure, that of the "blue" copper protein isolated from cucumber seedlings. This method makes use of crystallographic phases determined from measurements made at several wavelengths and has recently been made technically feasible through the use of intense, polychromatic synchrotron radiation together with accurate data collection from multiwire electronic area detectors. In contrast with all of the conventional methods of solving protein structures, which require either multiple isomorphous derivatives or coordinates of a similar structure for molecular replacement, this technique allows direct solution of the classical "phase problem" in x-ray crystallography. MAD phase assignment should be particularly useful for determining structures of small to medium-sized metalloproteins for which isomorphous derivatives are difficult or impossible to make. The structure of this particular protein provides new insights into the spectroscopic and redox properties of blue copper proteins, an important class of metalloproteins widely distributed in nature.

Amino Acid Sequence↗

PubMed1986

Crystal structure analyses of reduced (CuI) poplar plastocyanin at six pH values.

The structure of poplar plastocyanin in the reduced (CuI) state has been determined and refined, using counter data recorded from crystals at pH 3.8, 4.4, 5.1, 5.9, 7.0 and 7.8 (resolution 1.9 A, 1.9 A, 2.05 A, 1.7 A, 1.8 A and 2.15 A; the final residual R value was 0.15, 0.15, 0.16, 0.17, 0.16 and 0.15, respectively). The molecular and crystal structure of the protein is substantially the same in the reduced state as in the oxidized state. The refinements of the structures of the six forms of the reduced protein could therefore be commenced with a model derived from the known structure of CuII-plastocyanin. The refinements were made by reciprocal space least-squares calculations interspersed with inspections of electron-density difference maps. Precautions were taken to minimize any bias of the results of the refinements in the direction of the starting model. The most significant differences among the structures of the reduced protein at the six pH values, or between them and the structure of the oxidized protein, are concentrated at the Cu site. In the reduced protein at high pH (pH 7.8), the CuI atom is co-ordinated by the N delta(imidazole) atoms of His37 and His87, the S gamma(thiolate) atom of Cys84, and the S delta(thioether) atom of Met92, just as in CuII-plastocyanin. The distorted tetrahedral geometry and the unusually long Cu-S(Met92) bond are retained. The only effects of the change in oxidation state are a lengthening of the two Cu-N(His) bonds by about 0.1 A, and small changes in two bond angles involving the Cu-S(Cys) bond. The high-pH form of reduced plastocyanin accordingly meets all the requirements for efficient electron transfer. As the pH is lowered, the Cu atom and the four Cu-binding protein side-chains appear to undergo small but concerted movements in relation to the rest of the molecule. At low pH (pH 3.8), the CuI atom is trigonally co-ordinated by N delta(His37), S gamma(Cys84) and S delta(Met92). The fourth Cu-ligand bond is broken, the Cu atom making only a van der Waals' contact with the imidazole ring of His87. The trigonal geometry of the Cu atom strongly favours CuI, so that this form of the protein should be redox-inactive. This is known to be the case.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗

PubMed1986

The crystal structure of mercury-substituted poplar plastocyanin at 1.9-A resolution.

The crystal structure of Hg(II)-plastocyanin has been determined and refined at a resolution of 1.9 A. The crystals were prepared by soaking crystals of Cu(II)-plastocyanin from poplar leaves (Populus nigra var. italica) in a solution of a mercuric salt. Replacement of the Cu(II) atom in plastocyanin by Hg(II) causes only minor changes in the geometry of the metal site, and there are few significant changes elsewhere in the molecule. It is concluded that, as in the case of the native protein, the geometry of the metal site is determined by the polypeptide. The weak metal-S(methionine) bond found in Cu(II)-plastocyanin remains weak in Hg(II)-plastocyanin. The "flip" of a proline side chain close to the metal site from a C gamma-exo conformation in Cu(II)-plastocyanin to a C gamma-endo conformation in Hg(II)-plastocyanin suggests that this region of the molecule is particularly flexible. Crystallographic evidence for the close similarity of the Hg(II)- and Cu(II)-plastocyanin structures was originally obtained from electron density difference maps at 2.5-A resolution. The refinement of the structure was begun with a set of atomic coordinates taken from the structure of Cu(II)-plastocyanin. A Hg(II) atom was substituted for the Cu(II) atom, and the side chains of 6 residues in the vicinity of the metal site were omitted. Three series of stereochemically restrained least-squares refinement calculations were interspersed with two stages of model adjustment followed by phase extension. Fifty-nine water molecules were located. The final structure has a crystallographic residual R = 0.16.

Copper↗

PubMed1984

The crystal structure of poplar apoplastocyanin at 1.8-A resolution. The geometry of the copper-binding site is created by the polypeptide.

The three-dimensional structure of apoplastocyanin from poplar leaves (Populus nigra var. italica) has been determined by x-ray diffraction at 1.8-A resolution. The structure closely resembles that of the holoprotein. In particular, the positions of the copper-binding residues in the apo- and holoproteins differ by only 0.1-0.3 A. This indicates that the irregular geometry of the "type 1" copper site is imposed upon the metal atom by the polypeptide moiety. A 180 degrees rotation of one solvent-exposed histidine imidazole ring about C beta-C gamma appears to facilitate access to the copper site. The close structural similarity between apo-, Cu-(II)-, and Cu(I)-plastocyanin was initially demonstrated by means of electron density difference maps. Two series of restrained least squares refinement calculations for apoplastocyanin, originating from different sets of atomic positional parameters, were carried out in parallel. Both refinements converged to the same model which, when fully refined, had a residual R = 0.16. Forty-two water molecules were located during the refinement.

Apoproteins↗

PubMed1983

Structure of oxidized poplar plastocyanin at 1.6 A resolution.

The structure of poplar plastocyanin in the oxidized (CuII) state at pH 6.0 has been refined, using 1.6 A resolution counter data. The starting co-ordinates were obtained from the 2.7 A electron density map computed with phases derived by the multiple isomorphous replacement method. The model was refined successively by constrained real space, unrestrained reciprocal space, and restrained reciprocal space least-squares methods. The final residual R value is 0.17 for 8285 reflections (I greater than 2 sigma (I)). It is estimated that the root-mean-square standard deviation of the atomic positions is 0.1 A when averaged over all atoms, and 0.05 A for the Cu ligand atoms alone. The refined structure retains all the essential features of the 2.7 A model. The co-ordination geometry of the copper atom is confirmed as being distorted tetrahedral. The two Cu-N(His) bonds, 2.10 and 2.04 A, are within the range normally found in low molecular weight CuII complexes with Cu-N(imidazole) bonds. The Cu-S(Cys) bond, 2.13 A, is also normal, but the Cu-S(Met) bond, 2.90 A, is sufficiently long to raise important questions about its significance. The hydrogen-bonding and secondary structure can now be assigned confidently. Forty-four water molecules are included in the final model. Repetition of the refinement, using new data to 1.9 A resolution recorded from crystals at pH 4.2, has led to a residual R value of 0.16 for 6060 reflections (I greater than sigma (I)). There are few significant changes in the structure of poplar CuII-plastocyanin between pH 6.0 and pH 4.2. In particular, the geometry of the copper site is not affected. The observed changes in redox behaviour of plastocyanin at low pH are therefore unlikely to be connected with structural changes in the oxidized form of the protein. A number of features of the molecular structure appear to be directly related to the function of plastocyanin as an electron carrier in photosynthesis. Comparison between the known amino acid sequences of 67 plant plastocyanins reveals 52 conserved and 11 conservatively substituted residues in a total of 99. If three algal plastocyanin sequences are included in the comparison, there are still 26 conserved and 12 conservatively substituted residues. In many cases, the importance of these residues in determining the tertiary structure can be rationalized.

Amino Acid Sequence↗