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H J Dyson

Publications and source records attributed to H J Dyson.

At least 73 records · Page 4Linked to original sources
PubMed1997

Chemical shift dispersion and secondary structure prediction in unfolded and partly folded proteins.

The intrinsic chemical shift dispersion for 15N, 1HN, 13C(alpha), 1H(alpha), 13C(beta) and 13CO resonances has been evaluated utilizing complete resonance assignment data for unfolded apomyoglobin, together with two other unfolded and five folded proteins, obtained from the literature. The dispersion of 13C(alpha), 1H(alpha), and 13C(beta) resonances for the unfolded proteins is poor, whereas the dispersion of 15N, 1HN and 13CO is much greater, reflecting the sensitivity of these nuclei to the nature of the neighboring amino acid in the primary sequence. By contrast, the dispersion of the 13C(alpha), 1H(alpha), and 13C(beta) nuclei are much greater in the folded proteins, reflecting the well-known dependence of the environments of these nuclei on secondary and tertiary structure. These differences in chemical shift dispersion dictate differences in strategies for resonance assignment in unfolded proteins compared with those most commonly used for folded proteins. Strategies utilizing the superior chemical shift dispersion of the 15N, 1HN and, in particular, the 13CO nuclei, are indicated for use with unfolded or partially folded proteins.

Animals↗
PubMed1997

Solution structure of the KIX domain of CBP bound to the transactivation domain of CREB: a model for activator:coactivator interactions.

The nuclear factor CREB activates transcription of target genes in part through direct interactions with the KIX domain of the coactivator CBP in a phosphorylation-dependent manner. The solution structure of the complex formed by the phosphorylated kinase-inducible domain (pKID) of CREB with KIX reveals that pKID undergoes a coil-->helix folding transition upon binding to KIX, forming two alpha helices. The amphipathic helix alphaB of pKID interacts with a hydrophobic groove defined by helices alpha1 and alpha3 of KIX. The other pKID helix, alphaA, contacts a different face of the alpha3 helix. The phosphate group of the critical phosphoserine residue of pKID forms a hydrogen bond to the side chain of Tyr-658 of KIX. The structure provides a model for interactions between other transactivation domains and their targets.

Amino Acid Sequence↗
PubMed1997

Structure of the recombinant full-length hamster prion protein PrP(29-231): the N terminus is highly flexible.

The prion diseases seem to be caused by a conformational change of the prion protein (PrP) from the benign cellular form PrPC to the infectious scrapie form PrPSc; thus, detailed information about PrP structure may provide essential insights into the mechanism by which these diseases develop. In this study, the secondary structure of the recombinant Syrian hamster PrP of residues 29-231 [PrP(29-231)] is investigated by multidimensional heteronuclear NMR. Chemical shift index analysis and nuclear Overhauser effect data show that PrP(29-231) contains three helices and possibly one short beta-strand. Most striking is the random-coil nature of chemical shifts for residues 30-124 in the full-length PrP. Although the secondary structure elements are similar to those found in mouse PrP fragment PrP(121-231), the secondary structure boundaries of PrP(29-231) are different from those in mouse PrP(121-231) but similar to those found in the structure of Syrian hamster PrP(90-231). Comparison of resonance assignments of PrP(29-231) and PrP(90-231) indicates that there may be transient interactions between the additional residues and the structured core. Backbone dynamics studies done by using the heteronuclear [1H]-15N nuclear Overhauser effect indicate that almost half of PrP(29-231), residues 29-124, is highly flexible. This plastic region could feature in the conversion of PrPC to PrPSc by template-assisted formation of beta-structure.

Amino Acid Sequence↗
PubMed1997

Populating the equilibrium molten globule state of apomyoglobin under conditions suitable for structural characterization by NMR.

Conditions have been determined under which the equilibrium molten globule state of apomyoglobin is stable and remains monomeric for periods of time sufficient for the application of three-dimensional heteronuclear NMR experiments. The quality of initial two-dimensional NMR spectra suggests that sequence-specific assignments can be made for a majority of the protein resonances under these conditions. A pH titration of the protein followed using two-dimensional 1H-15N correlation experiments indicates that the equilibrium intermediate undergoes fast exchange on the chemical shift time scale with the unfolded state and intermediate time scale exchange with the native state, and suggests a strategy to assist with backbone resonance assignments. The conditions and techniques described may be applicable to the characterization of other equilibrium folding intermediates.

Apoproteins↗
PubMed1997

Contribution of increased length and intact capping sequences to the conformational preference for helix in a 31-residue peptide from the C terminus of myohemerythrin.

In order to examine the effects of chain length on the propensity of short peptides to form helix-like structures in aqueous solution, we have studied a peptide of 31 residues consisting of the C-terminal sequence (residues 88-118) of the four-helix bundle protein myohemerythrin from Themiste zostericola. This peptide, termed MDC, represents the final two elements of secondary structure in the protein, the D-helix and the C-terminal loop sequence, together with a five-residue sequence at the N terminus corresponding to the linker between the C- and D-helices. An N-capping sequence, VDAKNV, immediately precedes the D-helix sequence, and a C-capping sequence, VNHIKGT, corresponding to the alphaL termination motif, occurs at the C-terminal end. The effect of replacement of a cysteine residue in the middle of the sequence with an alanine was explored by the comparison of the MDC peptide and a 16-residue peptide representing the sequence of the D-helix alone, both containing the change Cys99Ala. Significant changes in the NMR and CD spectra were seen for both peptides compared to the wild-type sequence. A comparison of the fluorescence spectra of the wild-type and Cys99Ala peptides indicated that a specific interaction between the side chains of Cys 99 and Trp 102 acts to quench the fluorescence of the tryptophan ring and probably contributes a component that distorts the CD spectrum of the wild-type peptide at approximately 220-235 nm. The effect of an increase in the length of the peptide, with the incorporation of capping sequences derived from the native sequence, was explored by NMR and CD spectroscopy of the 31-residue and 16-residue peptides in aqueous solution and in TFE/water mixtures. Evidence for the formation of a significant population of helical conformers in the region of the MDC peptide corresponding to the D-helix was observed in aqueous solution using CD and NMR spectroscopy. The C-terminal 10 residues of the MDC peptide behave in solution in a manner identical to that of a 10-residue peptide with the same sequence; a highly specific local interaction between an aromatic ring and a glycine amide proton appears to be retained in the longer peptide. Upon addition of trifluoroethanol (TFE), significant shifts are observed in a number of resonances in the NMR spectrum, and both chemical shifts and NOEs provide evidence for a higher population of helix in the D-helix region of the peptide in TFE. However, TFE is unable to promote the propagation of helix beyond the N-cap or alphaL termination motifs, and the specific local interaction observed in the C-terminal sequence is retained in TFE. The CD spectrum in TFE shows an increase in the proportion of helix, to an overall maximum of approximately 55% helix at 50% v/v TFE, corresponding to approximately 100% helix in the D-helix sequence of the peptide, since the N and C termini of the MDC peptide are not helical according to the NMR spectra. The high proportion of helix observed in the D-helix sequence of the longer MDC peptide demonstrates that the presence of intact capping sequences can constrain the peptide conformational ensemble to resemble that seen in the native protein. A compendium of results from this and previous peptide studies has also led to a novel observation, the existence of a correlation between the amide proton chemical shift and temperature coefficient.

Amino Acid Sequence↗
PubMed1997

Effects of buried charged groups on cysteine thiol ionization and reactivity in Escherichia coli thioredoxin: structural and functional characterization of mutants of Asp 26 and Lys 57.

To investigate the role of Asp 26 and Lys 57, two conserved, buried residues, in the redox mechanism of Escherichia coli thioredoxin (Trx), three mutant proteins, Asp 26 --> Ala (D26A), Lys 57 --> Met (K57M), and the double mutant D26A/K57M, were prepared, replacing the charged amino acids with hydrophobic residues with similar sizes. Both the oxidized (Trx-S2) and reduced [Trx-(SH)2] forms of the mutant thioredoxins are fully folded and similar in overall structure to the wild-type protein (wt). The structure of the active site hydrophobic surface is unchanged by the mutation of Asp 26 and Lys 57, since DNA polymerase activity in the 1:1 complex of the T7 gene 5 protein and mutant Trx-(SH)2 shows similar Kd values (approximately 5 nM) for both mutants and wt. In contrast, redox reactions involving thioredoxin as a catalyst of the reduction of disulfides or oxidation of dithiols are strongly affected by the mutations. In the reaction of Trx-S2 with thioredoxin reductase at pH 8.0, the kcat/Km value for the D26A mutant is decreased by a factor of 10 from that of wt, while the value for the D26A/K57M mutant is reduced 40-fold. The activity of Trx-(SH)2 as a protein disulfide reductase was measured with insulin, using fluorescence to detect oxidation of thioredoxin. At 15 degrees C and pH 8.0, both the D26A and K57M mutants showed 5--10-fold decreases in rates of reaction compared to those of the wild type, and the pH-rate profiles for the mutants were shifted 1 (K57M) and 2 (D26A) units to higher pH compared with the wt curve. NMR measurements for the three mutant proteins indicate that the proteins have the same global fold as that of the wild type, although changes in the chemical shifts of a number of resonances indicate local structural changes in the active site region. The resonances of oxidized D26A and D26A/K57M are pH-independent between pH 6.0 and 10.0, confirming the identification of the active site group titrating with a pKa of 7.5 in wt Trx-S2 as Asp 26. A profound change in the pKa of Asp 26, from 7.5 in the wild type to 9.4 in the mutant, is observed for K57M Trx-S2. The pH-dependent behavior of the resonances is affected in all mutant Trx-(SH)2 proteins. A single pKa shifted to higher values is observed on both the Cys 32 and Cys 35 Cbeta resonances. Ultraviolet absorbance measurements (A240) as a function of pH for wt Trx-(SH)2 demonstrate that the cysteine thiols titrate with apparent pK(a)s of about 7.1 and 9.9. The mutant proteins each show a single transition in the A240 measurements, with a midpoint at pH 7.8-8.0, consistent with the NMR results. The change in absorbance at 240 nm with increasing pH indicates that the number of thiols titrating in each mutant is greater than one but less than two. It is clear that both thiol pK(a)s have been significantly shifted by the mutations. The Cys 32 pKa is moved from 7.1 in wt to 7.8-8.0 in the mutants. The value of the Cys 35 pKa either is indistinguishable from that of Cys 32, thus accounting for more than one thiol titrating in the UV absorbance measurements or else is shifted to much higher pHs (> 10) where its transition is masked in both UV and NMR measurements by the effects of ionization of the tyrosine residues and unfolding of the protein. Our results strongly suggest that the buried Asp 26 carboxyl and Lys 57 epsilon-amino groups significantly affect the pK(a)s of the active site thiols, particularly that of the exposed low-pKa thiol Cys 32, thereby enhancing the rates of thiol-disulfide reactions at physiological pH.

Amino Acid Sequence↗
PubMed1997

Structure-based design of a constrained peptide mimic of the HIV-1 V3 loop neutralization site.

Antigenic variation among different HIV-1 isolates has been a major problem in the development of an effective vaccine against AIDS. Peptide vaccines incorporating structural elements common to groups of viral isolates, such as the clade subtypes of HIV-1, hold promise; however, the design of such immunogens has been hampered by the lack of specific structural information on the viral proteins to be targeted. As part of a structure-based approach to this problem, we report the design and characterization of a conformationally restricted peptide analog (Aib142) of a highly conserved HIV-1 clade-B sequence from the third variable loop of the membrane glycoprotein gp120. The design strategy incorporates peptide conformational data derived from crystal structure analysis of an MN-isolate peptide (RP142) in complex with the Fab fragment (Fab59.1) of a broadly neutralizing antibody. The synthetic peptide (Aib142) replaces an alanine residue within the V3 loop epitope sequence GPGRAF by the conformationally restricted helicogenic alpha-aminoisobutyryl residue. As expected, the crystal structure of the Fab 59.1-Aib142 complex at 2.8 A resolution shows that the peptide interacts very similarly with the neutralizing antibody. Proton nuclear magnetic resonance (NMR) studies indicate that the free Aib142 peptide is indeed more ordered in solution with a conformational preference that corresponds to the X-ray structure of its Fab-bound form. Aib142 thus represents the first step in the design of conformationally constrained peptide analogs built to mimic biologically relevant structural forms of HIV-1 neutralization sites.

AIDS Vaccines↗
PubMed1997

Folding propensities of peptide fragments of myoglobin.

Myoglobin has been studied extensively as a paradigm for protein folding. As part of an ongoing study of potential folding initiation sites in myoglobin, we have synthetized a series of peptides covering the entire sequence of sperm whale myoglobin. We report here on the conformation preferences of a series of peptides that cover the region from the A helix to the FG turn. Structural propensities were determined using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution, trifluoroethanol, and methanol. Peptides corresponding to helical regions in the native protein, namely the B, C, D, and E helices, populate the alpha region of (phi, psi) space in water solution but show no measurable helix formation except in the presence of trifluoroethanol. The F-helix sequence has a much lower propensity to populate helical conformations even in TFE. Despite several attempts, we were not successful in synthesizing a peptide corresponding to the A-helix region that was soluble in water. A peptide termed the AB domain was constructed spanning the A- and B-helix sequences. The AB domain is not soluble in water, but shows extensive helix formation throughout the peptide when dissolved in methanol, with a break in the helix at a site close to the A-B helix junction in the intact folded myoglobin protein. With the exception of one local preference for a turn conformation stabilized by hydrophobic interactions, the peptides corresponding to turns in the folded protein do not measurably populate beta-turn conformations in water, and the addition of trifluoroethanol does not enhance the formation of either helical or turn structure. In contrast to the series of peptides described here, either studies of peptides from the GH region of myoglobin show a marked tendency to populate helical structures (H), nascent helical structures (G), or turn conformations (GH peptide) in water solution. This region, together with the A-helix and part of the B-helix, has been shown to participate in an early folding intermediate. The complete analysis of conformational properties of isolated myoglobin peptides supports the hypothesis that spontaneous secondary structure formation in local regions of the polypeptide may play an important role in the initiation of protein folding.

Amino Acid Sequence↗
PubMed1997

Absence of a stable intermediate on the folding pathway of protein A.

The B-domain of protein A has one of the simplest protein topologies, a three-helix bundle. Its folding has been studied as a model for elementary steps in the folding of larger proteins. Earlier studies suggested that folding might occur by way of a helical hairpin intermediate. Equilibrium hydrogen exchange measurements indicate that the C-terminal helical hairpin could be a potential folding intermediate. Kinetic refolding experiments were performed using stopped-flow circular dichroism and NMR hydrogen-deuterium exchange pulse labeling. Folding of the entire molecule is essentially complete within the 6 ms dead time of the quench-flow apparatus, indicating that the intermediate, if formed, progresses rapidly to the final folded state. Site-directed mutagenesis of the isoleucine residue at position 16 was used to generate a variant protein containing tryptophan (the 116 W mutant). The formation of the putative folding intermediate was expected to be favored in this mutant at the expense of the native folded form, due to predicted unfavorable steric interactions of the bulky tryptophan side chain in the folded state. The 116 W mutant refolds completely within the dead time of a stopped-flow fluorescence experiment. No partly folded intermediate could be detected by either kinetic or equilibrium measurements. Studies of peptide fragments suggest that the protein A sequence has an intrinsic propensity to form a helix II/helix III hairpin. However, its stability appears to be marginal (of the order of 1/2 kT) and it could not be an obligatory intermediate on a defined folding pathway. These results explicitly demonstrate that the protein A B domain folds extremely rapidly by an apparent two-state mechanism without formation of stable partly folded intermediates. Similar mechanisms may also be involved in the rapid folding of subdomains of larger proteins to form the compact molten globule intermediates that often accumulate during the folding process.

Amino Acid Sequence↗
PubMed1996

Gene synthesis, high-level expression and assignment of backbone 15N and 13C resonances of soybean leghemoglobin.

A synthetic gene for apoleghemoglobin-a from soybean, optimized for expression in Escherichia coli has been designed and synthesized by a recursive polymerase chain reaction technique. The protein has been expressed with high efficiency and a purification protocol has been developed. The holoprotein is readily reconstituted by the addition of heme. 15N- and 15N,13C-labeled samples were produced and backbone 15N and 13C assignments were determined by 2D and 3D NMR spectroscopy. Comparison of the chemical shifts of 13C(alpha) and 13CO with random coil shifts revealed a pattern of secondary structure which correlates well with the one previously derived from homonuclear NMR data and low-resolution X-ray crystallography.

Amino Acid Sequence↗
PubMed1996

NMR solution structure of Cu(I) rusticyanin from Thiobacillus ferrooxidans: structural basis for the extreme acid stability and redox potential.

The solution structure of the Cu(I) form of the rusticyanin from Thiobacillus ferrooxidans has been calculated from a total of 1979 distance and dihedral angle constraints derived from 1H, 13C and 15N NMR spectra. The structures reveal two beta-sheets, one of six strands and one of seven strands that are tightly packed in a beta-barrel or beta-sandwich arrangement, and a short helix that extends on the outside of one of the sheets to form a second hydrophobic core. The copper coordination sphere is composed of the standard type I ligands (His2CysMet) in a distorted tetrahedral arrangement. The copper-binding site is located within a hydrophobic region at one end of the molecule, surrounded by a number of aromatic rings and hydrophobic residues. This configuration probably contributes to the acid stability of the copper site, since close association of the aromatic rings with the histidine ligands would sterically hinder their dissociation from the copper. An electrostatic analysis based on a comparison of the structures of rusticyanin and French bean plastocyanin shows that factors determining the high redox potential of rusticyanin include contributions from charged side-chains and from the disposition of backbone peptide dipoles, particularly in the 81 to 86 region of the sequence and the ligand cysteine residue. These interactions should also contribute to the acid stability by inhibiting protonation of His143.

Azurin↗
PubMed1996

Replacement of Trp28 in Escherichia coli thioredoxin by site-directed mutagenesis affects thermodynamic stability but not function.

Escherichia coli thioredoxin contains two tryptophan residues (Trp28 and Trp31) situated close to the active site disulfide/dithiol. In order to probe the structural and functional roles of tryptophan in the mechanism of E. coli thioredoxin (Trx), we have replaced Trp28 with alanine using site-directed mutagenesis and expressed the mutant protein W28A in E. coli. Changes in the behavior of the mutant protein compared with the wild-type protein have been monitored by a number of physical and spectroscopic techniques and enzyme assays. As expected, removal of a tryptophan residue causes profound changes in the fluorescence spectrum of thioredoxin, particularly for the reduced protein (Trx-(SH)2), and to a lesser extent for the oxidized protein (Trx-S2). These results show that the major contribution to the strongly quenched fluorescence of Trx-S2 in both wild-type and mutant proteins is from Trp31, whereas the higher fluorescence quantum yield of Trx-(SH)2 in the wild-type protein is dominated by the emission from Trp28. The fluorescence, CD, and 1H NMR spectra are all indicative that the mutant protein is fully folded at pH 7 and room temperature, and, despite the significance of the change, from a tryptophan in close proximity to the active site to an alanine, the functions of the protein appear to be largely intact. W28A Trx-S2 is a good substrate for thioredoxin reductase, and W28A Trx-(SH)2 is as efficient as wild-type protein in reduction of insulin disulfides. DNA polymerase activity exhibited by the complex of phage T7 gene 5 protein and Trx-(SH)2 is affected only marginally by the W28A substitution, consistent with the buried position of Trp28 in the protein. However, the thermodynamic stability of the molecule appears to have been greatly reduced by the mutation: guanidine hydrochloride unfolds the protein at a significantly lower concentration for the mutant than for wild type, and the thermal stability is reduced by about 10 degrees C in each case. The stability of each form of the protein appears to be reduced by the same amount, an indication that the effect of the mutation is identical in both forms of the protein. Thus, despite its close proximity to the active site, the Trp28 residue of thioredoxin is not apparently essential to the electron transfer mechanism, but rather contributes to the stability of the protein fold in the active site region.

Amino Acid Sequence↗
PubMed1996

Direct measurement of the aspartic acid 26 pKa for reduced Escherichia coli thioredoxin by 13C NMR.

Because of interference from the pH-dependent behavior of nearby groups in the active site of Escherichia coli thioredoxin, the pKa of the buried carboxyl group of the aspartic acid at position 26 has been difficult to quantitate. We report a direct measurement of this pKa using an NMR method utilizing the correlation between the C beta H proton resonances and the 13CO of the titrating carboxyl group. The experiments show unequivocally that the pKa is 7.3-7.5, rather than the value of 9 or greater recently proposed by Wilson, N. A., et al. [(1995) Biochemistry 34, 8931-8939]. The assignment of the titrating resonances to Asp 26 is unambiguous: the values of the C beta H chemical shifts correspond exactly to those of Asp 26, and their titration in the pH range 5.7-10.0 is the same as that observed previously for the proton resonances alone. In addition, the chemical shift of the carboxyl 13C resonance at pH 5.7 is upfield of those of the other carboxyl and carboxamide resonances, diagnostic for a protonated carboxyl group. The resonances assigned to Asp 26 are the only ones that titrate in the pH range 5.7-10.5. None of the other aspartate and glutamate residues in the molecule are titrated in this pH range, consistent with their positions on the surface of the molecule. The pKa measured for Asp 26 in reduced thioredoxin is identical within experimental error to that measured in the oxidized form of the protein. This is significant for the reductive mechanism of thioredoxin: the buried salt bridged/hydrogen-bonded side chains of Asp 26 and Lys 57 are likely to contribute to the facility of the reaction by providing a convenient source and sink for protons in the hydrophobic environment of the complex between thioredoxin and its substrates.

Amino Acid Sequence↗
PubMed1996

Solution conformation of an immunogenic peptide derived from the principal neutralizing determinant of the HIV-2 envelope glycoprotein gp125.

BACKGROUND: The conformational preferences of a number of peptides with sequences related to the envelope glycoproteins of HIV-1 have been investigated in the past few years. Similar studies have not been made for HIV-2, which is a distinct virus with similar physiological effects to those of HIV-1. The discovery of common structural features would be a promising route to the design of immunogens for generally effective HIV vaccines. We present the results of an NMR conformational study of a sequence deriving from the V3 loop of HIV-2. RESULTS: Three synthetic immunogenic peptides were studied, of 12, 22 and 39 amino acids in length, all containing a central Met-Ser-Gly-Arg sequence conserved among a number of HIV-2 isolates. In addition, the 39-mer contained a disulfide bond between cysteine residues close to the ends of the molecule, forming a loop that is thought to comprise an important structural and immunological component of the intact glycoprotein. All three peptides display well defined beta-turns in the Met-Ser-Gly-Arg sequence, independent of the integrity of the disulfide bond. No other conformational preferences for folded conformations were found for the peptides. CONCLUSIONS: The presence of a beta-turn in the Met-Ser-Gly-Arg sequence is strikingly similar to the behavior seen for the corresponding principal neutralizing determinant sequence from gp120 of HIV-1 and argues, in the absence of information of the three-dimensional structure of the intact proteins, for a similarity in the structure of this region that could be exploited in the design of synthetic peptide vaccines generally effective against HIV infections.

AIDS Vaccines↗
PubMed1996

Insights into protein folding from NMR.

NMR has emerged as an important tool for studies of protein folding because of the unique structural insights it can provide into many aspects of the folding process. Applications include measurements of kinetic folding events and structural characterization of folding intermediates, partly folded states, and unfolded states. Kinetic information on a time scale of milliseconds or longer can be obtained by real-time NMR experiments and by quench-flow hydrogen-exchange pulse labeling. Although NMR cannot provide direct information on the very rapid processes occurring during the earliest stages of protein folding, studies of isolated peptide fragments provide insights into likely protein folding initiation events. Multidimensional NMR techniques are providing new information on the structure and dynamics of protein folding intermediates and both partly folded and unfolded states.

Animals↗
PubMed1995

Proton sharing between cysteine thiols in Escherichia coli thioredoxin: implications for the mechanism of protein disulfide reduction.

Proton sharing between acidic groups has been observed in the active sites of several enzymes, including bacteriorhodopsin, aspartic proteases, and ribonuclease HI. We here report NMR observations suggestive of proton sharing between cysteine thiols in the active site of the oxidation-reduction enzyme thioredoxin. The pKas of the two cysteine thiols in the Escherichia coli protein are removed from the expected value of 8.4 by approximately 1 pH unit in either direction, upward and downward. Further, the C beta resonances of both residues show clearly the effects of both of these pKas, indicating that the titrations of the two thiol groups are intimately linked. This behavior strongly suggests that the low pKa ascribed to the deprotonation of the Cys 32 thiol most likely arises through the interaction and close approach of the thiol of Cys 35, with the thiolate anion of Cys 32 stabilized through the sharing of the remaining thiol proton, nominally attached to Cys 35. These observations provide a rationale for the mediation of active site pH control, an important aspect of the mechanism of thioredoxin and other proteins with catalytic thioredoxin domains, such as protein disulfide isomerases.

Carbon Isotopes↗
PubMed1995

Gene synthesis, high-level expression, and mutagenesis of Thiobacillus ferrooxidans rusticyanin: His 85 is a ligand to the blue copper center.

An artificial gene of the blue copper protein rusticyanin from Thiobacillus ferrooxidans was constructed from eight overlapping oligonucleotides in a recursive "one-pot" polymerase chain reaction. The gene was placed behind the T7/lacOR promoter of pET24a and expressed in Escherichia coli as a soluble protein. A purification scheme involving a pH titration step, cation-exchange chromatography, and reverse-phase HPLC separation provided yields of the apoprotein ranging from 70 to 100 mg/L of cell culture; reconstitution with Cu(II) is quantitative at pH 3.4-5.5. The redox reactions and the electronic absorption and EPR spectra of the recombinant Cu(II)-rusticyanin and NMR spectra of the reduced holoprotein are indistinguishable from those of the protein derived from T. ferrooxidans. Rusticyanin possesses the phylogenetically conserved carboxy-terminal loop of three copper ligands (Cys 138, His 143, and Met 148), but the identity of the fourth ligand was not clear from sequence homology to other blue copper proteins. To address this question directly, we have prepared two site-specific mutants where two of the proposed ligands, Asp 73 and His 85, have been replaced with alanine. The Asp73Ala mutant retained the electronic properties of the wild-type blue copper center (absorption maxima at 452, 597, and 750 nm), whereas the His85Ala variant gave rise to a green type 1 copper protein (absorption maxima at 455 and 618 nm).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗