A study on the role of evolutionarily invariant leucine 32 of cytochrome c

To investigate the role of evolutionarily invariant leucine 32 of horse cytochrome c, analogs of residues 28-38, (28-38), each containing a substituted amino acid at positions 32 or 35 were synthesized using Merrifield's method. Position 35 is leucine in horse cytochrome c but replaced by nonpolar amino acids in some species. The ability of the analogs to bind to the two-fragment complex of ferri- or ferro heme fragment (1-25)H and apofragment (39-104) was measured using gel filtration and equilibrium dialysis. Replacement of leucine 32 with isoleucine, for example, increased the dissociation constant by more than 400-fold for the ferrous complex. In contrast, replacement of leucine 35 with isoleucine seems to increase it only by a small degree. Since both leucine 32 and leucine 35 are completely buried within the structure, hydrophobic interaction would not explain this striking difference. However, thermodynamic analyses and absorption spectra of the ferric complex have indicated that replacement with norvaline of leucine 32 increases both delta H and delta S (more positive) associated with formation of an intermediate three-fragment complex and decreases both delta H and delta S (more negative) associated with transformation from the intermediate to the ground state, resulting in weakening the methionine 80--S--heme-Fe bond formed in the latter step. Taking the results together with the fragment exchange studies on the ferrous complex and available evidence, we suggest that the interaction involving leucine 32 would be coupled not only with the methionine 80--S--heme-Fe bond but also with the energy state of other distant residues such as tryptophan 59, generating extra energy for modulating the binding of the complex, i.e. the force of folding. In contrast, leucine 35 would be less important even if it were involved in such coupling.     

Structure-function studies on bacteriorhodopsin. VIII. Substitutions of the membrane-embedded prolines 50, 91, and 186: the effects are determined by the substituting amino acids

To study their role in the structure and function of bacteriorhodopsin, three prolines, presumed to be in the membrane-embedded alpha-helices, have been individually replaced as follows: Pro-50 and Pro-91 each by Gly and Ala and Pro-186 by Ala, Gly, and Val. The mutants of Pro-50 and Pro-91 all showed normal chromophore and proton pumping. However, the rates of regeneration of the chromophore in Pro-50----Ala, Pro-91----Ala and ----Gly with all-trans-retinal were about 30-fold slower than that in the wild-type, whereas the chromophore regeneration rate in Pro-50----Gly was 10-fold faster than in the wild-type. While, Pro-186----Ala regenerated the wild-type chromophore, the mutants Pro-186----Val and Pro-186----Gly showed large blue shifts (about 80 nm) in the chromophore regenerated with all-trans-retinal and showed no apparent dark-light adaptation. Pro-186----Gly first regenerated the wild-type chromophore with 13-cis-retinal which was thermally unstable and rapidly converted to the blue-shifted chromophore obtained with all-trans-retinal. High salt concentration restored the wild-type purple chromophore in the Pro-186----Gly mutant. Thus, in this mutant, the protein interconverts between two conformational states. Pro-186----Ala and Pro-186----Gly showed about 65%, whereas Pro-186----Val showed 10-20% of the normal proton pumping.     

The rate and structural consequences of proline cis-trans isomerization in calbindin D9k: NMR studies of the minor (cis-Pro43) isoform and the Pro43Gly mutant

The EF-hand calcium-binding protein, calbindin D9k, exists in solution in the calcium-loaded state, as a 1:3 equilibrium mixture of two isoforms, the result of cis-trans isomerism at the Gly42-Pro43 peptide bond [Chazin et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2195-2198]. Nuclear magnetic resonance (NMR) studies of the minor (cis-Pro43) isoform and the Pro43----Gly mutant are reported here. The rate of cis----trans isomerization at the Pro43 peptide bond in the wild-type protein was determined by line-shape analysis at elevated temperatures, using a sample in which all amino acids, except Ser and Val, were deuterated. The cis----trans rate is calculated to be 0.2 s-1 at 25 degrees C, corresponding to a free energy of activation, delta G, of 77 kJ/mol. The complete sequence-specific 1H NMR assignments of the cis-Pro43 isoform and the Pro43----Gly mutant in the calcium-loaded state have been obtained by using standard methods combined with comparisons to the previously assigned major (trans-Pro43) isoform. This has permitted detailed comparative analysis of 1H NMR chemical shifts, backbone scalar coupling constants, and nuclear Overhauser effects. The minor isoform has a global fold that is identical with that of the major isoform. Structural changes imposed by cis-trans isomerization at Pro43 are highly localized to the linker loop (containing Pro43) that joins the two EF hands. The Pro43----Gly mutant has a global fold that is identical with the wild-type protein, but does not exhibit conformational heterogeneity. Only very limited structural differences are observed between mutant and wild-type protein, and these are also highly localized to the linker loop. The ion-binding properties of the mutant, as determined by 43Ca and 113Cd NMR, are found to be very similar to the wild-type protein. These results provide crucial evidence that justifies the calculation of high-resolution three-dimensional structures of the Pro43Gly mutant, rather than of the conformationally heterogeneous wild-type protein.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

Effect of cavity-modulating mutations on the stability of Escherichia coli ribonuclease HI.

The size of the cavity around Ser68 of Escherichia coli ribonuclease HI was modulated by amino acid substitutions to examine the effects on the stability of the enzyme. Five mutant proteins, Ser68----Gly, Ser68----Ala, Ser68----Thr, Ser68----Val and Ser68----Leu, were constructed. Each of the mutant proteins exhibited at least 40% of the enzyme activity of the wild-type protein. The stabilities of the mutant proteins were determined from urea-denaturation and thermal-denaturation curves. Among the five mutations, only the Ser----Val mutation resulted in an increase in the stability of the enzyme. The melting temperature, tm, at pH 3.0 of the mutant protein Ser68----Val was increased by 1.9 degrees C. Its free-energy change of unfolding in the absence of urea, delta G(H2O), and the midpoint of the denaturation curve, [D]1/2, were also increased by 5.4 kJ/mol and 0.18 M, respectively. The increase in the stability of the enzyme is probably due to the filling of the cavity space around Ser68 by valine. However, the mutation of Ser68 to glycine or leucine residues resulted in a considerable decrease in stability. In these cases, some conformational changes occur, as suggested by the CD and 1H-NMR spectra of these mutant proteins.     

Structure and stability effects of the mutation of glycine 34 to serine in Rhodobacter capsulatus cytochrome c(2)

Gly 34 and the adjacent Pro 35 of Rhodobacter capsulatus cytochrome c(2) (or Gly 29 and Pro 30 in vertebrate cytochrome c) are highly conserved side chains among the class I c-type cytochromes. The mutation of Gly 34 to Ser in Rb. capsulatus cytochrome c(2) has been characterized in terms of physicochemical properties and NMR in both redox states. A comparison of the wild-type cytochrome c(2), the G34S mutation, and the P35A mutation is presented in the context of differences in chemical shifts, the differences in NOE patterns, and structural changes resulting from oxidation of the reduced cytochrome. G34S is substantially destabilized relative to wild-type (2.2 kcal/mol in the oxidized state) but similarly destabilized relative to P35A. Nevertheless, differences in terms of the impact of the mutations on specific structural regions are found when comparing G34S and P35A. Although available data indicates that the overall secondary structure of G34S and wild-type cytochrome c(2) are similar, a number of both perturbations of hydrogen bond networks and interactions with internal waters are found. Thus, the impact of the mutation at position 35 is propagated throughout the cytochrome but with alterations at defined sites within the molecule. Interestingly, we find that the substitution of serine at position 34 results in a perturbation of the heme beta meso and the methyl-5 protons. This suggests that the hydroxyl and beta carbon are positioned away from the solvent and toward the heme. This has the consequence of preferentially stabilizing the oxidized state in G34S, thus, altering hydrogen bond networks which involve the heme propionate, internal waters, and key amino acid side chains. The results presented provide important new insights into the stability and solution structure of the cytochrome c(2).     

Specificity determinants of rat tissue kallikrein probed by site-directed mutagenesis.

Site-specific mutagenesis was employed to study structure-function relationships at the substrate binding site of rat tissue kallikrein. Four kallikrein mutants, the Pro219 deletion (P219del), the 34-38 loop Tyr-Tyr-Phe-Gly to Ile-Asn mutation [YYFG(34-38)IN], the Trp215----Gly exchange (W215G) and the double mutant with Tyr99----His and Trp215----Gly exchange (Y99H:W215G) were created by site-directed mutagenesis to probe their function in substrate binding. The mutant proteins were expressed in Escherichia coli at high levels and analyzed by Western blot. These mutant enzymes were purified to apparent homogeneity. Each migrated as a single band on SDS-PAGE, with slightly lower molecular mass (36 kDa) than that of the native enzyme, (38 kDa) because of their lack of glycosylation. The recombinant kallikreins are immunologically identical to the native enzyme, displaying parallelism with the native enzyme in a direct radioimmunoassay for rat tissue kallikrein. Kinetic analyses of Km and kcat using fluorogenic peptide substrates support the hypothesis that the Tyr99-Trp215 interaction is a major determinant for hydrophobic P2 specificity. The results suggest an important role for the 34-38 loop in hydrophobic P3 affinity and further show that Pro219 is essential to substrate binding and efficient catalysis of tissue kallikrein.     

Mutations in Ser174 and the glycine-rich sequence (Gly149, Gly150, and Thr156) in the beta subunit of Escherichia coli H(+)-ATPase.

A sequence motif in the beta subunit of Escherichia coli F1 (Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr, residue 149-156, where conserved residues are underlined) is one of the glycine-rich sequences found in many nucleotide binding proteins. In this study, we constructed a plasmid carrying all the F0F1 genes. This plasmid gave the highest membrane ATPase activity so far reported. Substitution of beta Gly149 by Ser suppressed the effect of the beta Ser174----Phe mutation (defective H(+)-ATPase), but beta Gly150----Ser substitution did not have this effect. A single mutation (beta Gly149----Ser or beta Gly150----Ser) gave active enzyme with altered divalent cation dependency and azide sensitivity: the beta Gly149----Ser mutant enzyme had 100-fold lower azide sensitivity and essentially no Ca(2+)-dependent activity, but had the wild-type level of Mg(2+)-dependent activity with active oxidative phosphorylation. Introduction of a beta Gly149----Ser or beta Gly150----Ser mutation with the beta Ser174----Phe mutation also lowered the Ca(2+)-dependent activity and azide sensitivity. Consistent with our previous findings (Takeyama, M., Ihara, K., Moriyama, Y., Noumi, T., Ida, K., Tomioka, N., Itai, A., Maeda, M., and Futai, M. (1990) J. Biol. Chem. 265, 21279-21284), a beta Thr156----Ala or Cys mutation impaired ATPase activity, suggesting that the hydroxyl moiety at position 156 is essential for the catalytic activity. The possible location of the catalytic site including divalent cation binding site(s) is discussed.     

Stabilization of lambda repressor against thermal denaturation by site-directed Gly----Ala changes in alpha-helix 3

Oligonucleotide-directed mutagenesis has been used to replace alpha-helical glycines in the N-terminal domain of lambda repressor with alanines. Since alanine is a significantly better helix-forming residue than glycine, these changes were predicted to have a stabilizing effect. We show that the Gly46----Ala substitution, the Gly48----Ala substitution, and the double substitution increase the melting temperature of the N-terminal domain by 3-6 degrees.     

The structural bases of antibiotic resistance in the clinically derived mutant beta-lactamases TEM-30, TEM-32, and TEM-34

Widespread use of beta-lactam antibiotics has promoted the evolution of beta-lactamase mutant enzymes that can hydrolyze ever newer classes of these drugs. Among the most pernicious mutants are the inhibitor-resistant TEM beta-lactamases (IRTs), which elude mechanism-based inhibitors, such as clavulanate. Despite much research on these IRTs, little is known about the structural bases of their action. This has made it difficult to understand how many of the resistance substitutions act as they often occur far from Ser-130. Here, three IRT structures, TEM-30 (R244S), TEM-32 (M69I/M182T), and TEM-34 (M69V), are determined by x-ray crystallography at 2.00, 1.61, and 1.52 A, respectively. In TEM-30, the Arg-244 --> Ser substitution (7.8 A from Ser-130) displaces a conserved water molecule that usually interacts with the beta-lactam C3 carboxylate. In TEM-32, the substitution Met-69 --> Ile (10 A from Ser-130) appears to distort Ser-70, which in turn causes Ser-130 to adopt a new conformation, moving its O gamma further away, 2.3 A from where the inhibitor would bind. This substitution also destabilizes the enzyme by 1.3 kcal/mol. The Met-182 --> Thr substitution (20 A from Ser-130) has no effect on enzyme activity but rather restabilizes the enzyme by 2.9 kcal/mol. In TEM-34, the Met-69 --> Val substitution similarly leads to a conformational change in Ser-130, this time causing it to hydrogen bond with Lys-73 and Lys-234. This masks the lone pair electrons of Ser-130 O gamma, reducing its nucleophilicity for cross-linking. In these three structures, distant substitutions result in accommodations that converge on the same point of action, the local environment of Ser-130.     

Catalytic triad residue mutation (Asp156----Gly) causing familial lipoprotein lipase deficiency. Co-inheritance with a nonsense mutation (Ser447----Ter) in a Turkish family

We studied the molecular basis of familial Type I hyperlipoproteinemia in two brothers of Turkish descent who had normal plasma apolipoprotein C-II levels and undetectable plasma post-heparin lipoprotein lipase (LPL) activity. We cloned the cDNAs of LPL mRNA from adipose tissue biopsies obtained from these individuals by the polymerase chain reaction and directional cloning into M13 vectors. Direct sequencing of pools of greater than 2000 cDNA clones indicates that their LPL mRNA contains two mutations: a missense mutation changing codon 156 from GAU to GGU predicting an Asp156----Gly substitution and a nonsense mutation changing the codon for Ser447 from UCA to UGA, a stop codon, predicting a truncated LPL protein that contains 446 instead of 448 amino acid residues. Both patients were homozygous for both mutations. Analysis of genomic DNAs of the patients and their family members by the polymerase chain reaction, restriction enzyme digestion (the GAT----GGT mutation abolishes a TaqI restriction site), and allele-specific oligonucleotide hybridization confirms that the patients were homozygous for these mutations at the chromosomal level, and the clinically unaffected parents and sibling were true obligate heterozygotes for both mutations. In order to examine the functional significance of the mutations in this family, we expressed wild type and mutant LPLs in vitro using a eukaryotic expression vector. Five types of LPL proteins were produced in COS cells by transient transfection: (i) wild type LPL, (ii) Asp156----Gly mutant, (iii) Ser447----Ter mutant, (iv) Gly448----Ter mutant, and (v) Asp156----Gly/Ser447----Ter double mutant. Both LPL immunoreactive mass and enzyme activity were determined in the culture media and intracellularly. Immunoreactive LPLs were produced in all cases. The mutant LPLs, Asp156----Gly and Asp156----Gly/Ser447----Ter, were devoid of enzyme activity, indicating that the Asp156----Gly mutation is the underlying defect for the LPL deficiency in the two patients. The two mutant LPLs missing a single residue (Gly448) or a dipeptide (Ser447-Gly448) from its carboxyl terminus had normal enzyme activity. Thus, despite its conservation among all mammalian LPLs examined to date, the carboxyl terminus of LPL is not essential for enzyme activity. We further screened 224 unrelated normal Caucasians for the Ser447----Ter mutation and found 36 individuals who were heterozygous and one individual who was homozygous for this mutation, indicating that it is a sequence polymorphism of no functional significance. Human LPL shows high homology to hepatic triglyceride lipase and pancreatic lipase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of proline residues in the structure and function of a membrane transport protein

By use of site-directed mutagenesis, each prolyl residue in the lac permease of Escherichia coli at positions 28 (putative helix I), 31 (helix I), 61 (helix II), 89 (helix III), 97 (helix III), 123 (helix IV), 192 (putative hydrophilic region 7), 220 (helix VII), 280 (helix VIII), and 327 [helix X; Lolkema, J. S., et al. (1988) Biochemistry 27, 8307] was systematically replaced with Gly, Ala, or Leu or deleted by truncation of the C-terminus [i.e., Pro403 and Pro405; Roepe, P.D., et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3992]. Replacements were chosen on the basis of side-chain helical propensity: Gly, like Pro, is thought to be a "helix breaker", while Ala and Leu are "helix makers". With the exception of Pro28, each prolyl residue can be replaced with Gly or Ala, and Pro403 and -405 can be deleted with the C-terminal tail, and significant lac permease activity is retained. In contrast, when Pro28 is replaced with Gly, Ala, or Ser, lactose transport is abolished, but permease with Ser28 binds p-nitrophenyl alpha-D-galactopyranoside and catalyzes active transport of beta-galactopyranosyl-1-thio-beta-D- galactopyranoside. Replacement of Pro28, -31, -123, -280, or -327 with Leu abolishes lactose transport, while replacement of Pro61, -89, -97, or -220 with Leu has relatively minor effects. None of the alterations in permease activity is due to inability of the mutant proteins to insert into the membrane or to diminished lifetimes after insertion, since the concentration of each mutant permease in the membrane is comparable to that of wild-type permease as judged by immunological analyses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Point mutation of alanine (31) to valine prohibits the folding of reduced lysozyme by sulfhydryl-disulfide interchange

In the preceding paper in this issue, we described the overproduction of one mutant chicken lysozyme in Escherichia coli. Since this lysozyme contained two amino acid substitutions (Ala31----Val and Asn106----Ser) in addition to an extra methionine residue at the NH2-terminus, the substituted amino acid residues were converted back to the original ones by means of oligonucleotide-directed site-specific mutagenesis and in vitro recombination. Thus, four kinds of chicken lysozyme [Met-1Val31Ser106-, Met-1Ser106-, Met-1Val31- and Met-1 (wild type)] were expressed in E. coli. From the results of folding experiments of the reduced lysozymes by sulfhydryl-disulfide interchange at pH 8.0 and 38 degrees C, followed by the specific activity measurements of the folded enzymes, the following conclusions can be drawn: (i) an extra methionine residue at the NH2-terminus reduces the folding rate but does not affect the lysozyme activity of the folded enzyme; (ii) the substitution of Asn106 by Ser decreases the activity to 58% of that of intact native lysozyme without changing the folding rate; and (iii) the substitution of Ala31 Val prohibits the correct folding of lysozyme. Since the wild type enzyme (Met-1-lysozyme) was activated in vitro without loss of specific activity, the systems described in this study (mutagenesis, overproduction, purification and folding of inactive mutant lysozymes) may be useful in the study of folding pathways, expression of biological activity and stability of lysozyme.     

Probing the structure of the linker connecting the reductase and heme domains of cytochrome P450BM-3 using site-directed mutagenesis.

Cytochrome P450BM-3 is a catalytically self-sufficient fatty acid hydroxylase containing one equivalent each of heme, FMN, and FAD. The heme and flavins reside in separate domains connected by a linker peptide. In an earlier study (Govindaraj S, Poulos T, 1995, Biochemistry 34:11221-11226), we found that the length but not the sequence of the linker connecting the heme and reductase domains is important for enzyme activity. In the present study, residues in the linker were replaced with Pro and Gly to probe the role that regular secondary structure plays in linker function. The rate of flavin-to-heme electron transfer and the fatty acid hydroxylase activities of the glycine and proline substitution mutants, including a six-proline substitution, did not change significantly relative to wild-type enzyme. These results indicate that the linker does not adopt any regular secondary structure essential for activity and that the length of the linker is the critical feature that controls flavin-to-heme electron transfer.     

Ligand dynamics in an electron transfer protein. Picosecond geminate recombination of carbon monoxide to heme in mutant forms of cytochrome c

Substitution of the heme coordination residue Met-80 of the electron transport protein yeast iso-1-cytochrome c allows external ligands like CO to bind and thus increase the effective redox potential. This mutation, in principle, turns the protein into a quasi-native photoactivable electron donor. We have studied the kinetic and spectral characteristics of geminate recombination of heme and CO in a series of single M80X (X = Ala, Ser, Asp, Arg) mutants, using femtosecond transient absorption spectroscopy. In these proteins, all geminate recombination occurs on the picosecond and early nanosecond time scale, in a multiphasic manner, in which heme relaxation takes place on the same time scale. The extent of geminate recombination varies from >99% (Ala, Ser) to approximately 70% (Arg), the latter value being in principle low enough for electron injection studies. The rates and extent of the CO geminate recombination phases are much higher than in functional ligand-binding proteins like myoglobin, presumably reflecting the rigid and hydrophobic properties of the heme environment, which are optimized for electron transfer. Thus, the dynamics of CO recombination in cytochrome c are a tool for studying the heme pocket, in a similar way as NO in myoglobin. We discuss the differences in the CO kinetics between the mutants in terms of the properties of the heme environment and strategies to enhance the CO escape yield. Experiments on double mutants in which Phe-82 is replaced by Asp or Gly as well as the M80D substitution indicate that such steric changes substantially increase the motional freedom-dissociated CO.     

Examination of the functional roles of 5 highly conserved residues in the cytochrome b subunit of the bc1 complex of Rhodobacter sphaeroides.

The cytochrome b subunit of the bc1 complexes contains two cytochrome components (bL and bH) and is the locus of both a quinol-oxidizing site (Qo or Qz) and a quinone-reducing site (Qi or Qc). Sequence alignments of this subunit from over 20 eukaryotic and prokaryotic species have revealed a remarkable degree of conservation, including approximately 20 totally conserved residues. In this paper, site-directed mutagenesis has been used to examine the structural or functional roles of 5 of these highly conserved residues, Gly48, Gln58, Ser102, Phe104, and Pro202, all predicted to be within transmembrane alpha-helical segments. The mutants were made in the bc1 complex of Rhodobacter sphaeroides, a photosynthetic bacterium. The ability to use spectroscopic, electrochemical, and flash-induced kinetic methods allows the mutants to be analyzed for influences both on cytochrome spectra and thermodynamic properties and on the kinetics of specific electron transfer reactions. The results show that none of the 5 residues is absolutely essential. Substitution of aspartate or valine for Gly48 results in the loss of photosynthetic growth. The G48V mutant assembles a bc1 complex, but with modified cytochromes bH and bL, and a dysfunctional quinone reductase (Qc) site; an alanine is tolerated at this position. Possibly, a small residue is important here for heme packing. Gln58 and Ser102 are the only highly conserved polar residues predicted to be within the transmembrane spans, apart from the histidines which are heme axial ligands. Neither Gln58 nor Ser102 is essential for assembly or function of the bc1 complex, although substitution of other amino acids in these positions does cause subtle, but measurable changes. Phe104 lies midway between the axial ligands to cytochromes bL and bH and can be modeled to project in the space separating the two hemes. Replacement of this highly conserved aromatic residue by isoleucine has no measurable influence on the rate of electron transfer through the cytochrome b chain containing the two hemes. Finally, Pro202 is a totally conserved proline which is in the middle of transmembrane helix D, in between the 2 histidines which provide ligands to the hemes. No major inhibition of electron transfer resulted from replacing this proline by a leucine, although subtle changes in spectra of the b cytochromes and their electrochemical properties were noted.     

Polyproline, beta-turn helices. Novel secondary structures proposed for the tandem repeats within rhodopsin, synaptophysin, synexin, gliadin, RNA polymerase II, hordein, and gluten

Seven proteins each contain 8 to 52 tandem repeats of a unique class of oligopeptide. The consensus peptide for each is rhodopsin Tyr Pro Pro Gln Gly synaptophysin Tyr Gly Pro Gln Gly synexin Tyr Pro Pro Pro Pro Gly gliadin Tyr Pro Pro Pro Gln Pro RNA polymerase II Tyr Ser Pro Thr Ser Pro Ser hordein Phe Pro Gln Gln Pro Gln Gln Pro gluten Tyr Pro Thr Ser Pro Gln Gln Gly Tyr Although there is obvious variation of sequence and of length, the penta- to nonapeptides share an initial Tyr (or Phe) and have high Pro contents and abundant Gly, Gln, and Ser. We have evaluated helical models that both recognize the uniqueness of these sequence repeats and accommodate variations on the basic theme. We have developed a group of related helical models for these proteins with about three oligopeptide repeats per turn of 10-20 A. These models share several common features: Most of the phi dihedral angles are -54 degrees, to accommodate Pro at all positions except the first (Tyr). Except for the beta-turns, most psi dihedral angles are near +140 degrees as found in polyproline. Each oligopeptide has at least one beta-turn; several have two. Some contain a cis-Tyr, Pro peptide bond; a few have a cis-bond plus one beta-turn. Tyr side chains vary from totally exposed to buried within the helices and could move to accommodate either external hydrophobic interactions or phosphorylation. The several related structures seem to be readily interconverted without major change in the overall helical parameters, and therein may lie the key to their functions.     

Nuclear magnetic resonance studies of amino acids and proteins. Side-chain mobility of methionine in the crystalline amino acid and in crystalline sperm whale (Physeter catodon) myoglobin

We have obtained deuterium (2H) nuclear magnetic resonance (NMR) spectra and spin-lattice relaxation times (T1) of L-[epsilon-2H3]methionine, L-[epsilon-2H3]methionine in a D,L lattice, and [S-methyl-2H3]methionine in the crystalline solid state, as a function of temperature, in addition to obtaining 2H T1 and line-width results as a function of temperature on [epsilon-2H3]methionine-labeled sperm whale (Physeter catodon) myoglobins by using the method of magnetic ordering [Rothgeb, T. M., & Oldfield, E. (1981) J. Biol. Chem. 256, 1432-1446]. The results indicate that in the L-amino acid, methyl rotation having an activation energy (delta E) of 8.3 +/- 1 kJ dominates T1 at low temperatures (less than or equal to 10 degrees C), while at higher temperatures an additional large-amplitude side-chain motion occurs which causes changes in the 2H NMR line shape and T1. This motion is inhibited in the D,L lattice, indicating that lattice effects may have a strong effect on the mobility of anhydrous amino acids in the solid state. Further substitution at S delta to form the sulfonium salt [S-methyl-2H3]-methionine causes a large increase in delta E, to 15.9 +/- 2 kJ, a value comparable to the 14-16 kJ found in valine and leucine, which contain the structurally similar isopropyl moiety. These results suggest that the very low barriers to methyl rotation in the methionine side chain are due to long C-S bond lengths and the presence of only two substituents on sulfur, while the anomalous high-temperature behavior is due to a lattice-packing effect. 2H T1 results with methionine-labeled myoglobin are complex, reflecting the presence of fast large-amplitude side-chain motions, in addition to rapid methyl rotation. Our data indicate that Met-55 and Met-131 are motionally inequivalent in crystalline cyanoferrimyoglobin, in contrast to solution NMR results. We have also recorded 13C cross-polarization "magic-angle" sample-spinning NMR spectra of [epsilon-13C]methionine-labeled crystalline cyanoferrimyoglobin (at 37.7 MHz, corresponding to a magnetic field strength of 3.52 T) and of the same protein in aqueous solution. Cross-polarization transfer rates and proton rotating-frame relaxation time results again indicate that Met-55 and Met-131 are motionally inequivalent in the solid state, and the TCH data indicate that Met-55 is more solidlike. However, we find that 13C chemical shifts in solution and those in the crystalline solid state are in very close agreement, suggesting that the average solution and crystal conformations are the same, in the area of Met-55 and Met-131.     

Single amino acid substitutions in the B870 alpha and beta light-harvesting polypeptides of Rhodobacter capsulatus. Structural and spectral effects.

To obtain information on the structural and functional role of highly conserved amino acid residues in the B870 alpha and beta light-harvesting polypeptides of Rhodobacter capsulatus, site-directed mutagenesis was performed. 18 mutants with single amino acid substitutions at nine different positions in the B870 antenna polypeptides were prepared in a B800-850-lacking strain. The characterization of the resulting phenotypes was based on a quantification of the core-complex elements (reaction center, light-harvesting polypeptides, bacteriochlorophyll a and carotenoid) and the core-complex spectral characteristics (absorption maximum, absorption coefficient and fluorescence intensity). These data generally showed that strong structural effects were caused by the amino acid substitutions. Thus, the three tryptophan exchanges at the position alpha 8 resulted in either the absence of a core complex (alpha Trp8----Leu), the absence of the core antenna (alpha Trp8----Ala) or a reduction in the carotenoid content (alpha Trp8----Tyr). Likewise, the mutants alpha Pro13Gly (i.e. alpha Pro13----Gly), beta Gly10Val and alpha Phe23Ala demonstrated an abnormal protein/pigment ratio in the core antenna, while a drastically reduced antenna size resulted from the amino acid exchange beta Arg45Asp. In contrast to the structural effects, the absorption maxima and the fluorescence intensities of the mutant antennae differed only slightly from the wild type. The strongest blue shift of the bacteriochlorophyll a (8-11 nm) was induced by substitutions of the Trp at position alpha 43 (alpha Trp43----Ala, Leu or Tyr). Contrary to the other spectral effects, the absorption coefficient of bacteriochlorophyll a was strongly influenced by the amino acid substitutions and varied by 1.6-times less (beta Arg45Asp) and 1.3-times greater (alpha Phe25Ala) than normal. The antenna-free mutant, alpha Trp8Ala, yielded a high rate of B800-850 revertants during phototrophic growth, indicating a direct energy transfer from the B800-850 antenna to the reaction center in these strains. Although conditions for growth were generally observed to influence phenotypic expression, the structural as well as spectral effects were demonstrated to differ to the greatest extent between chemotrophically grown and phototrophically grown cells.     

Synthetic study on the structure-activity relationship of sperm activating peptides from the jelly coat of sea urchin eggs

Various analogue peptides with substitution and deletion of amino acid residues have been synthesized by liquid phase method for Sperm Activating Peptides from the jelly coat of sea urchin eggs. The deletion of C-terminal Gly reduced the activity to about 1/3000, while removal of N-terminal Gly reduced the activity to 1/10. The residues Ser5 and Asp3 were replaced by Lys without significant loss of activity. Substitution of Phe2 by Gly, Ala or Pro markedly reduced the activity by the factor of 10(4)-10(6), in contrast to Tyr-substitution retaining almost full activity, indicating the essential role of the aromatic residue in exerting the activity. Substitutions, Asp3 to Glu and Gly10 to Pro, increased the activity 5-fold and 500-fold, respectively.