Intramolecular catalysis of a proline isomerization reaction in the folding of dihydrofolate reductase.

The cis/trans isomerization of the peptide bond preceding proline residues in proteins can limit the rate at which a protein folds to its native conformation. Mutagenic analyses of dihydrofolate reductase (DHFR) from Escherichia coli show that this isomerization reaction can be intramolecularly catalyzed by a side chain from an amino acid which is distant in sequence but adjacent in the native conformation. The guanidinium NH2 nitrogen of Arg 44 forms one hydrogen bond to the imide nitrogen and a second to the carbonyl oxygen of Pro 66 in wild-type DHFR. Replacement of Arg 44 with Leu results in a change of the nature of the two slow steps in refolding from being limited by the acquisition of secondary and/or tertiary structure to being limited by isomerization. The simultaneous replacement of Pro 66 with Ala (i.e., the Leu 44/Ala 66 double mutant) eliminates this isomerization reaction and once again makes protein folding the limiting process. Apparently, one or both of the hydrogen bonds between Arg 44 and Pro 66 accelerate the isomerization of the Gln 65-Pro 66 peptide bond. The replacement of Arg 44 with Leu affects the kinetics of the slow folding reactions in a fashion which indicates that the crucial hydrogen bonds form in the transition states for the rate-limiting steps in folding.     

Parallel channels and rate-limiting steps in complex protein folding reactions: prolyl isomerization and the alpha subunit of Trp synthase,a TIM barrel protein

A kinetic folding mechanism for the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli, involving four parallel channels with multiple native, intermediate and unfolded forms, has recently been proposed. The hypothesis that cis/trans isomerization of several Xaa-Pro peptide bonds is the source of the multiple folding channels was tested by measuring the sensitivity of the three rate-limiting phases (tau(1), tau(2), tau(3)) to catalysis by cyclophilin, a peptidyl-prolyl isomerase. Although the absence of catalysis for the tau(1) (fast) phase leaves its assignment ambiguous, our previous mutational analysis demonstrated its connection to the unique cis peptide bond preceding proline 28. The acceleration of the tau(2) (medium) and tau(3) (slow) refolding phases by cyclophilin demonstrated that cis/trans prolyl isomerization is also the source of these phases. A collection of proline mutants, which covered all of the remaining 18 trans proline residues of alphaTS, was constructed to obtain specific assignments for these phases. Almost all of the mutant proteins retained the complex equilibrium and kinetic folding properties of wild-type alphaTS; only the P217A, P217G and P261A mutations caused significant changes in the equilibrium free energy surface. Both the P78A and P96A mutations selectively eliminated the tau(1) folding phase, while the P217M and P261A mutations eliminated the tau(2) and tau(3) folding phases, respectively. The redundant assignment of the tau(1) phase to Pro28, Pro78 and Pro96 may reflect their mutual interactions in non-random structure in the unfolded state. The non-native cis isomers for Pro217 and Pro261 may destabilize an autonomous C-terminal folding unit, thereby giving rise to kinetically distinct unfolded forms. The nature of the preceding amino acid, the solvent exposure, or the participation in specific elements of secondary structure in the native state, in general, are not determinative of the proline residues whose isomerization reactions can limit folding.     

Nmr studies of the molecular conformations in the linear oligopeptides H-(L-Ala)n-L-Pro-OH

The molecular conformations of the linear oligopeptides H-(L -Ala)_n-L -Pro-OH, with n = 1,2 and 3, have been investigated. ¹³C nmr observation of the equilibrium between the cis and trans forms of the Ala-Pro peptide bond indicated the occurrence of nonrandom conformations in solutions of these flexible peptides. The formation of the nonrandom species containing the cis form of the Ala-Pro bond was found to depend on the deprotonation of the carboxylic acid group of proline, the solvent, and the ionic strength in aqueous solution. The influence of intramolecular hydrogen bonding on the relative conformational energies of the species containing the cis and trans Ala-Pro peptide bond was studied by comparison of the peptides H-(Ala)_n-Pro-OH with analogous molecules where hydrogen bond formation was excluded by the covalent structure. In earlier work a hydrogen bond between the protonated terminal carboxylic acid group and the carbonyl oxygen of the penultimate amino acid residue had been suggested to stabilize conformations including trans proline. For the systems described here this hypothesis can be ruled out, since the cis:trans ratio is identical for molecules with methyl ester protected and free protonated terminal carboxylic acid groups of proline. Direct evidence for hydrogen bond formation between the deprotonated terminal carboxylic acid group and the amide proton of the penultimate amino acid residue in the molecular species containing cis proline was obtained from ¹H nmr studies. However, the cis:trans ratio of the Ala-Pro bond was not affected by N-methylation of the penultimate amino acid residue, which prevents formation of this hydrogen bond. Overall the experimental observations lead to the conclusion that the relative energies of the peptide conformations including cis or trans proline are mainly determined by intramolecular electrostatic interactions, whereas in the molecules considered, intramolecular hydrogen bonding is a consequence of specific peptide backbone conformations rather than a cause for the occurrence of energetically favored species. Independent support for this conclusion was obtained from model consideration which indicated that electrostatic interactions between the terminal carboxylic acid group and the carbonyl oxygen of the penultimate amino acid residue could indeed account for the observed relative conformational energies of the species containing cis and trans proline, respectively.     

Site-directed mutagenesis of Pro327 in the lac permease of Escherichia coli

By use of oligonucleotide-directed, site-specific mutagenesis, Pro327 in the lac permease of Escherichia coli has been replaced with Ala, Gly, or Leu. Permease with Ala at position 327 catalyzes lactose/H+ symport in a manner indistinguishable from wild-type permease. Permease with Gly at position 327, on the other hand, exhibits about one-tenth the activity of wild-type permease but catalyzes lactose accumulation to essentially the same steady-state level as wild-type permease. Finally, permease with Leu at position 327 is completely inactive. The results demonstrate that there is no relationship between permease activity and the helix-breaking (Pro and Gly) or helix-making (Ala and Leu) properties of the residue at position 327. It is suggested that it is primarily a chemical property of the side chain at position 327 (i.e., bulk, hydropathy, and/or ability to hydrogen bond) that is critical for activity and that neither cis/trans isomerization of Pro327 nor the presence of a kink at this position is important.     

Isomerization of the Xaa-Pro peptide bond induced by ionic interactions of arginine

Inclusion of Arg or Pro residues in proteins and peptides has been proved to play an essential role in biochemical functions through ionic interactions, conformational transitions, and formation of turns as well. In this study we present the conformational properties of the Ac-Arg-Ala-Pro (1), Ac-Arg-Ala-Pro-NH₂ (2), Ac-Arg-Pro-Asp-NH₂ (3), and Ac-Arg-Pro-Asp (4) tripeptides, using ¹H-nmr spectroscopy and molecular dynamics. These peptides were modeled with the aim of studying the role of the Arg-guanidinium to carboxylate ionic interactions on the Xaa-Pro peptide bond isomerization. It was found with 1 and 4 that arginine preferentially interacts with the C-terminal carboxylate group, even though the β-carboxylate is also accessible. This tendency of the Arg moiety was found to induce the cis disposition of the Ala-Pro peptide bond in 1. It was also confirmed that the Arg…Asp side chain-side chain ionic interaction in 3 plays a key role in backbone folding and structural stabilization through a type I β-turn. The nmr pattern for 3 showed a remarkable similarity with that for various Arg-Tyr-Asp containing peptides, a sequence that is crucial for the adhesion properties of the Leishmania gp63 glycoprotein. © 1996 John Wiley & Sons, Inc.     

Peptidyl prolyl cis/trans-isomerases: comparative reactivities of cyclophilins, FK506-binding proteins, and parvulins with fluorinated oligopeptide and protein substrates

Peptidyl prolyl cis/trans-isomerases catalyze the cis-trans isomerization of prolyl bonds in oligopeptides and various folding states of proteins. The proline residue in PPIase substrates at the P1' subsite, which follows the isomerizing peptide bond, appears to be the common recognition element for all subfamilies of this enzyme class. The molecular principles that govern substrate specificity at the P1' subsite were analyzed using 4-fluoroproline-containing tetrapeptide 4-nitroanilides and barstar Cys40Ala/Cys82Ala/Pro27Ala/Pro48-->4-fluoroproline quadruple variants. Generally, PPIase catalysis demonstrated stereospecificity for monofluoro substitutions at the 4-position of the pyrrolidine ring. However, the replacement of hydrogens with fluoro atoms did not impair productive interactions for the majority of PPIase-substrate complexes. Comparison of specificity constants for oligopeptide and protein substrates revealed striking differences in the 4-fluoroproline substituent effects between members of the PPIase families. Introduction of 4(R)-fluoroproline resulted in an oligopeptide substrate completely resistant to catalytic effects of FKBP-like PPIases. By contrast, the 4(R)-fluoroproline barstar variant demonstrated only slightly reduced or even better catalytic susceptibility when compared to the parent barstar Cys40Ala/Cys82Ala/Pro27Ala/Pro48 substrate. On the other hand, Suc-Ala-Ser-4(S)-FPro-Phe-pNA exhibits a discriminating specificity toward the prototypic parvulin, the Escherichia coli Par10. The E. coli trigger factor, in the extreme, catalyzes Cys40Ala/Cys82Ala/Pro27Ala/4-F(2)Pro48 with a more than 20-fold higher efficiency when compared to the proline-containing congener. These findings support the combined subsite concept for PPIase catalysis in which the positioning of a substrate in the active cleft must activate a still unknown number of remote subsites in the transition state of the reaction. The number of critical subsites was shown to vary between the PPIase families.     

The structures of the horseradish peroxidase C-ferulic acid complex and the ternary complex with cyanide suggest how peroxidases oxidize small phenolic substrates

We have solved the x-ray structures of the binary horseradish peroxidase C-ferulic acid complex and the ternary horseradish peroxidase C-cyanide-ferulic acid complex to 2.0 and 1.45 A, respectively. Ferulic acid is a naturally occurring phenolic compound found in the plant cell wall and is an in vivo substrate for plant peroxidases. The x-ray structures demonstrate the flexibility and dynamic character of the aromatic donor binding site in horseradish peroxidase and emphasize the role of the distal arginine (Arg(38)) in both substrate oxidation and ligand binding. Arg(38) hydrogen bonds to bound cyanide, thereby contributing to the stabilization of the horseradish peroxidase-cyanide complex and suggesting that the distal arginine will be able to contribute with a similar interaction during stabilization of a bound peroxy transition state and subsequent O-O bond cleavage. The catalytic arginine is additionally engaged in an extensive hydrogen bonding network, which also includes the catalytic distal histidine, a water molecule and Pro(139), a proline residue conserved within the plant peroxidase superfamily. Based on the observed hydrogen bonding network and previous spectroscopic and kinetic work, a general mechanism of peroxidase substrate oxidation is proposed.     

Pyruvate carboxylase: isolation of the biotin-containing tryptic peptide and the determination of its primary sequency

The biotin-containing tryptic peptides of pyruvate carboxylase from sheep, chicken, and turkey liver mitochondria have been isolated and their primary structures determined. The amino acid sequences of the 19 residue peptides from chicken and turkey are identical and share a common sequence of 14 residues around biocytin with the 24-residue peptide isolated from sheep. The sequences obtained were: residue 1 → 11 Avian: Gly Ala Pro Leu Val Leu Ser Ala Met Biocytin Met Sheep: Gly Gln Pro Leu Val Leu Ser Ala Met Biocytin Met residues 12 → 19 or 24 Avian: Glu Thr Val Val Thr Ala Pro Arg Sheep: Glu Thr Val Val Thr Ser Pro Val Thr Glu Gly Val Arg A sensitive radiochemical assay for biotin was developed based on the tight binding of biotin by avidin. The ability of zinc sulfate to precipitate, without dissociating, the avidin-biotin complex provided a convenient procedure for separating free and bound biotin, and hence, for back-titrating a standard amount of avidin with [¹⁴C]biotin.     

Substrate specificity of aminopeptidase M: evidence that the commercial preparation is contaminated by dipeptidyl aminopeptidase IV and prolidase

Commercial preparations of aminopeptidases M split Gly-Pro-beta-naphthylamide (Gly-Pro-2-NNap) into Gly-Pro and beta-naphthylamine, and Ala-Pro into Ala and Pro. The activities on Gly-Pro-2-NNap and Ala-Pro were completely inhibited by diisopropyl phosphorofluoridate (DFP) and p-chloromercuribenzoate (PCMB), respectively. When the substrate specificity was analyzed with tuftsin, Thr and Lys-Pro-Arg were released, and then Lys-Pro-Arg was split into Lys-Pro and Arg. Thereafter, slow liberation of Lys and Pro from Lys-Pro took place. The DFP-treated enzyme released only Thr from tuftsin and no hydrolysis of Lys-Pro-Arg was observed. With the enzyme treated with PCMB, tuftsin was converted into Thr and Lys-Pro-Arg, followed by the liberation of Arg, but no release of Lys and Pro was observed, contrary to the case of the untreated-enzyme. These results show that commercial aminopeptidase M contains dipeptidyl aminopeptidase IV and prolidase. Contamination by dipeptidyl aminopeptidase IV was confirmed by an immunological method.     

The amino acids involved in the distinct carbohydrate specificities between macrophage galactose-type C-type lectins 1 and 2 (CD301a and b) of mice

Binding specificities of mouse macrophage galactose-type C-type lectin 1 (MGL1/CD301a) and 2 (MGL2/CD301b) toward various oligosaccharides were compared by frontal affinity chromatography. MGL1 preferentially bound oligosaccharides containing Lewis(X) (Le(X)) trisaccharides among 111 oligosaccharides tested, whereas MGL2 preferentially bound globoside Gb4. The important amino acids for the preferential bindings were investigated by pair-wise site-directed mutagenesis at positions 61, 89, 97, 100, 110-113, 115, 124, and 125 in the soluble recombinant carbohydrate recognition domains (CRD) prepared in Escherichia coli and purified with galactose-Sepharose. Mutations of Val, Ala, Thr, and Phe at positions 61, 89, 111 and 125 on MGL1 CRD caused reductions in Le(X) binding. Mutations of MGL2 CRD at Leu, Arg, Arg, and Tyr at positions 61, 89, 115 and 125 were implicated in the preference for beta-GalNAc. Le(X) binding was observed with MGL2 mutants of Arg89Ala and Arg89Ala/Ser111Thr. MGL1 mutants of Ala89Arg and Ala89Arg/Pro115Arg showed beta-GalNAc bindings. Molecular modeling illustrated potential direct molecular interactions of Leu61, Arg89, and His109 in MGL2 CRD with GalNAc.     

A novel method of analyzing proline synonymous codons in E. coli

Proline is a special imino acid in protein and the isomerization of the prolyl peptide bond has notable biological significance and influences the final structure of protein greatly, so the correlation between proline synonymous codon usage and local amino acid, the correlation between proline synonymous codon usage and the isomerization of the prolyl peptide bond were both investigated in the Escherichia coli genome by using a novel method based on information theory. The results show that in peptide chain, the residue at the first position C-terminal influences the usage of proline synonymous codon greatly and proline synonymous codons contain some factors influencing the isomerization of the prolyl peptide bond.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Crystal structure of cyclophilin A complexed with a binding site peptide from the HIV-1 capsid protein.

The cellular protein, cyclophilin A (CypA), is incorporated into the virion of the type 1 human immunodeficiency virus (HIV-1) via a direct interaction with the capsid domain of the viral Gag polyprotein. We demonstrate that the capsid sequence 87His-Ala-Gly-Pro-Ile-Ala92 (87HAGPIA92) encompasses the primary cyclophilin A binding site and present an X-ray crystal structure of the CypA/HAGPIA complex. In contrast to the cis prolines observed in all previously reported structures of CypA complexed with model peptides, the proline in this peptide, Pro 90, binds the cyclophilin A active site in a trans conformation. We also report the crystal structure of a complex between CypA and the hexapeptide HVGPIA, which also maintains the trans conformation. Comparison with the recently determined structures of CypA in complexes with larger fragments of the HIV-1 capsid protein demonstrates that CypA recognition of these hexapeptides involves contacts with peptide residues Ala(Val) 88, Gly 89, and Pro 90, and is independent of the context of longer sequences.     

A nonessential role for Arg 55 in cyclophilin18 for catalysis of proline isomerization during protein folding

The protein folding process is often in vitro rate‐limited by slow cis‐trans proline isomerization steps. Importantly, the rate of this process in vivo is accelerated by prolyl isomerases (PPIases). The archetypal PPIase is the human cyclophilin 18 (Cyp18 or CypA), and Arg 55 has been demonstrated to play a crucial role when studying short peptide substrates in the catalytic action of Cyp18 by stabilizing the transition state of isomerization. However, in this study we show that a R55A mutant of Cyp18 is as efficient as the wild type to accelerate the refolding reaction of human carbonic anhydrase II (HCA II). Thus, it is evident that the active‐site located Arg 55 is not required for catalysis of the rate‐limiting prolyl cis‐trans isomerization steps during the folding of a protein substrate as HCA II. Nevertheless, catalysis of cis‐trans proline isomerization in HCA II occurs in the active‐site of Cyp18, since binding of the inhibitor cyclosporin A abolishes rate acceleration of the refolding reaction. Obviously, the catalytic mechanisms of Cyp18 can differ when acting upon a simple model peptide, four residues long, with easily accessible Pro residues compared with a large protein molecule undergoing folding with partly or completely buried Pro residues. In the latter case, the isomerization kinetics are significantly slower and simpler mechanistic factors such as desolvation and/or strain might operate during folding‐assisted catalysis, since binding to the hydrophobic active site is still a prerequisite for catalysis.     

Alpha-helix stability in proteins. I. Empirical correlations concerning substitution of side-chains at the N and C-caps and the replacement of alanine by glycine or serine at solvent-exposed surfaces.

The importance of amino acid side-chains in helix stability has been investigated by making a series of mutations at the N-caps, C-caps and internal positions of the solvent-exposed faces of the two alpha-helices of barnase. There is a strong positional and context dependence of the effect of a particular amino acid on stability. Correlations have been found that provide insight into the physical basis of helix stabilization. The relative effects of Ala and Gly (or Ser) may be rationalized on the basis of solvent-accessible surface areas: burial of hydrophobic surface stabilizes the protein as does exposure to solvent of unpaired hydrogen bond donors or acceptors in the protein. There is a good correlation between the relative stabilizing effects of Ala and Gly at internal positions with the total change in solvent-accessible hydrophobic surface area of the folded protein on mutation of Ala----Gly. The relationship may be extended to the N and C-caps by including an extra term in hydrophilic surface area for the solvent exposure of the non-intramolecularly hydrogen-bonded main-chain CO, NH or protein side-chain hydrogen bonding groups. The requirement for solvent exposure of the C-cap main-chain CO groups may account for the strong preference for residues having positive phi and psi angles at this position, since this alpha L-conformation results in the largest solvent exposure of the C-terminal CO groups. Glycine in an alpha L-conformation results in the greatest exposure of these CO groups. Further, the side-chains of His, Asn, Arg and Lys may, with positive phi and psi-angles, form a hydrogen bond with the backbone CO of residue in position C -3 (residues are numbered relative to the C-cap). The preferences at the C-cap are Gly much greater than His greater than Asn greater than Arg greater than Lys greater than Ala approximately Ser approximately greater than Asp. The preferences at the N-cap are determined by hydrogen bonding of side-chains or solvent to the exposed backbone NH groups and are: Thr approximately Asp approximately Ser greater than Gly approximately Asn greater than Gln approximately Glu approximately His greater than Ala greater than Val much greater than Pro. These general trends may be obscured when mutation allows another side-chain to become a surrogate cap.(ABSTRACT TRUNCATED AT 400 WORDS)     

Conformational preferences and prolyl cis–trans isomerization of phosphorylated Ser/Thr‐Pro motifs

The conformational study on Ac‐pSer‐Pro‐NHMe and Ac‐pThr‐Pro‐NHMe peptides has been carried out using hybrid density functional methods with the implicit solvation reaction field theory at the B3LYP/ 6‐311++G(d,p)//B3LYP/6‐31+G(d) level of theory in the gas phase and in solution (chloroform and water). For both pSer‐Pro and pThr‐Pro peptides in the gas phase and in chloroform, the most preferred conformation has the α‐helical structure for the pSer/pThr residue, the down‐puckered polyproline I structure for the Pro residue, and the cis prolyl peptide bond between the two residues, in which two hydrogen bonds between the phosphate oxygens with the backbone N? H groups seem to play a role. However, the trans conformations that have a single hydrogen bond of the phosphate oxygen with either of two backbone N? H groups become most preferred for both peptides in water. This is because the hydration free energy of the anionic oxygen of the phosphate group is expected to dramatically decrease for the cis conformation upon formation of the hydrogen bond with the backbone N? H groups. These calculated results are consistent with the observations by NMR and IR experiments, suggesting the existence of hydrogen bonds between the charged phosphoryl group and the backbone amide protons in solution. The calculated cis populations of 14.7 and 14.2% and rotational barriers of 19.87 and 20.57 kcal/mol to the cis‐to‐trans isomerization for pSer‐Pro and pThr‐Pro peptides in water, respectively, are consistent with the observed values for pSer‐Pro and pThr‐Pro containing peptides from NMR experiments. However, the hydrogen bond between the prolyl nitrogen and the following amide N? H group, which was suggested to be capable of catalyzing the prolyl isomerization, does not play a role in stabilizing the preferred transition state for the pSer/pThr‐Pro peptides in water. Instead, the amide hydrogen of the NHMe group is involved in a bifurcated hydrogen bond with the anionic oxygen and phosphoester oxygen of the phosphate group. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 330–339, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Conformational studies of the interdomain linker peptides in the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

Two peptides (PEP1, 26 residues, and PEP2, 22 residues) were synthesized with amino acid sequences identical to two of the long segments of polypeptide chain rich in alanine, proline, and charged amino acids that link the lipoyl domains together in the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli. The circular dichroism and 400-MHz 1H NMR spectra of the peptides indicated that they lacked regular secondary structure. Even in the presence of 45% (v/v) hexafluoroisopropanol, they appeared to acquire a helical content of only 23-25%. However, 13C NMR spectroscopy revealed that the Ala-Pro peptide bonds were all (> 95%) in the trans configuration, compared with a value of 87% for the Ala-Pro bond in the model peptide AAPA, which is a recurrent sequence motif in PEP1 and PEP2. Likewise in peptides representing the N- and C-terminal halves of peptide PEP2, the Ala-Pro bonds were again all (> 95%)-trans, suggesting that peptide length is the essential determinant of the cis:trans ratio. Antisera were raised against peptides PEP2 and PEP3, the latter representing a third interdomain segment of polypeptide chain (Radford, S. E., Laue, E. D., Perham, R. N., Martin, S. R., and Appella, E. (1989a) J. Biol. Chem. 264, 767-775). Despite extensive sequence similarity among peptides PEP1, PEP2, and PEP3, only limited immunological cross-reactivity was observed, which suggests that the antigenic epitope(s) in the peptides are different and distinct. It is likely that these peptides are representative of a class of inter-domain linkers or spacers found in a wide variety of proteins and endowed with varying degrees of flexibility and stiffness to match their particular biological purpose.     

Prolyl isomerization as a molecular timer in phage infection

Prolyl cis-trans isomerizations are intrinsically slow reactions and known to be rate-limiting in many protein folding reactions. Here we report that a proline is used as a molecular timer in the infection of Escherichia coli cells by the filamentous phage fd. The phage is activated for infection by the disassembly of the two N-terminal domains, N1 and N2, of its gene-3-protein, which is located at the phage tip. Pro213, in the hinge between N1 and N2, sets a timer for the infective state. The timer is switched on by cis-to-trans and switched off by the unusually slow trans-to-cis isomerization of the Gln212-Pro213 peptide bond. The switching rate and thus the infectivity of the phage are determined by the local sequence around Pro213, and can be tuned by mutagenesis.     

Effects of proline mutations on the folding of staphylococcal nuclease

Effects of proline isomerizations on the equilibrium unfolding and kinetic refolding of staphylococcal nuclease were studied by circular dichroism in the peptide region (225 nm) and fluorescence spectra of a tryptophan residue. For this purpose, four single mutants (P11A, P31A, P42A, and P56A) and four multiple mutants (P11A/P47T/P117G, P11A/P31A/P47T/P117G, P11A/P31A/P42A/P47T/P117G, and P11A/P31A/P42A/P47T/P56A/P117G) were constructed. These mutants, together with the single and double mutants for Pro47 and Pro117 constructed in our previous study, cover all six proline sites of the nuclease. The P11A, P31A, and P42A mutations did not change the stability of the protein remarkably, while the P56A mutation increased protein stability to a small extent by 0.5 kcal/mol. The refolding kinetics of the protein were, however, affected remarkably by three of the mutations, namely, P11A, P31A, and P56A. Most notably, the amplitude of the slow phase of the triphasic refolding kinetics of the nuclease observed by stopped-flow circular dichroism decreased by increasing the number of the proline mutations; the slow phase disappeared completely in the proline-free mutant (P11A/P31A/P42A/P47T/P56A/P117G). The kinetic refolding reactions of the wild-type protein assessed in the presence of Escherichia coli cyclophilin A showed that the slow phase was accelerated by cyclophilin, indicating that the slow phase was rate-limited by cis-trans isomerization of the proline residues. Although the fast and middle phases of the refolding kinetics were not affected by cyclophilin, the amplitude of the middle phase decreased when the number of the proline mutations increased; the percent amplitudes for the wild-type protein and the proline-free mutants were 43 and 13%, respectively. In addition to these three phases detected with stopped-flow circular dichroism, a very fast phase of refolding was observed with stopped-flow fluorescence, which had a shorter dead time (3.6 ms) than the stopped-flow circular dichroism. The following conclusions were drawn. (1) The effects of the P11A, P31A, and P56A mutations on the refolding kinetics indicate that the isomerizations of the three proline residues are rate-limiting, suggesting that the structures around these residues (Pro11, Pro31, and Pro56) may be organized at an early stage of refolding. (2) The fast phase corresponds to the refolding of the native proline isomer, and the middle phase whose amplitude has decreased when the number of proline mutations was increased may correspond to the slow refolding of non-native proline isomers. The occurrence of the fast- and slow-refolding reactions together with the slow phase rate-limited by the proline isomerization suggests that there are parallel folding pathways for the native and non-native proline isomers. (3) The middle phase did not completely disappear in the proline-free mutant. This suggests that the slow-folding isomer is produced not only by the proline isomerizations but also by another conformational event that is not related to the prolines. (4) The very fast phase detected with the fluorescent measurements suggests that there is an intermediate at a very early stage of kinetic refolding.     

Crystal structures of E. coli CcmG and its mutants reveal key roles of the N-terminal beta-sheet and the fingerprint region

CcmG, also designated DsbE, functions as a periplasmic protein thiol:disulfide oxidoreductase and is required for cytochrome c maturation. Here we report the crystal structures of Escherichia coli CcmG and its two mutants, P144A and the N-terminal fifty seven-residue deletion mutant, and two additional deletion mutants were studied by circular dichroism. Structural comparison of E. coli CcmG with its deletion mutants reveals that the N-terminal beta-sheet is essential for maintaining the folding topology and consequently maintaining the active-site structure of CcmG. Pro144 and Glu145 are key residues of the fingerprint region of CcmG. Pro144 is in cis-configuration, and it makes van der Waals interactions with the active-site disulfide Cys80-Cys83 and forms a C--H...O hydrogen bond with Thr82, helping stabilize the active-site structure. Glu145 forms a salt-bridge and hydrogen-bond network with other residues of the fingerprint region and with Arg158, further stabilizing the active-site structure. The cis-configuration of Pro144 makes the backbone nitrogen and oxygen of Ala143 exposed to solvent, favorable for interacting with binding partners. The key role of cis-Pro144 is verified by the P144A mutant, which contains trans-Ala144 and displays redox property changes. Structural comparison of E. coli CcmG with the recently reported structure of CcmG in complex with the N-terminal domain of DsbD reveals that Tyr141 undergoes conformational changes upon binding DsbD. A cis-proline located at the N-terminus of the first beta-strand of the betabetaalpha motif of the thioredoxin-like domain is a conserved structural feature of the thioredoxin superfamily.