Proline isomerization during refolding of ribonuclease A is accelerated by the presence of folding intermediates

The trans----cis isomerization of Pro 93 was measured during refolding of bovine ribonuclease A. This isomerization is slow (tau = 500 s) under marginally stable folding conditions of 2.0 M GdmCl, pH 6, at 10 degrees C. However, it is strongly accelerated (tau = 100 s) in samples which, prior to isomerization, had been converted to a folding intermediate by a 15 s refolding pulse under strongly native conditions (0.8 M ammonium sulfate, 0 degree C). The results demonstrate that extensive folding is possible before Pro 93 isomerizes to its native cis state and that the presence of structural folding intermediates leads to a marked increase in the rate of subsequent proline isomerization.     

Nmr studies of the rates of proline cis–trans isomerization in oligopeptides

¹H-Nmr was used to measure the rate of cis–trans interconversion of X-Pro bonds in linear and cyclic oligopeptides. k(cis → trans) = 2.5 × 10^?3 s^?1 at 25°C was found for the zwitterionic form of H-Ala-Pro-OH, in good agreement with earlier measurements. Replacement of Ala by Phe, Tyr, or Trp resulted in a 10-fold slower interconversion rate, whereas after substitution of Ala by His or Glu, the rate decreased only slightly. Independent of the residues X, the interconversion rate was increased by a factor of ca. 20 when the peptide chain was elongated by addition of Ala to the C-terminal Pro. An additional increase by a factor of 6 was observed when going from the protected linear peptide CF₃CO-Gly-Gly-Pro-Ala-OCH₃ to the closely related cyclic compound c[-Gly-Gly-Pro-Gly-Ala-]. These data are evaluated with regard to their possible use in future studies on the role of X-Pro cis–trans isomerization in the kinetics of protein folding.     

An examination of the involvement of proline peptide isomerization in protein folding.

The proline peptide isomerization model of protein folding predicts that the fraction of denatured polypeptide chains which rapidly fold should be quantitatively related to the numbers of cis and trans proline residues in the folded polypeptide conformation. However, we find that neither the comparative nor the absolute kinetic pattern for folding of the homologous proteins, tuna heart and horse heart ferricytochrome c which differ by one proline residue, is compatible with the quantitative predictions of the proline peptide isomerization model.     

Coupling between trans/cis proline isomerization and protein stability in staphylococcal nuclease.

The nucleases A produced by two strains of Staphylococcus aureus, which have different stabilities, differ only in the identity of the single amino acid at residue 124. The nuclease from the Foggi strain of S. aureus (by convention nuclease WT), which contains His124, is 1.9 kcal.mol-1 less stable (at pH 5.5 and 20 degrees C) than the nuclease from the V8 strain (by convention nuclease H124L), which contains Leu124. In addition, the population of the trans conformer at the Lys116-Pro117 peptide bond, as observed by NMR spectroscopy, is different for the two variants: about 15% for nuclease WT and 9% for nuclease H124L. In order to improve our understanding of the origin of these differences, we compared the properties of WT and H124L with those of the H124A and H124I variants. We discovered a correlation between effects of different residues at this position on protein stability and on stabilization of the cis configuration of the Lys116-Pro117 peptide bond. In terms of free energy, approximately 17% of the increase in protein stability manifests itself as stabilization of the cis configuration at Lys116-Pro117. This result implies that the differences in stability arise mainly from structural differences between the cis configurational isomers at Pro117 of the different variants at residue 124. We solved the X-ray structure of the cis form of the most stable variant, H124L, and compared it with the published high-resolution X-ray structure of the cis form of the most stable variant, WT (Hynes TR, Fox RO, 1991, Proteins Struct Funct Genet 10:92-105). The two structures are identical within experimental error, except for the side chain at residue 124, which is exposed in the models of both variants. Thus, the increased stability and changes in the trans/cis equilibrium of the Lys116-Pro117 peptide bond observed in H124L relative to WT are due to subtle structural changes that are not observed by current structure determination technique. Residue 124 is located in a helix. However, the stability changes are too large and follow the wrong order of stability to be explained simply by differences in helical propensity. A second site of conformational heterogeneity in native nuclease is found at the His46-Pro47 peptide bond, which is approximately 80% trans in both WT and H124L. Because proline to glycine substitutions at either residue 47 or 117 remove the structural heterogeneity at that position and increase protein stability, we determined the X-ray structures of H124L + P117G and H124L + P47G + P117G and the kinetic parameters of H124L, H124L + P47G, H124L + P117G, and H124L + P47G + P117G. The individual P117G and P47G mutations cause decreases in nuclease activity, with kcat affected more than Km, and their effects are additive. The P117G mutation in nuclease H124L leads to the same local conformational rearrangement described for the P117G mutant of WT (Hynes TR, Hodel A, Fox RO, 1994, Biochemistry 33:5021-5030). In both P117G mutants, the loop formed by residues 112-117 is located closer to the adjacent loop formed by residues 77-85, and residues 115-118 adopt a type I' beta-turn conformation with the Lys116-Gly117 peptide bond in the trans configuration, as compared with the parent protein in which these residues have a typeVIa beta-turn conformation with the Lys116-Pro117 peptide bond in the cis configuration. Addition of the P47G mutation appears not to cause any additional structural changes. However, the electron density for part of the loop containing this peptide bond was not strong enough to be interpreted.     

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.     

The folding process of acylphosphatase from Escherichia coli is remarkably accelerated by the presence of a disulfide bond

The acylphosphatase from Escherichia coli (EcoAcP) is the first AcP so far studied with a disulfide bond. A mutational variant of the enzyme lacking the disulfide bond has been produced by substituting the two cysteine residues with alanine (EcoAcP mutational variant C5A/C49A, mutEcoAcP). The native states of the two protein variants are similar, as shown by far-UV and near-UV circular dichroism and dynamic light-scattering measurements. From unfolding experiments at equilibrium using intrinsic fluorescence and far-UV circular dichroism as probes, EcoAcP shows an increased conformational stability as compared with mutEcoAcP. The wild-type protein folds according to a two-state model with a very fast rate constant (k_F^H2O = 72,600 s^− 1), while mutEcoAcP folds ca 1500-fold slower, via the accumulation of a partially folded species. The correlation between the hydrophobicity of the polypeptide chain and the folding rate, found previously in the AcP-like structural family, is maintained only when considering the mutant but not the wild-type protein, which folds much faster than expected from this correlation. Similarly, the correlation between the relative contact order and the folding rate holds only for mutEcoAcP. The correlation also holds for EcoAcP, provided the relative contact order value is recalculated by considering the disulfide bridge as an alternate path for the backbone to determine the shortest sequence separation between contacting residues. These results indicate that the presence of a disulfide bond in a protein is an important determinant of the folding rate and allows its contribution to be determined in quantitative terms.     

Molecular Mechanism for Attractant Signaling to DHMA by E. coli Tsr

The attractant chemotaxis response of Escherichia coli to norepinephrine requires that it be converted to 3,4-dihydroxymandelic acid (DHMA) by the monoamine oxidase TynA and the aromatic aldehyde dehydrogenase FeaB. DHMA is sensed by the serine chemoreceptor Tsr, and the attractant response requires that at least one subunit of the periplasmic domain of the Tsr homodimer (pTsr) has an intact serine-binding site. DHMA that is generated in vivo by E. coli is expected to be a racemic mixture of the (R) and (S) enantiomers, so it has been unclear whether one or both chiral forms are active. Here, we used a combination of state-of-the-art tools in molecular docking and simulations, including an in-house simulation-based docking protocol, to investigate the binding properties of (R)-DHMA and (S)-DHMA to E. coli pTsr. Our studies computationally predicted that (R)-DHMA should promote a stronger attractant response than (S)-DHMA because of a consistently greater-magnitude piston-like pushdown of the pTsr α-helix 4 toward the membrane upon binding of (R)-DHMA than upon binding of (S)-DHMA. This displacement is caused primarily by interaction of DHMA with Tsr residue Thr156, which has been shown by genetic studies to be critical for the attractant response to L-serine and DHMA. These findings led us to separate the two chiral species and test their effectiveness as chemoattractants. Both the tethered cell and motility migration coefficient assays validated the prediction that (R)-DHMA is a stronger attractant than (S)-DHMA. Our study demonstrates that refined computational docking and simulation studies combined with experiments can be used to investigate situations in which subtle differences between ligands may lead to diverse chemotactic responses.     

Probing E. coli SSB protein-DNA topology by reversing DNA backbone polarity

Escherichia coli single-strand (ss) DNA binding protein (SSB) is an essential protein that binds ssDNA intermediates formed during genome maintenance. SSB homotetramers bind ssDNA in two major modes, differing in occluded site size and cooperativity. The (SSB)₃₅ mode in which ssDNA wraps, on average, around two subunits is favored at low [NaCl] and high SSB/DNA ratios and displays high unlimited, nearest-neighbor cooperativity forming long protein clusters. The (SSB)₆₅ mode, in which ssDNA wraps completely around four subunits of the tetramer, is favored at higher [NaCl] (>200 mM) and displays limited low cooperativity. Crystal structures of E. coli SSB and Plasmodium falciparum SSB show ssDNA bound to the SSB subunits (OB folds) with opposite polarities of the sugar phosphate backbones. To investigate whether SSB subunits show a polarity preference for binding ssDNA, we examined EcSSB and PfSSB binding to a series of (dT)₇₀ constructs in which the backbone polarity was switched in the middle of the DNA by incorporating a reverse-polarity (RP) phosphodiester linkage, either 3′-3′ or 5′-5′. We find only minor effects on the DNA binding properties for these RP constructs, although (dT)₇₀ with a 3′-3′ polarity switch shows decreased affinity for EcSSB in the (SSB)₆₅ mode and lower cooperativity in the (SSB)₃₅ mode. However, (dT)₇₀ in which every phosphodiester linkage is reversed does not form a completely wrapped (SSB)₆₅ mode but, rather, binds EcSSB in the (SSB)₃₅ mode with little cooperativity. In contrast, PfSSB, which binds ssDNA only in an (SSB)₆₅ mode and with opposite backbone polarity and different topology, shows little effect of backbone polarity on its DNA binding properties. We present structural models suggesting that strict backbone polarity can be maintained for ssDNA binding to the individual OB folds if there is a change in ssDNA wrapping topology of the RP ssDNA.     

Green‐lighting green fluorescent protein: Faster and more efficient folding by eliminating a cis–trans peptide isomerization event

Wild‐type green fluorescent protein (GFP) folds on a time scale of minutes. The slow step in folding is a cis–trans peptide bond isomerization. The only conserved cis‐peptide bond in the native GFP structure, at P89, was remodeled by the insertion of two residues, followed by iterative energy minimization and side chain design. The engineered GFP was synthesized and found to fold faster and more efficiently than its template protein, recovering 50% more of its fluorescence upon refolding. The slow phase of folding is faster and smaller in amplitude, and hysteresis in refolding has been eliminated. The elimination of a previously reported kinetically trapped state in refolding suggests that X‐P89 is trans in the trapped state. A 2.55 Å resolution crystal structure revealed that the new variant contains only trans‐peptide bonds, as designed. This is the first instance of a computationally remodeled fluorescent protein that folds faster and more efficiently than wild type.     

The mutagenic effect of accelerated heavy ions on E. coli cells

In studying E. coli mutation rate as a function of dose of different types of ionizing radiation it was found that mutagenic efficiency of helium ions (LET-22, 54 and 72 keV/microns) was higher than that of gamma-rays. As LET increased the mutagenic efficiency decreased. The mutation rate for all types of radiation under study was both a power function and a linear-quadratic function of dose.     

Trx-NAP 5融合蛋白在大肠杆菌中的表达及其活性检测

目的:用大肠杆菌表达获得重组线虫抗凝血肽5(rNAP 5),为研究开发NAP5的功能与应用提供原料来源。方法:将扩增的NAP5基因经BamHⅠ和HindⅢ双酶切后与表达载体pET-32a连接。构建好的重组表达质粒转化至大肠杆菌BL21(DE3)后,分别经IPTG和乳糖诱导表达,并探讨诱导表达条件,分析表达产物的可溶性情况。表达产物经镍亲和纯化后,用凝血酶原时间(PT)和活化部分凝血活酶时间(aPTT)检测体外抗凝活性。结果:成功构建了pET-32a/NAP5表达载体,IPTG和乳糖均能诱导目的蛋白在大肠杆菌BL21(DE3)中高效地可溶性表达。优化条件下每升LB培养基可获可溶性目的融合蛋白量达65.3mg。纯化的蛋白能明显延长PT及aPTT,7.0mg/L的蛋白平均约延长5.09倍aPTT,2.55倍PT。结论:在大肠杆菌中成功表达了具有很好生物活性的Trx-NAP5融合蛋白,为研究开发NAP5的功能与应用奠定了基础。     

Factors influencing the presence and concentration of E. coli O157 and E. coli in farm waste on six cattle farms in North-West England

Aims:  To investigate the factors influencing the presence and burden of Escherichia coli O157 in farm wastes.
Methods and Results:  Wastes from six cattle farms were screened for the presence and concentration of E. coli O157 and E. coli on three occasions over a year and waste management data were collected. Sixty-three of 878 (7·1%) samples were positive for verocytotoxigenic Escherichia coli O157 and 664/875 (75·9%) for E. coli with detectable levels greater in fresh waste than in stored waste, pasture or dirty water.
Conclusions:  The turning/stirring of stored waste and the use of more than one store (allowing longer storage times) reduced the proportion of E. coli O157 positive samples. The presence of E. coli O157 significantly reduced from a high prevalence found in fresh faeces and stored waste to lower proportions in dirty water and pasture samples. Escherichia coli O157 was only detected on pasture when waste was spread from contaminated stores the day before sampling. A high prevalence of positive E. coli O157 samples were detected when cattle were re-housed.
Significance and Impact of the Study:  These findings help to support the importance of treating and storing farm waste, as well as providing evidence for the level of dilution of E. coli O157 from fresh waste to recently spread pastures.     

RNA polymerase drives ribonucleotide excision DNA repair in E. coli

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Spatial engineering of E. coli with addressable phase-separated RNAs

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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.     

Phosphoinositide (PI) hydrolysis by phospholipase C is accelerated by heat-stable (ST) E. coli enterotoxin in rat intestine

Abstract A partially purified E. coli heat-stable (ST) enterotoxin stimulated the phosphoinositide (PI)-specific phospholipase C (PLC) activity of the rat intestine. The effect of ST on PI-specific PLC was significant compared with the control. The importance of PI-specific phospholipase C as a potential agent to promote calcium translocation across the plasma membrane, was discussed in this communication.