Franz X. Schmid

Two exposed amino acid residues confer thermostability on a cold shock protein

Thermophilic organisms produce proteins of exceptional stability. To understand protein thermostability at the molecular level we studied a pair of cold shock proteins, one of mesophilic and one of thermophilic origin, by systematic mutagenesis. Although the two proteins differ in sequence at 12 positions, two surface-exposed residues are responsible for the increase in stability of the thermophilic protein (by 15.8 kJ mol−1 at 70 °C). 11.5 kJ mol−1 originate from a predominantly electrostatic contribution of Arg 3 and 5.2 kJ mol−1 from hydrophobic interactions of Leu 66 at the carboxy terminus. The mesophilic protein could be converted to a highly thermostable form by changing the Glu residues at positions 3 and 66 to Arg and Leu, respectively. The variation of surface residues may thus provide a simple and powerful approach for increasing the thermostability of a protein.

A ribosome-associated peptidyl-prolyl cis/trans isomerase identified as the trigger factor.

Peptidyl‐prolyl cis/trans isomerases (PPIases) are enzymes that catalyse protein folding both in vitro and in vivo. We isolated a peptidyl‐prolyl cis/trans isomerase (PPIase) which is specifically associated with the 50S subunit of the Escherichia coli ribosome. This association was abolished by adding at least 1.5 M LiCl. Sequencing the N‐terminal amino acids in addition to three proteolytic fragments totalling 62 amino acids revealed that this PPIase is identical to the E.coli trigger factor. A comparison of the amino acid sequence of trigger factor with those of other PPIase families shows little similarities, suggesting that trigger factor may represent an additional family of PPIases. Trigger factor was purified to homogeneity on a preparative scale from E.coli and its enzymatic properties were studied. In its activity towards oligopeptide substrates, the trigger factor resembles the FK506‐binding proteins (FKBPs). Additionally, the pattern of subsite specificities with respect to the amino acid preceding proline in Suc‐Ala‐Xaa‐Pro‐Phe‐4‐nitroanilides is reminiscent of FKBPs. However, the PPIase activity of the trigger factor was not inhibited by either FK506 or by cyclosporin A at concentrations up to 100 microM. In vitro, the trigger factor catalysed the proline‐limited refolding of a variant of RNase T1 much better than all other PPIases that have been examined so far.

The GroE chaperonin machine is a major modulator of the CIRCE heat shock regulon of Bacillus subtilis

Class I heat‐inducible genes in Bacillus subtilis consist of the heptacistronic dnaK and the bicistronic groE operon and form the CIRCE regulon. Both operons are negatively regulated at the level of transcription by the HrcA repressor interacting with its operator, the CIRCE element. Here, we demonstrate that the DnaK chaperone machine is not involved in the regulation of HrcA and that the GroE chaperonin exerts a negative effect in the post‐transcriptional control of HrcA. When expression of the groE operon was turned off, the dnaK operon was significantly activated and large amounts of apparently inactive HrcA repressor were produced. Overproduction of GroEL, on the other hand, resulted in decreased expression of the dnaK operon. Introduction of the hrcA gene and its operator into Escherichia coli was sufficient to elicit a transient heat shock response, indicating that no additional Bacillus‐specific gene(s) was needed. As in B.subtilis, the groEL gene of E.coli negatively influenced the activity of HrcA. HrcA could be overproduced in E.coli, but formed inclusion bodies which could be dissolved in 8 M urea. Upon removal of urea, HrcA had a strong tendency to aggregate, but aggregation could be suppressed significantly by the addition of GroEL. Purified HrcA repressor was able specifically to retard a DNA fragment containing the CIRCE element, and the amount of retarded DNA was increased significantly in the presence of GroEL. These results suggest that the GroE chaperonin machine modulates the activity of the HrcA repressor and therefore point to a novel function of GroE as a modulator of the heat shock response.

Prolyl Isomerases: Role in Protein Folding

Publisher Summary This chapter discusses the function of prolyl isomerases in protein folding. The importance of prolyl isomerization reactions as slow, rate-limiting steps of folding, and their interdependence with other events in protein folding are described. Some experimental data on the catalysis by prolyl isomerases of various slow in vitro protein folding reactions are reviewed. The enzymatic functions of prolyl isomerases in vitro are fairly well characterized. They catalyze cis-trans isomerizations of Xaa-Pro bonds in small peptides and some proline-limited steps in the slow folding of several proteins. The characterization of the molecular nature of rate-limiting steps is a major aim in the elucidation of the folding mechanism of proteins. Folding reactions that involve prolyl isomerization are identified by measuring their kinetic properties and by comparing them with the properties of prolyl isomerization in short peptides. The chapter also explores the results that possibly suggest a role for prolyl isomerases in cellular folding and a close inter-relationship with disulfide bond formation. The efficiencies of various prolyl isomerases as catalysts of the slow-folding reactions of different proteins under varying folding conditions are tabulated.

Cooperation of enzymatic and chaperone functions of trigger factor in the catalysis of protein folding

The trigger factor of Escherichia coli is a prolyl isomerase and accelerates proline‐limited steps in protein folding with a very high efficiency. It associates with nascent polypeptide chains at the ribosome and is thought to catalyse the folding of newly synthesized proteins. In its enzymatic mechanism the trigger factor follows the Michaelis–Menten equation. The unusually high folding activity of the trigger factor originates from its tight binding to the folding protein substrate, as reflected in the low Km value of 0.7 μM. In contrast, the catalytic constant kcat is small and shows a value of 1.3 s−1 at 15°C. An unfolded protein inhibits the trigger factor in a competitive fashion. The isolated catalytic domain of the trigger factor retains the full prolyl isomerase activity towards short peptides, but in a protein folding reaction its activity is 800‐fold reduced and no longer inhibited by an unfolded protein. Unlike the prolyl isomerase site, the polypeptide binding site obviously extends beyond the FKBP domain. Together, this suggests that the good substrate binding, i.e. the chaperone property, of the intact trigger factor is responsible for its high efficiency as a catalyst of proline‐limited protein folding.

The SurA periplasmic PPIase lacking its parvulin domains functions in vivo and has chaperone activity

The Escherichia coli periplasmic peptidyl‐prolyl isomerase (PPIase) SurA is involved in the maturation of outer membrane porins. SurA consists of a substantial N‐terminal region, two iterative parvulin‐like domains and a C‐terminal tail. Here we show that a variant of SurA lacking both parvulin‐like domains exhibits a PPIase‐independent chaperone‐like activity in vitro and almost completely complements the in vivo function of intact SurA. SurA interacts preferentially (>50‐fold) with in vitro synthesized porins over other similarly sized proteins, leading us to suggest that the chaperone‐like function of SurA preferentially facilitates maturation of outer membrane proteins.

Kinetic coupling between protein folding and prolyl isomerization. I. Theoretical models

Kinetic models were developed to describe the influence of prolyl peptide bond isomerization on the kinetics of reversible protein folding for cases in which structural intermediates do not occur. In the simulations, the number of prolyl residues and the relative rates of folding and isomerization were varied. The experimentally observed rate constants were found to be identical with the intrinsic rate constants of folding and isomerization only when folding remains much faster than prolyl isomerization throughout the transition region. When the rate of folding becomes similar to or lower than the rate of isomerization, the observed kinetic parameters are complex functions of all microscopic rate constants. In particular, the observed folding rates in the transition region decrease with the number of prolyl residues. Pseudo two-state kinetics with single folding and unfolding reactions are observed in several cases, although the apparent folding rates depend strongly on prolyl isomerization reactions in the unfolded chain. This virtual simplicity can easily lead to misinterpretation of kinetic data. Additional phases can be resolved when refolding is started from the fast-folding species (UF). The coupling between folding and prolyl peptide bond isomerization also modifies the dependence on denaturant concentration of the apparent rate constants of folding. We suggest several tests to detect and characterize the contributions of folding and isomerization steps to the observed folding kinetics.

Detection of an early intermediate in the folding of ribonuclease A by protection of amide protons against exchange

A new method is presented for detecting structural intermediates at early stages of protein folding. The principle is to label with 3H the exchangeable amide protons of the unfolded protein and then to choose refolding conditions (pH, temperature) where rapid exchange-out will occur unless folding intermediates protect some protons. The label which would be retained after folding if there were no folding intermediates is calculated and subtracted to give the number of protected protons. Folding is allowed to go to completion and the number of protected protons which are stable to exchange for 6 to 13 hours after folding is determined. The slow-folding class of unfolded ribonuclease A has been chosen for study. The folding conditions are: 10 °C, pH 6 to 8, 0.2 to 2.5 m-guanidine · HCl. A large fraction (13) of the stable protons of native RNAase A is protected during folding. This fraction drops rapidly with increasing guanidine · HCl molarity as folding intermediates are destabilized. At 2.5 m-guanidine · HCl, pH 6, only 1.5 protons are protected although folding goes to completion and there is little effect of guanidine · HCl on the exchange kinetics of the stable protons in native RNAase A. This result provides a negative control. A second control is given by RNAase S, for which no protected protons are found after folding. We conclude that the S-peptide moiety of RNAase A is needed for stability of the early folding intermediate. The number of protected protons also decreases with pH in the range pH 6 to 8: this result is expected since the exchange step is base-catalyzed while the folding kinetics are independent of pH between 6 and 8. There are two conclusions about the kinetic mechanism of folding. 1. (1) The structural basis for protection against exchange appears to be formation of an H-bonded structure, since exclusion of water from the tertiary structure occurs later, as monitored by tyrosine absorbance. It appears that at least part of the H-bonded backbone of RNAase A is formed at an early stage in folding. The intermediate is stabilized by the S-peptide moiety of RNAase A. 2. (2) Folding of RNAase A is a sequential process, with kinetically observable intermediates, in these conditions.

The effects of ionic strength on protein stability: the cold shock protein family.

Continuum electrostatic models are used to examine in detail the mechanism of protein stabilization and destabilization due to salt near physiological concentrations. Three wild-type cold shock proteins taken from mesophilic, thermophilic, and hyperthermophilic bacteria are studied using these methods. The model is validated by comparison with experimental data collected for these proteins. In addition, a number of single point mutants and three designed sequences are examined. The results from this study demonstrate that the sensitivity of protein stability toward salt is correlated with thermostability in the cold shock protein family. The calculations indicate that the mesophile is stabilized by the presence of salt while the thermophile and hyperthermophile are destabilized. A decomposition of the salt influence at a residue level permits identification of regions of the protein sequences that contribute toward the observed salt-dependent stability. This model is used to rationalize the effect of various point mutations with regard to sensitivity toward salt. Finally, it is demonstrated that designed cold shock protein variants exhibit electrostatic properties similar to the natural thermophilic and hyperthermophilic proteins.

Semisynthesis of a Homogeneous Glycoprotein Enzyme: Ribonuclease C: Part 2

Active RNase glycoprotein from three pieces: The glycoprotein enzyme ribonuclease C, which contains a complex saccharide N-glycan, was synthesized by sequential native chemical ligation. An optimized ligation and isolation protocol allowed the efficient assembly and refolding of the 124 amino acid enzyme.