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Summary Trigger factor (TF) is the first molecular chaperone interacting cotranslationally with virtually all nascent polypeptides synthesized by the ribosome in bacteria. Thermal adaptation of chaperone function was investigated in TFs from the Antarctic psychrophile Pseudoalteromonas haloplanktis, the mesophile Escherichia coli and the hyperthermophile Thermotoga maritima. This series covers nearly all temperatures encountered by bacteria. Although structurally homologous, these TFs display strikingly distinct properties that are related to the bacterial environmental temperature. The hyperthermophilic TF strongly binds model proteins during their folding and protects them from heat-induced misfolding and aggregation. It decreases the folding rate and counteracts the fast folding rate imposed by high temperature. It also functions as a carrier of partially folded proteins for delivery to downstream chaperones ensuring final maturation. By contrast, the psychrophilic TF displays weak chaperone activities, showing that these functions are less important in cold conditions because protein folding, misfolding and aggregation are slowed down at low temperature. It efficiently catalyses prolyl isomerization at low temperature as a result of its increased cellular concentration rather than from an improved activity. Some chaperone properties of the mesophilic TF possibly reflect its function as a cold shock protein in E. coli.

Summary Filamentous phage use the two N-terminal domains of their gene-3-proteins to initiate infection of Escheri- chia coli. One domain interacts with a pilus, and then the other domain binds to TolA at the cell surface. In phage fd, these two domains are tightly associated with each other, which renders the phage robust but non-infectious, because the TolA binding site is inaccessible. Activation for infection requires partial unfolding, domain disassembly and prolyl isomeriza- tion. Phage IKe infects E. coli less efficiently than phage fd. Unlike in phage fd, the pilus- and TolA- bindingdomainsofphageIKeareindependentofeach other in stability and folding. The site for TolA binding is thus always accessible, but the affinity is very low. The structures of the two domains, analysed by X-ray crystallography and by NMR spectroscopy, revealed a unique fold for the N-pilus-binding domain and a con- served fold for the TolA-binding domain. The absence of an activation mechanism as in phage fd and the low affinity for TolA probably explain the low infectivity of phage IKe. They also explain why, in a previous co-evolution experiment with a mixture of phage fd and phage IKe, all hybrid phage adopted the superior infection mechanism of phage fd.

When they infect Escherichia coli cells, the filamentous phages IF1 and fd first interact with a pilus and then target TolA as their common receptor. They use the domains N2 and N1 of their gene-3-proteins (G3P) for these interactions but differ in the mechanism of infection. In G3P of phage IF1, N1 and N2 are independent modules that are permanently binding-active. G3P of phage fd is usually in a closed state in which N1 and N2 are tightly associated. The TolA binding site is thus inaccessible and the phage incompetent for infection. Partial unfolding and prolyl isomerization must occur to abolish the domain interactions and expose the TolA binding site. This complex mechanism of phage fd could be changed to the simple infection mechanism of phage IF1 by reprogramming its G3P following physicochemical rules of protein stability. The redesigned phage fd was robust and as infectious as wild-type phage fd.

PpiD is a periplasmic folding helper protein of Escherichia coli. It consists of an N-terminal helix that anchors PpiD in the inner membrane near the SecYEG translocon, followed by three periplasmic domains. The second domain (residues 264-357) shows homology to parvulin-like prolyl isomerases. This domain is a well folded, stable protein and follows a simple two-state folding mechanism. In its solution structure, as determined by NMR spectroscopy, it resembles most closely the first parvulin domain of the SurA protein, which resides in the periplasm of E. coli as well. A previously reported prolyl isomerase activity of PpiD could not be reproduced when using improved protease-free peptide assays or assays with refolding proteins as substrates. The parvulin domain of PpiD interacts, however, with a proline-containing tetrapeptide, and the binding site, as identified by NMR resonance shift analysis, colocalized with the catalytic sites of other parvulins. In its structure, the parvulin domain of PpiD resembles most closely the inactive first parvulin domain of SurA, which is part of the chaperone unit of this protein and presumably involved in substrate recognition.

Seven in one blow: The efficient formation of mixed disulfides on the thiol-rich fusion protein A followed by subsequent intein cleavage gave the fragment B with all seven cysteines protected against oxidation. The native chemical ligation of B with synthetic glycopeptide thioesters provides glycoproteins.

Aktives RNase-Glycoprotein aus drei Segmenten: Das Glycoprotein-Enzym Ribonuclease C mit einem komplexen nonasaccharidischen N-Glycan wurde durch sequenzielle native chemische Ligation synthetisiert. Durch optimierte Ligations- und Isolierungsbedingungen konnte das aus 124 Aminosauren bestehende Enzym effizient aufgebaut und zuruckgefaltet werden.

Sieben auf einen Streich: Durch die effiziente Bildung von gemischten Disulfiden an dem thiolreichen Fusionsprotein A konnte nach Inteinabspaltung das Cys-Fragment B mit sieben oxidationsgeschutzten Cysteinen erhalten werden. Die native chemische Ligation von B mit synthetischen Glycopeptidthioestern ergibt Glycoproteine.

Unfolded ribonuclease A consists of a mixture of fast-folding (UF) and slow-folding species (US); US folds slowly because it contains non-native proline isomers. The folding kinetics of US have been determined by measuring the changes in tyrosine fluorescence upon refolding. Under conditions where the energy of stabilization of the native protein is small (in marginally native conditions) the fluorescence-detected kinetics of folding coincide with the ones measured by other probes. However, when refolding is carried out under conditions where the native protein is highly stabilized (in strongly native conditions), the fluorescence changes become progressively slower than folding monitored by tyrosine absorbance or by 2′CMP binding. Under these conditions the fluorescence-detected kinetics of folding show properties of proline isomerization reactions. In addition they match proline isomerization measured during refolding of Us by a two-step assay for non-native proline isomers. The following conclusions are drawn. (1) There is a kinetic intermediate between US and the native enzyme in the folding pathway of RNase A. This has been shown by the divergent progress curves monitored by different probes. Although the intermediate contains non-native proline isomers, it is folded and hence sensitive to agents that affect the stability of native proteins. (2) Fluorescence-detected folding closely follows proline isomerization during folding. Refolding of US is not blocked by incorrect prolinc isomers, but US is able to fold to a structural intermediate before proline isomerization occurs. (3) The quantum yield of the fluorescence of native RNase A depends on the precise spatial location of the tyrosine side chains with respect to their hydrogen bond acceptors and to the disulfide bonds. Therefore the native fluorescence is not regained until all essential proline peptide bonds are in the right configuration.

Unfolded ribonuclease A (RNase A) consists of a mixture of fast refolding (UF) and slow-refolding (US) species. The slow UF⇌US equilibration reaction is rate-limited by proline peptide bond isomerization. Investigations of the dependence on temperature of the UF⇌US equilibrium have led to conflicting results and different molecular interpretations. Here the dependence on temperature of the UF: US ratio was reinvestigated by using a new assay for the fast-folding molecules UF. Between 0°C and 60°C the proportion of UF present in unfolded RNase A at 6M guanidine · HCl was found to be independent of temperature. Consequently, no conclusions can be drawn regarding the role and importance of particular prolines in the UF⇌US transition solely from these results.

The superior hemodynamic performance of the pulmonary autograft in aortic position is expected to reflect complete regression of hypertrophy and improved ventricular function. We evaluated and compared early and midterm transthoracic color-Doppler echocardiography (TTE) and magnetic resonance imaging (MRI) assessment concerning left ventricular (LV) function, LV mass regression, and performance of the semilunar valves. A total of 42 consecutive patients, mean age 36 +/- 6 years (range 15 to 56 years), were studied. TTE and MRI were performed preoperatively, at discharge, and at 6 and 12 months postoperatively. Left ventricular diameters and function and LV wall thickness and mass were assessed. There was no early and one late postoperative death. Maximum and mean LV outflow gradients were significantly reduced (p = 0.0001 and p = 0.0001, respectively). There was a significant reduction in left ventricular mass to near normal for all patients (p = 0.001) seen after 6 months. This was paralleled by significant reductions in the interventricular septum and posterior wall thickness, but difference was slight during further follow-up. There was a strong correlation between the results obtained by TTE and MRI for LV mass and ejection fraction (r = 0.86 and 0. 87, respectively). The pulmonary autograft operation gives excellent results that are translated into greater, rapid, and near complete regression of LV hypertrophy. Magnetic resonance imaging is a sensitive and noninvasive methodology that provides reliable visualization and quantification of ventricular performance.

In the cold-shock protein CspB from Bacillus subtilis three exposed Phe residues (F15, F17, and F27) are essential for its function in binding to single-stranded nucleic acids. Usually, the hydrophobic Phe side chains are buried in folded proteins. We asked here whether the exposition of the essential Phe residues could be a cause for the very low conformational stability of CspB. Urea-induced and heat-induced equilibrium unfolding transitions were measured for three mutants of CspB, where Phe 15, Phe 17, and Phe 27 were individually replaced by alanine. Unexpectedly, all three mutations strongly destabilized CspB. The aromatic side chains of Phe 15, Phe 17, and Phe 27 in the active site are thus important for both binding to nucleic acids and conformational stability. There is no compromise between function and stability in the active site. Model calculations indicate that, although they are partially exposed to solvent, all three Phe residues nevertheless lose accessible surface upon folding, and this should favor the native state. A different result is obtained with the F38A variant. Phe 38 is hyperexposed in native CspB, and its substitution by Ala is in fact stabilizing.

Regulation of the heat shock response in bacteria has been studied extensively inEscherichia coli where heat shock genes are classified into three classes and where each class is regulated by a different alternate sigma factor.Bacillus subtilis serves as a second model bacterium to study regulation of the heat shock response in detail. Here, four classes of heat shock genes have been described so far where two are controlled by two different repressor proteins and the third by the alternate sigma factorσB. Class I heat shock genes consists of two operons, the heptacistronicdnaK and the bicistronicgroE operon. Transcription of thednaK operon is complex involving two promoters, premature termination of transcription, mRNA processing and different stabilities of the processed transcripts to ensure the appropriate amounts of heat shock proteins under different growth conditions. The translation product of thehrcA gene, the first gene of thednaK operon, binds to an operator designated CIRCE element, and its activity is modulated by the GroE chaperonin system. We assume that the HrcA protein, uponde novo synthesis and upon dissociation from its operator, is present in an inactive form and has to be activated by the GroE chaperonin system resulting in an HrcA-GroE reaction cycle. Induction of class I heat shock genes occurs by the appearance of denatured proteins within the cytoplasm which titrate the GroE system. This results in accumulation of inactive HrcA repressor and thereby in induction of class I heat shock genes. Upon removal of the non-native proteins from the cytoplasm, the GroE chaperonin will interact with HrcA and promote folding into its active conformation resulting in turning off of class I heat shock genes. This mechanism ensures adequate adjustment of class I heat shock proteins depending on their actual need.

Die Proteinfaltung verlauft in einem Netzwerk von Faltungshelfern. Neue Polypeptide und Antikorper gehoren zu den Schwerpunkten im Protein Engineering. Nach einer Darstellung der Mechanismen der negativen Regulation der neuronalen Genexpression in nicht-neuronalem Gewebe beschliest ein Uberblick zum aktuellen Stand der Genomforschung die diesjahrige Auswahl.

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 Cys 2-Cys 10 disulfide bond in ribonuclease T1 was broken by substituting Cys 2 and Cys 10 by Ser and Asn, respectively, as present in ribonuclease F1. This C2S/C10N variant resembles the wild-type protein in structure and in catalytic activity. Minor structural changes were observed by 2-dimensional NMR in the local environment of the substituted amino acids only. The thermodynamic stability of ribonuclease T1 is strongly reduced by breaking the Cys 2-Cys 10 bond, and the free energy of denaturation is decreased by about 10 kJ/mol. The folding mechanism is not affected, and the trans to cis isomerizations of Pro 39 and Pro 55 are still the rate-limiting steps of the folding process. The differences in the time courses of unfolding and refolding are correlated with the decrease in stability: the folding kinetics of the wild-type protein and the C2S/C10N variant become indistinguishable when they are compared under conditions of identical stability. Apparently, the Cys 2-Cys 10 disulfide bond is important for the stability but not for the folding mechanism of ribonuclease T1. The breaking of this bond has the same effect on stability and folding kinetics as adding 1 M guanidinium chloride to the wild-type protein.

Kinetic intermediates in protein folding are short-lived and therefore difficult to detect and to characterize. In the folding of polypeptide chains with incorrect isomers of Xaa-Pro peptide bonds the final rate-limiting transition to the native state is slow, since it is coupled to prolyl isomerization. Incorrect prolyl isomers thus act as effective traps for folding intermediates and allow their properties to be studied more easily. We employed this strategy to investigate the mechanism of slow folding of ribonuclease T1. In our experiments we use a mutant form of this protein with a single cis peptide bond at proline 39. During refolding, protein chains with an incorrect trans proline 39 can rapidly form extensive secondary structure. The CD signal in the amide region is regained within the dead-time of stopped-flow mixing (15 ms), indicating a fast formation of the single alpha-helix of ribonuclease T1. This step is correlated with partial formation of a hydrophobic core, because the fluorescence emission maximum of tryptophan 59 is shifted from 349 nm to 325 nm within less than a second. After about 20 s of refolding an intermediate is present that shows about 40% enzymatic activity compared to the completely refolded protein. In addition, the solvent accessibility of tryptophan 59 is drastically reduced in this intermediate and comparable to that of the native state as determined by acrylamide quenching of the tryptophan fluorescence. Activity and quenching measurements have long dead-times and therefore we do not know whether enzymatic activity and solvent accessibility also change in the time range of milliseconds. At this stage of folding at least part of the beta-sheet structure is already present, since it hosts the active site of the enzyme. The trans to cis isomerization of the tyrosine 38-proline 39 peptide bond in the intermediate and consequently the formation of native protein is very slow (tau = 6,500 s at pH 5.0 and 10 degrees C). It is accompanied by an additional increase in tryptophan fluorescence, by the development of the fine structure of the tryptophan emission spectrum, and by the regain of the full enzymatic activity. This indicates that the packing of the hydrophobic core, which involves both tryptophan 59 and proline 39, is optimized in this step. Apparently, refolding polypeptide chains with an incorrect prolyl isomer can very rapidly form partially folded intermediates with native-like properties.

The role of carbohydrate chains for the structure, function, stability, and folding of glycoproteins has been investigated using invertase as a model. The protein is encoded by several different genes, and its carbohydrate moiety is heterogeneous. Both properties complicate physicochemical comparisons. Here we used the temperature-sensitive sec18 secretion mutant of yeast with a single invertase gene (SUC2). This mutant produces the carbohydrate-free internal invertase, the core-glycosylated form, and, at the permissive temperature, the fully glycosylated external enzyme, all with identical protein moieties. The core-glycosylated enzyme resembles the nascent glycoprotein chain that folds in the endoplasmic reticulum. Therefore, it may be considered a model for the in vivo folding of glycoproteins. In addition, because of its uniform glycosylation, it can be used to investigate the state of association of native invertase. Glycosylation is found to stabilize the protein with respect to thermal denaturation and chaotropic solvent components; the stabilizing effect does not differ for the external and the core-glycosylated forms. Unlike the internal enzyme, the glycosylated forms are protected from aggregation. Native internal invertase is a dimer (115 kDa) whereas the core-glycosylated enzyme is a mixture of dimers, tetramers, and octamers. This implies that core-glycosylation is necessary for oligomerization to tetramers and octamers. Dimerization is required and sufficient to generate enzymatic activity; further association does not alter the specific activity of core-glycosylated invertase, suggesting that the active sites of invertase are not affected by the association of the dimeric units. Reconstitution of the glycosylated and nonglycosylated forms of the enzyme after preceding guanidine denaturation depends on protein concentration. The maximum yield (approximately 80%) is obtained at pH 6-8 and protein concentrations < or = 4 micrograms/mL for the nonglycosylated and < or = 40 for the glycosylated forms of the enzyme. The lower stability of the internal enzyme is reflected by a narrower pH range of reactivation and enhanced aggregation. As indicated by the sigmoidal reactivation kinetics at low protein concentration both folding and association are rate-determining.

The complexity of protein folding originates in large part from the huge amount of rotations about single bonds that are possible in a polypeptide chain. Even when only the backbone is considered, rotations about two single bonds can occur at every Cα atom. The enormous number of conformations that are thus possible for the backbone of an unfolded protein have long been considered as a major problem for protein folding (Levinthal, 1968).

Trypsin pulses, applied after varying times of refolding, have been employed to probe the accessibility of the polypeptide chain of ribonuclease A during the process of refolding. The increase in resistance against proteolytic cleavage was measured by activity assays and by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The sites of cleavage which become inaccessible in the course of refolding are located in the 31 - 39 chain segment of the ribonuclease chain. Protection of this region against attack by trypsin is attained on the major slow refolding pathway in parallel with the formation of a native-like folded, active intermediate, when refolding is carried out under conditions which strongly stabilize the folded state. Under conditions of marginal stability intermediates are not observed during refolding and the formation of trypsin-resistant molecules occurs with the same kinetics as the generation of native ribonuclease. In the native protein the 31 - 39 region of the ribonuclease chain largely forms a loop structure, which is located at the surface of the molecule. Our results indicate that this part of the sequence is still accessible at early stages of refolding, when a hydrogen-bonded network is formed. It is structured and hence does not become inaccessible until the formation of the overall folded native or native-like structure. This suggests that the 31 - 39 region of the ribonuclease chain is not important for early steps which direct the pathway of refolding.