F X Schmid

University of Bayreuth , Bayreuth, Bavaria, Germany

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Publications (213)843.71 Total impact

  • Johanna R Koch, Franz X Schmid
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    ABSTRACT: Mia40 catalyzes oxidative protein folding in mitochondria. It contains a unique catalytic CPC dithiol flanked by a hydrophobic groove, and unlike other oxidoreductases, it forms long-lived mixed disulfides with substrates. We show that this distinctive property originates neither from particular properties of mitochondrial substrates nor from the CPC motif of Mia40. The catalytic cysteines of Mia40 display unusually low chemical reactivity, as expressed in conventional pK values and reduction potentials. The stability of the mixed disulfide intermediate is coupled energetically with hydrophobic interactions between Mia40 and the substrate. Based on these properties, we suggest a mechanism for Mia40, where the hydrophobic binding site is employed to select a substrate thiol for forming the initial mixed disulfide. Its long lifetime is used to retain partially folded proteins in the mitochondria and to direct folding towards forming the native disulfide bonds.
    ACS Chemical Biology 07/2014; · 5.44 Impact Factor
  • Philipp A M Schmidpeter, Franz X Schmid
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    ABSTRACT: The cellular CT10 regulator of kinase protein (c-CrkII) transmits signals from oncogenic tyrosine kinases to cellular targets. NMR studies had suggested that in chicken c-CrkII a native-state prolyl cis/trans isomerization is involved in signal propagation. Corresponding evidence for the closely related human c-CrkII was not obtained. Here we analyzed the kinetics of folding and substrate binding of the two homologs and found that cis/trans isomerization of Pro238 determines target binding in chicken but not in human c-CrkII. A reciprocal mutational analysis uncovered residues that determine the isomeric state at Pro238 and transmit it to the binding site for downstream target proteins. Transfer of these key residues to human c-CrkII established a regulatory proline switch in this protein as well. We suggest that Pro238 isomerization extends the life time of the signaling-active state of c-CrkII and thereby functions as a long-term molecular storage device.
    ACS Chemical Biology 02/2014; · 5.44 Impact Factor
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    ABSTRACT: To initiate infection of Escherichia coli, phage fd uses its gene-3-protein (G3P) to bind first to an F pilus and then to the TolA protein at the cell surface. G3P is normally auto-inhibited, because a tight interaction between the two N-terminal domains N1 and N2 buries the TolA binding site. Binding of N2 to the pilus activates G3P by initiating long-range conformational changes that are relayed to the domain interface and to a proline timer. We discovered that the 23-28 loop of the N1 domain is critical for propagating these conformational signals. The analysis of the stability and the folding dynamics of G3P variants with a shortened loop, combined with TolA interaction studies and phage infection experiments reveal how the contact between the N2 domain and the 23-28 loop of N1 is energetically linked with the interdomain region and the proline timer and how it affects phage infectivity. Our results illustrate how conformational transitions and prolyl cis/trans isomerization can be coupled energetically and how conformational signals to and from prolines can be propagated over long distances in proteins.
    Journal of Molecular Biology 01/2014; · 3.91 Impact Factor
  • Johanna R Koch, Franz X Schmid
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    ABSTRACT: Mia40 catalyses the oxidative folding of disulphide-containing proteins in the mitochondria. The folding pathway is directed by the formation of the first mixed disulphide between Mia40 and its substrate. Here, we employ Cox17 to elucidate the molecular determinants of this reaction. Mia40 engages initially in a dynamic non-covalent enzyme-substrate complex that forms and dissociates within milliseconds. Cys36 of Cox17 forms the mixed disulphide in an extremely rapid reaction that is limited by the preceding complex formation with Mia40. Cys36 reacts much faster than the three other cysteines of Cox17, because it neighbours three hydrophobic residues. Mia40 binds preferentially to hydrophobic regions and the dynamic nature of the non-covalent complex allows rapid reorientation for an optimal positioning of the reactive cysteine. Mia40 thus uses the unique proximity between its substrate-binding site and the catalytic disulphide to select a particular cysteine for forming the critical initial mixed disulphide.
    Nature Communications 01/2014; 5:3041. · 10.02 Impact Factor
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    ABSTRACT: Parvulins are small prolyl isomerases and serve as catalytic domains of folding enzymes. SurA from the periplasm of Escherichia coli consists of an inactive (Par1) and an active (Par2) parvulin domain as well as a chaperone domain. In the absence of the chaperone domain, the folding activity of Par2 is virtually abolished. We created a chimeric protein by inserting the chaperone domain of SlyD, an unrelated folding enzyme from the FKBP family, into a loop of the isolated Par2 domain of SurA. This increased its folding activity 450-fold to a value higher than the activity of SurA, in which Par2 is linked with its natural chaperone domain. In the presence of both the natural and the foreign chaperone domain, the folding activity of Par2 was 1500-fold increased. Related and unrelated chaperone domains thus are similarly efficient in enhancing the folding activity of the prolyl isomerase Par2. A sequence analysis of various chaperone domains suggests that clusters of exposed methionine residues in mobile chain regions might be important for a generic interaction with unfolded protein chains. This binding is highly dynamic to allow frequent transfer of folding protein chains between chaperone and catalytic domains.
    Journal of Molecular Biology 07/2013; · 3.91 Impact Factor
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    ABSTRACT: Abstract SlyD is a bacterial two-domain protein that functions as a molecular chaperone, a prolyl cis/trans isomerase, and a nickel binding protein. This review summarizes recent findings about the molecular enzyme mechanism of SlyD. The chaperone function located in one domain of SlyD is involved in twin-arginine translocation and increases the catalytic efficiency of the prolyl cis/trans isomerase domain in protein folding by two orders of magnitude. The C-terminal tail of SlyD binds Ni2+ ions and supplies them for the maturation of [NiFe] hydrogenases. A combined biochemical and biophysical analysis revealed the molecular basis of the delicate interplay of the different domains of SlyD for an optimal function.
    Biological Chemistry 03/2013; · 2.68 Impact Factor
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    ABSTRACT: Infection of Escherichia coli by the filamentous phage fd starts with the binding of the N2 domain of the phage gene-3-protein to an F pilus. This interaction triggers partial unfolding of the gene-3-protein, cis/trans isomerization at Pro213, and domain disassembly, thereby exposing its binding site for the ultimate receptor TolA. The trans proline sets a molecular timer to maintain the binding-active state long enough for the phage to interact with TolA. We elucidated the changes in structure and local stability that lead to partial unfolding and thus to the activation of the gene-3-protein for phage infection. Protein folding and TolA binding experiments were combined with real-time NMR spectroscopy, amide hydrogen exchange measurements and phage infectivity assays. In combination, the results provide a molecular picture of how a local unfolding reaction couples with prolyl isomerization not only to generate the activated state of a protein but also to maintain it for an extended time.
    Journal of Biological Chemistry 03/2013; · 4.65 Impact Factor
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    ABSTRACT: Folding enzymes often use distinct domains for the interaction with a folding protein chain and for the catalysis of intrinsically slow reactions such as prolyl cis/trans isomerization. Here we investigated the refolding reaction of ribonuclease T1 in the presence of the prolyl isomerase SlyD from Escherichia coli to examine how this enzyme catalyzes the folding of molecules with an incorrect trans proline isomer and how it modulates the conformational folding of the molecules with the correct cis proline. The kinetic analysis suggests that prolyl cis-trans isomerization in the SlyD-bound state shows a rate near 100 s-1 and is thus more than 10exp4-fold accelerated, relative to the uncatalyzed reaction. As a consequence of its fast binding and efficient catalysis, SlyD retards the conformational folding of the protein molecules with the correct cis isomer, because it promotes the formation of the species with the incorrect trans isomer. In the presence of 1 microM SlyD or higher, protein molecules with cis and trans prolyl isomers refold with identical rates, because SlyD-catalyzed cis/trans equilibration is faster than conformational folding. The cis or trans state of a particular proline is thus no longer a determinant for the rate of folding.
    Journal of the American Chemical Society 02/2013; · 10.68 Impact Factor
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    ABSTRACT: Human aryl hydrocarbon receptor (AHR) interacting protein (AIP) and AIP like 1 (AIPL1) are cochaperones of Hsp90 which share 49% sequence identity. Both proteins contain an N-terminal FKBP-like prolyl peptidyl isomerase (PPIase) domain followed by a Tetratricopeptide repeat (TPR) domain. In addition, AIPL1 harbors a unique C-terminal proline-rich domain (PRD). Little is known about the functional relevance of the individual domains and how these contribute to the association with Hsp90. In this study, we show that these cochaperones differ from other Hsp90-associated PPIase as their FKBP domains are enzymatically inactive. Furthermore, in contrast to other large PPIases, AIP is inactive as a chaperone. AIPL1, however, exhibits chaperone activity and prevents the aggregation of non-native proteins. The unique proline-rich domain of AIPL1 is important for its chaperone function as its truncation severely affects the ability of AIPL1 to bind non-native proteins. Furthermore, the proline-rich domain decreased the affinity of AIPL1 for Hsp90, implying that this domain acts as a negative regulator of the Hsp90 interaction besides being necessary for efficient binding of AIPL1 to non-native proteins.
    Biochemistry 02/2013; · 3.38 Impact Factor
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    ABSTRACT: In general, β-lactamases of medically important Gram-negative bacteria are Sec-dependently translocated into the periplasm. In contrast, β-lactamases of Mycobacteria spp. (BlaC, BlaS) and the Gram-negative environmental bacteria Stenotrophomonas maltophilia (L2) and Xanthomonas campestris (Bla(XCC-1)) have been reported to be secreted by the twin-arginine translocation (Tat) system. Yersinia enterocolitica carries 2 distinct β-lactamase genes (blaA and blaB) encoding BlaA(Ye) and the AmpC-like β-lactamase BlaB, respectively. By using the software PRED-TAT for prediction and discrimination of Sec from Tat signal peptides, we identified a functional Tat signal sequence for Yersinia BlaA(Ye). The Tat-dependent translocation of BlaA(Ye) could be clearly demonstrated by using a Y. enterocolitica tatC-mutant and cell fractionation. Moreover, we could demonstrate a unique unusual temperature-dependent activity profile of BlaA(Ye) ranging from 15 to 60°C and a high 'melting temperature' (T(M)=44.3°) in comparison to the related Sec-dependent β-lactamase TEM-1 (20-50°C, T(M)=34.9°C). Strikingly, the blaA gene of Y. enterocolitica is present in diverse environmental Yersinia spp. and a blaA homolog gene could be identified in the closely related Photorhabdus asymbiotica (BlaA(Pa); 69% identity to BlaA(Ye)). For BlaA(Pa) of P. asymbiotica, we could also demonstrate Tat-dependent secretion. These results suggest that Yersinia BlaA-related β-lactamases may be the prototype of a large Tat-dependent β-lactamase family, which originated from environmental bacteria.
    International journal of medical microbiology: IJMM 12/2012; · 4.54 Impact Factor
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    ABSTRACT: The (βα)(8)-barrel is among the most ancient, frequent, and versatile enzyme structures. It was proposed that modern (βα)(8)-barrel proteins have evolved from an ancestral (βα)(4)-half-barrel by gene duplication and fusion. We explored whether the mechanism of protein folding has remained conserved during this long-lasting evolutionary process. For this purpose, potential primordial (βα)(8)-barrel proteins were constructed by the duplication of a (βα)(4) element of a modern (βα)(8)-barrel protein, imidazole glycerol phosphate synthase (HisF), followed by the optimization of the initial construct. The symmetric variant Sym1 was less stable than HisF and its crystal structure showed disorder in the contact regions between the half-barrels. The next generation variant Sym2 was more stable than HisF, and the contact regions were well resolved. Remarkably, both artificial (βα)(8)-barrels show the same refolding mechanism as HisF and other modern (βα)(8)-barrel proteins. Early in folding, they all equilibrate rapidly with an off-pathway species. On the productive folding path, they form closely related intermediates and reach the folded state with almost identical rates. The high energy barrier that synchronizes folding is thus conserved. The strong differences in stability between these proteins develop only after this barrier and lead to major changes in the unfolding rates. We conclude that the refolding mechanism of (βα)(8)-barrel proteins is robust. It evolved early and, apparently, has remained conserved upon the diversification of sequences and functions that have taken place within this large protein family.
    Journal of the American Chemical Society 07/2012; 134(30):12786-91. · 10.68 Impact Factor
  • Source
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    ABSTRACT: Filamentous phage use the two N-terminal domains of their gene-3-proteins to initiate infection of Escherichia 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 isomerization. Phage IKe infects E. coli less efficiently than phage fd. Unlike in phage fd, the pilus- and TolA-binding domains of phage IKe are independent of each 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 conserved 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.
    Molecular Microbiology 05/2012; 84(6):1124-38. · 4.96 Impact Factor
  • Anne-Juliane Geitner, Franz Xaver Schmid
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    ABSTRACT: Folding enzymes often use distinct domains for the binding of substrate proteins ("chaperone domains") and for the catalysis of slow folding reactions such as disulfide formation or prolyl isomerization. The human prolyl isomerase FKBP12 is a small single-domain protein without a chaperone domain. Its very low folding activity could previously be increased by inserting the chaperone domain from the homolog SlyD (sensitive-to-lysis protein D) of Escherichia coli. We now inserted three unrelated chaperone domains into human FKBP12: the apical domain of the chaperonin GroEL from E. coli, the chaperone domain of protein disulfide isomerase from yeast, or the chaperone domain of SurA from the periplasm of E. coli. All three conveyed FKBP12 with a high affinity for unfolded proteins and increased its folding activity. Substrate binding and release of the chimeric folding enzymes were found to be very fast. This allows rapid substrate transfer from the chaperone domain to the catalytic domain and ensures efficient rebinding of protein chains that were unable to complete folding. The advantage of having separate sites, first for generic protein binding and then for specific catalysis, explains why our construction of the artificial folding enzymes with foreign chaperone domains was successful.
    Journal of Molecular Biology 04/2012; 420(4-5):335-49. · 3.91 Impact Factor
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    ABSTRACT: Prolyl isomerases catalyze the cis/trans isomerization of peptide bonds preceding proline. Previously, we had determined the specificity toward the residue before the proline for cyclophilin-, FKBP-, and parvulin-type prolyl isomerases by using proline-containing oligopeptides and refolding proteins as model substrates. Here, we report the specificities of members of these three prolyl isomerase families for the residue following the proline, again in short peptide and in refolding protein chains. Human cyclophilin 18 and parvulin 10 from Escherichia coli show high activity, but low specificity, with respect to the residue following the proline. Human FKBP12 prefers hydrophobic residues at this position in the peptide assays and shows a very low activity in the protein folding assays. This activity was strongly improved, and the sequence specificity was virtually eliminated after the insertion of a chaperone domain into the prolyl isomerase domain of human FKBP12.
    Biochemistry 05/2011; 50(21):4796-803. · 3.38 Impact Factor
  • Stefan H Lorenz, Franz X Schmid
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    ABSTRACT: 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.
    Molecular Microbiology 03/2011; 80(3):827-34. · 4.96 Impact Factor
  • Gabriel Zoldák, Franz X Schmid
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    ABSTRACT: The SlyD (sensitive to lysis D) protein of Escherichia coli is a folding enzyme with a chaperone domain and a prolyl isomerase domain of the FK506 binding protein type. Here we investigated how the two domains and their interplay are optimized for function in protein folding. Unfolded protein molecules initially form a highly dynamic complex with the chaperone domain of SlyD, and they are then transferred to the prolyl isomerase domain. The turnover number of the prolyl isomerase site is very high and guarantees that, after transfer, prolyl peptide bonds in substrate proteins are isomerized very rapidly. The Michaelis constant of catalyzed folding reflects the substrate affinity of the chaperone domain, and the turnover number is presumably determined by the rate of productive substrate transfer from the chaperone to the prolyl isomerase site and by the intrinsic propensity of the refolding protein chain to leave the active site with the native prolyl isomer. The efficiency of substrate transfer is high because dissociation from the chaperone site is very fast and because the two sites are close to each other. Protein molecules that left the prolyl isomerase site with an incorrect prolyl isomer can rapidly be re-bound by the chaperone domain because the association rate is very high as well.
    Journal of Molecular Biology 02/2011; 406(1):176-94. · 3.91 Impact Factor
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    ABSTRACT: The filamentous phage fd uses its gene 3 protein (G3P) to target Escherichia coli cells in a two-step process. First, the N2 domain of G3P attaches to an F pilus, and then the N1 domain binds to TolA-C. N1 and N2 are tightly associated, rendering the phage robust but noninfectious because the binding site for TolA-C is buried at the domain interface. Binding of N2 to the F pilus initiates partial unfolding, domain disassembly, and prolyl cis-to-trans isomerization in the hinge between N1 and N2. This activates the phage, and trans-Pro213 maintains this state long enough for N1 to reach TolA-C. Phage IF1 targets I pili, and its G3P contains also an N1 domain and an N2 domain. The pilus-binding N2 domains of the phages IF1 and fd are unrelated, and the N1 domains share a 31% sequence identity. We show that N2 of phage IF1 mediates binding to the I pilus, and that N1 targets TolA. Crystallographic and NMR analyses of the complex between N1 and TolA-C indicate that phage IF1 interacts with the same site on TolA-C as phage fd. In IF1-G3P, N1 and N2 are independently folding units, however, and the TolA binding site on N1 is permanently accessible. Activation by unfolding and prolyl isomerization, as in the case of phage fd, is not observed. In IF1-G3P, the absence of stabilizing domain interactions is compensated for by a strong increase in the stabilities of the individual domains. Apparently, these closely related filamentous phages evolved different mechanisms to reconcile robustness with high infectivity.
    Journal of Molecular Biology 01/2011; 405(4):989-1003. · 3.91 Impact Factor
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    ABSTRACT: In the N2 domain of the gene-3-protein of phage fd, two consecutive beta-strands are connected by a mobile loop of seven residues (157-163). The stability of this loop is low, and the Asp160-Pro161 bond at its tip shows conformational heterogeneity with 90% being in the cis and 10% in the trans form. The refolding kinetics of N2 are complex because the molecules with cis or trans isomers at Pro161 both fold to native-like conformations, albeit with different rates. We employed consensus design to shorten the seven-residue irregular loop around Pro161 to a four-residue type I' turn without a proline. This increased the conformational stability of N2 by almost 10 kJ mol(-1) and abolished the complexity of the folding kinetics. Turn sequences obtained from in vitro selections for increased stability strongly resembled those derived from the consensus design. Two other type I' turns of N2 could also be stabilized by consensus design. For all three turns, the gain in stability originates from an increase in the rate of refolding. The turns form native-like structures early during refolding and thus stabilize the folding transition state. The crystal structure of the variant with all three stabilized turns confirms that the 157-163 loop was in fact shortened to a type I' turn and that the other turns maintained their type I' conformation after sequence optimization.
    Journal of Molecular Biology 06/2010; 399(2):331-46. · 3.91 Impact Factor
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    ABSTRACT: Class A beta-lactamases (M(r) approximately 29000) provide good models for studying the folding mechanism of large monomeric proteins. In particular, the highly conserved cis peptide bond between residues 166 and 167 at the active site of these enzymes controls important steps in their refolding reaction. In this work, we analyzed how conformational folding, reactivation, and cis/trans peptide bond isomerizations are interrelated in the folding kinetics of beta-lactamases that differ in the nature of the cis peptide bond, which involves a Pro167 in the BS3 and TEM-1 enzyme, a Leu167 in the NMCA enzyme, and which is missing in the PER-1 enzyme. The analysis of folding by spectroscopic probes and by the regain of enzymatic activity in combination with double-mixing procedures indicates that conformational folding can proceed when the 166-167 bond is still in the incorrect trans form. The very slow trans --> cis isomerization of the Glu166-Xaa167 peptide bond, however, controls the final step of folding and is required for the regain of the enzymatic activity. This very slow phase is absent in the refolding of PER-1, in which the Glu166-Ala167 peptide bond is trans. The double-mixing experiments revealed that a second slow kinetic phase is caused by the cis/trans isomerization of prolines that are trans in the folded proteins. The folding of beta-lactamases is best described by a model that involves parallel pathways. It highlights the role of peptide bond cis/trans isomerization as a kinetic determinant of folding.
    Biochemistry 04/2010; 49(19):4264-75. · 3.38 Impact Factor
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    ABSTRACT: A low-resistance membrane oxygenator [interventional lung assist (iLA), Novalung, Hirrlingen, Germany] has been placed in series with a pulsatile extracorporeal left ventricular assist device (LVAD, Berlin Heart EXCOR, Berlin, Germany) in 1 circuit in a patient with postcardiotomy cardiopulmonary failure subsequent to 5 days of extracorporeal life support (ECLS or ECMO). This concept offers an intermediate step between ECLS and mechanical ventilation in this particular patient population. Pulmonary function stabilized under the combined lung and ventricular support. As a result, mechanical ventilation became sufficient after 48 hours, and the iLA was then removed easily out of the circuit. In conclusion, this case demonstrates the feasibility of integrating a low-resistance membrane oxygenator for additional lung support in extracorporeal LVAD patients.
    ASAIO journal (American Society for Artificial Internal Organs: 1992) 03/2010; 56(3):270-2. · 1.39 Impact Factor

Publication Stats

5k Citations
843.71 Total Impact Points

Institutions

  • 1990–2014
    • University of Bayreuth
      • • Bayreuth Center for Molecular Biosciences (BZMB)
      • • Chair of Biochemistry
      Bayreuth, Bavaria, Germany
  • 1989–2009
    • Martin Luther University of Halle-Wittenberg
      • • Fachgruppe Biophysik
      • • Institut für Physik
      • • Institute of Biochemistry and Biotechnology
      Halle, Saxony-Anhalt, Germany
  • 2005–2008
    • Roche
      Bâle, Basel-City, Switzerland
  • 2002–2007
    • University Hospital Regensburg
      • Klinik und Poliklinik für Herz-, Thorax- und herznahe Gefäßchirurgie
      Regensburg, Bavaria, Germany
  • 1988–2007
    • Universität Regensburg
      • • Department of Cardiac, Thoracic and Vascular Surgery near the Heart
      • • Institut für Biophysik und physikalische Biochemie
      Ratisbon, Bavaria, Germany
  • 1987–2001
    • Johannes Gutenberg-Universität Mainz
      • Department of Cardiothoracic and Vascular Surgery
      Mainz, Rhineland-Palatinate, Germany
  • 1997
    • Max-Planck-Forschungsstelle für Enzymologie der Proteinfaltung
      Halle-on-the-Saale, Saxony-Anhalt, Germany
  • 1996
    • Deutsche Gesellschaft für Thorax-, Herz- und Gefäßchirurgie e.V. 
      Mayence, Rheinland-Pfalz, Germany