Search

Drosophila Toll receptors are involved in embryonic development and the immune response of adult flies. In both processes, the only known Toll receptor ligand is the human nerve growth factor-like cystine knot protein Spätzle. Here we present the crystal structure of a 1:1 (nonsignaling) complex of the full-length Toll receptor ectodomain (ECD) with the Spätzle cystine knot domain dimer. The ECD is divided into two leucine-rich repeat (LRR) domains, each of which is capped by cysteine-rich domains. Spätzle binds to the concave surface of the membrane-distal LRR domain, in contrast to the flanking ligand interactions observed for mammalian Toll-like receptors, with asymmetric contributions from each Spätzle protomer. The structure allows rationalization of existing genetic and biochemical data and provides a framework for targeting the immune systems of insects of economic importance, as well as a variety of invertebrate disease vectors.

VP1 is the major coat protein of murine polyomavirus and forms virus-like particles (VLPs) in vitro. VLPs consist of 72 pentameric VP1 subunits held together by a terminal clamp structure that is further stabilized by disulfide bonds and chelation of calcium ions. Yeast-derived VLPs (yVLPs) assemble intracellularly in vivo during recombinant protein production. These in vivo assembled yVLPs differ in several properties from VLPs assembled in vitro from bacterially produced pentamers. We found several intermolecular disulfide linkages in yVLPs involving 5 of the 6 cysteines of VP1 (Cys(115)-Cys(20), Cys(12)-Cys(20), Cys(16)-Cys(16), Cys(12)/ Cys(16)-Cys(115), and Cys(274)-Cys(274)), indicating a highly coordinated disulfide network within the in vivo assembled particles involving the N-terminal region of VP1. Cryoelectron microscopy revealed structured termini not resolved in the published crystal structure of the bacterially expressed VLP that appear to clamp the pentameric subunits together. These structural features are probably the reason for the observed higher stability of in vivo assembled yVLPs compared with in vitro assembled bacterially expressed VLPs as monitored by increased thermal stability, higher resistance to trypsin cleavage, and a higher activation enthalpy of the disassembly reaction. This high stability is decreased following disassembly of yVLPs and subsequent in vitro reassembly, suggesting a role for cellular components in optimal assembly.

The ionic liquid N-ethyl-N'-methyl imidazolium chloride (EMIMCl) has been described as being very efficient in promoting refolding of the recombinant plasminogen activator rPA. Our study reveals that molar concentrations of EMIMCl increase the solubility of native and unfolded proteins due to favorable interactions with amino acid side chains rather than favorably interacting with the peptide backbone. This delicate balance of favorable interactions with side chains and unfavorable interactions with the peptide backbone provides a molecular explanation of how EMIMCl suppresses protein aggregation and simultaneously promotes refolding. By contrast, high concentrations of EMIMCl denature proteins because of a reduced water content and strong favorable interactions with amino acid side chains. This denatured species is not soluble and aggregates because, in contrast to the classical denaturants, guanidine hydrochloride and urea, EMIMCl does not solubilize the peptide backbone., Structured Digital Abstract: PNP and PNP bind by molecular sieving (1, 2, 3, 4)., (© 2014 FEBS.)

For selective killing of tumor cells, there are many novel and promising therapeutic approaches like immunotoxins. However, on the long way to clinical application, especially in vitro approved biologicals often fail due to loss of target sensitivity and efficacy in vivo. This is mostly explained with degradation or penetration disability in vivo. Although, these problems are well known, until today, there are no in vitro systems for reliable monitoring and quantification of therapeutic efficacy in 3D tumor models and the direct comparison to results from 2D models. In this context, we developed a combined label-free impedimetric monitoring system using our self-developed planar interdigital electrode arrays and our unique microcavity array technology. Therefore, we could demonstrate the time and concentration dependent monitoring and quantification of therapeutic efficacy in a 2D and 3D mamma carcinoma model. In detail, we synthesized a novel modular immunotoxin B3(dsFv)-PE38 (B3-PE38) in which the antibody fragment and the protein toxin are polyionic linked together. We compared the efficacy of the immunotoxin B3-PE38, the toxin E8C-PE38 (PE38) and the small molecule chemotherapeutic paclitaxel. The impedimetric screening revealed the highest cytotoxicity for the immunotoxin B3-PE38 in the 2D model. More strikingly, the immunotoxin efficacy was substantially higher in the 3D model when compared to PE38 and paclitaxel even though having a considerably lower penetration capability than paclitaxel. So our novel impedimetric monitoring system offers the comparative efficacy quantification of novel therapeutics in 2D and 3D in vitro tumor models., (© 2013 Published by Elsevier B.V.)

The topology of the GCAP-2 homodimer was investigated by chemical cross-linking and high resolution mass spectrometry. Complementary conducted size-exclusion chromatography and analytical ultracentrifugation studies indicated that GCAP-2 forms a homodimer both in the absence and in the presence of Ca(2+). In-depth MS and MS/MS analysis of the cross-linked products was aided by (15)N-labeled GCAP-2. The use of isotope-labeled protein delivered reliable structural information on the GCAP-2 homodimer, enabling an unambiguous discrimination between cross-links within one monomer (intramolecular) or between two subunits (intermolecular). The limited number of cross-links obtained in the Ca(2+)-bound state allowed us to deduce a defined homodimeric GCAP-2 structure by a docking and molecular dynamics approach. In the Ca(2+)-free state, GCAP-2 is more flexible as indicated by the higher number of cross-links. We consider stable isotope-labeling to be indispensable for deriving reliable structural information from chemical cross-linking data of multi-subunit protein assemblies.

In response to advances in proteomics research and the use of proteins in medical and biotechnological applications, recombinant protein production and the design of specific protein characteristics and functions has become a widely used technology. In this context, protein fusion tags have been developed as indispensable tools for protein expression, purification, and the design of functionalized surfaces or artificially bifunctional proteins. Here we summarize how positively or negatively charged polyionic fusion peptides with or without an additional cysteine can be used as protein tags for protein expression and purification, for matrix-assisted refolding of aggregated protein, and for coupling of proteins either to technologically relevant matrices or to other proteins. In this context we used cysteine-containing polyionic fusion peptides for the design of immunotoxins. In general, polyionic fusion tags can be considered as a multifunctional module in protein technology.

The bifunctional hexokinase KlHxk1 is a key component of glucose-dependent signal transduction in Kluyveromyces lactis. KlHxk1 is phosphorylated in vivo and undergoes ATP-dependent autophosphorylation-inactivation in vitro. This study identifies serine-15 as the site of in vivo phosphorylation and serine-157 as the autophosphorylation-inactivation site. X-ray crystallography of the in vivo phosphorylated enzyme indicates the existence of a ring-shaped symmetrical homodimer carrying two phosphoserine-15 residues. In contrast, small-angle X-ray scattering and equilibrium sedimentation analyses reveal the existence of monomeric phosphoserine-15 KlHxk1 in solution. While phosphorylation at serine-15 and concomitant homodimer dissociation are likely to be involved in glucose signalling, mechanism and putative physiological significance of KlHxk1 inactivation by autophosphorylation at serine-157 remain to be established., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The protein disulfide isomerase (PDI) family member ERp46/endoPDI/thioredoxin domain-containing protein 5 is preferentially expressed in a limited number of tissues, where it may function as a survival factor for nitrosative stress in vivo. It is involved in insulin production as well as in adiponectin signaling and interacts specifically with the redox-regulatory endoplasmic reticulum proteins endoplasmic oxidoreductin 1α (Ero1α) and peroxiredoxin-4. Here, we show that ERp46, although lacking a PDI-like redox-inactive b'-thioredoxin domain with its hydrophobic substrate binding site, is able to bind to a large pool of peptides containing aromatic and basic residues via all three of its catalytic domains (a(0), a and a'), though the a(0) domain may contain the primary binding site. ERp46, which shows relatively higher activity as a disulfide-reductase than as an oxidase/isomerase in vitro compared to PDI and ERp57, possesses chaperone activity in vivo, a property also shared by the C-terminal a' domain. A crystal structure of the a' domain is also presented, offering a view of possible substrate binding sites within catalytic domains of PDI proteins., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Turnover of mRNA releases, in addition to the four regular nucleoside monophosphates, the methylated cap nucleotide in the form of 7-methylguanosine monophosphate (m(7)GMP) or diphosphate (m(7)GDP). The existence of pathways to eliminate the modified nucleotide seems likely, as its incorporation into nucleic acids is undesirable. Here we describe a novel 5' nucleotidase from Drosophila that cleaves m(7)GMP to 7-methylguanosine and inorganic phosphate. The enzyme, encoded by the predicted gene CG3362, also efficiently dephosphorylates CMP, although with lower apparent affinity; UMP and the purine nucleotides are poor substrates. The enzyme is inhibited by elevated concentrations of AMP and also cleaves m(7)GDP to the nucleoside and two inorganic phosphates, albeit less efficiently. CG3362 has equivalent sequence similarity to two human enzymes, cytosolic nucleotidase III (cNIII) and the previously uncharacterized cytosolic nucleotidase III-like (cNIII-like). We show that cNIII-like also displays 5' nucleotidase activity with a high affinity for m(7)GMP. CMP is a slightly better substrate but again with a higher K(m). The activity of cNIII-like is stimulated by phosphate. In contrast to cNIII-like, cNIII and human cytosolic nucleotidase II do not accept m(7)GMP as a substrate. We suggest that the m(7)G-specific nucleotidases protect cells against undesired salvage of m(7)GMP and its incorporation into nucleic acids.

VP1, the major coat protein of polyomavirus, assembles intracellularly to virus-like particles if expressed in eukaryotes. Here, the nonconventional yeast Kluyveromyces lactis was used for production of virus-like particles of murine polyomavirus. The heterologous gene of VP1 was integrated in the LAC4 locus of the GAL/LAC genes. Consequently the expression of VP1 is regulated by the interplay of the activator KlGal4p and inhibitor KlGal80p. This cloning strategy couples the production of VP1 to that of the enzyme β -galactosidase, allowing a fast alternative for monitoring the course of recombinant protein production by measuring the β -galactosidase activity. A Klgal80 knockout strain was generated for a constitutive expression of VP1 and a continuous VLP production. High-cell-density fermentation showed that (1) Kluyveromyces lactis is generally suitable for VLP production and (2) the Klgal80 knockout strain produces higher amounts of recombinant VP1. Furthermore, VLPs could be purified chromatographically to 87% (w/w) of total protein, and showed a homogeneous species of 45-nm particles and a high resistance against proteolysis compared to conventional in vitro assembled VLPs. This demonstrates the superior stability of virus-like particles produced in yeast.

A variety of approaches have been employed to generate binding proteins from non-antibody scaffolds. Utilizing a beta-sheet of the human ubiquitin for paratope creation we obtained binding proteins against tumor necrosis factor (TNF)-alpha. The bioactive form of this validated pharmacological target protein is a non-covalently linked homo-trimer. This structural feature leads to the observation of a certain heterogeneity concerning the binding mode of TNF-alpha binding molecules, for instance in terms of monomer/trimer specificity. We analyzed a ubiquitin-based TNF-alpha binder, selected by ribosome display, with a particular focus on its mode of interaction. Using enzyme-linked immunosorbent assays, specific binding to TNF-alpha with nanomolar affinity was observed. In isothermal titration calorimetry we obtained comparable results regarding the affinity and detected an exothermic reaction with one ubiquitin-derived binding molecule binding one TNF-alpha trimer. Using NMR spectroscopy and other analytical methods the 1:3 stoichiometry could be confirmed. Detailed binding analysis showed that the interaction is affected by the detergent Tween-20. Previously, this phenomenon was reported only for one other type of alternative scaffold-derived binding proteins--designed ankyrin repeat proteins--without further investigation. As demonstrated by size exclusion chromatography and NMR spectroscopy, the presence of the detergent increases the association rate significantly. Since the special architecture of TNF-alpha is known to be modulated by detergents, the access to the recognized epitope is indicated to be restricted by conformational transitions within the target protein. Our results suggest that the ubiquitin-derived binding protein targets a new epitope on TNF-alpha, which differs from the epitopes recognized by TNF-alpha neutralizing antibodies.

Pyruvate decarboxylase is a key enzyme in organisms whose energy metabolism is based on alcoholic fermentation. The enzyme catalyses the nonoxidative decarboxylation of 2-oxo acids in the presence of the cofactors thiamine diphosphate and magnesium ions. Pyruvate decarboxylase species from yeasts and plant seeds studied to date are allosterically activated by their substrate pyruvate. However, detailed kinetic studies on the enzyme from Neurospora crassa demonstrate for the first time the lack of substrate activation for a yeast pyruvate decarboxylase species. The quaternary structure of this enzyme species is also peculiar because it forms filamentous structures. The complex enzyme structure was analysed using a number of methods, including small-angle X-ray solution scattering, transmission electron microscopy, analytical ultracentrifugation and size-exclusion chromatography. These measurements were complemented by detailed kinetic studies in dependence on the pH., (Journal compilation © 2011 FEBS. No claim to original German government works.)

In the uropathogenic Escherichia coli strain F11, in silico genome analysis revealed the dicistronic iron uptake operon fetMP, which is under iron-regulated control mediated by the Fur regulator. The expression of fetMP in a mutant strain lacking known iron uptake systems improved growth under iron depletion and increased cellular iron accumulation. FetM is a member of the iron/lead transporter superfamily and is essential for iron uptake by the Fet system. FetP is a periplasmic protein that enhanced iron uptake by FetM. Recombinant FetP bound Cu(II) and the iron analog Mn(II) at distinct sites. The crystal structure of the FetP dimer reveals a copper site in each FetP subunit that adopts two conformations: CuA with a tetrahedral geometry composed of His(44), Met(90), His(97), and His(127), and CuB, a second degenerate octahedral geometry with the addition of Glu(46). The copper ions of each site occupy distinct positions and are separated by ∼1.3 Å. Nearby, a putative additional Cu(I) binding site is proposed as an electron source that may function with CuA/CuB displacement to reduce Fe(III) for transport by FetM. Together, these data indicate that FetMP is an additional iron uptake system composed of a putative iron permease and an iron-scavenging and potentially iron-reducing periplasmic protein.

Guanylate cyclase activating protein-2 (GCAP-2) is a Ca²⁺-binding protein of the neuronal calcium sensor (NCS) family. Ca²⁺-free GCAP-2 activates the retinal rod outer segment guanylate cyclases ROS-GC1 and 2. Native GCAP-2 is N-terminally myristoylated. Detailed structural information on the Ca²⁺-dependent conformational switch of GCAP-2 is missing so far, as no atomic resolution structures of the Ca²⁺-free state have been determined. The role of the myristoyl moiety remains poorly understood. Available functional data is incompatible with a Ca²⁺-myristoyl switch as observed in the prototype NCS protein, recoverin. For the homologous GCAP-1, a Ca²⁺-independent sequestration of the myristoyl moiety inside the proteins structure has been proposed. In this article, we compare the thermodynamic stabilities of myristoylated and non-myristoylated GCAP-2 in their Ca²⁺-bound and Ca²⁺-free forms, respectively, to gain information on the nature of the Ca²⁺-dependent conformational switch of the protein and shed some light on the role of its myristoyl group. In the absence of Ca²⁺, the stability of the myristoylated and non-myristoylated forms was indistinguishable. Ca²⁺ exerted a stabilizing effect on both forms of the protein, which was significantly stronger for myr GCAP-2. The stability data were corroborated by dye binding experiments performed to probe the solvent-accessible hydrophobic surface of the protein. Our results strongly suggest that the myristoyl moiety is permanently solvent-exposed in Ca²⁺-free GCAP-2, whereas it interacts with a hydrophobic part of the protein's structure in the Ca²⁺-bound state., (Copyright © 2011 The Protein Society.)

Protein disulfide isomerase (PDI) supports proinsulin folding as chaperone and isomerase. Here, we focus on how the two PDI functions influence individual steps in the complex folding process of proinsulin. We generated a PDI mutant (PDI-aba'c) where the b' domain was partially deleted, thus abolishing peptide binding but maintaining a PDI-like redox potential. PDI-aba'c catalyzes the folding of human proinsulin by increasing the rate of formation and the final yield of native proinsulin. Importantly, PDI-aba'c isomerizes non-native disulfide bonds in completely oxidized folding intermediates, thereby accelerating the formation of native disulfide bonds. We conclude that peptide binding to PDI is not essential for disulfide isomerization in fully oxidized proinsulin folding intermediates., (Copyright © 2011 The Protein Society.)

Because of their ability to degrade RNA, RNases are potent cytotoxins. The cytotoxic activity of most members of the RNase A superfamily, however, is abolished by the cytosolic ribonuclease inhibitor (RI). RNase A tandem enzymes, in which two RNase A molecules are artificially connected by a peptide linker, and thus have a pseudodimeric structure, exhibit remarkable cytotoxic activity. In vitro, however, these enzymes are still inhibited by RI. Here, we present the crystal structures of three tandem enzymes with the linker sequences GPPG, SGSGSG, and SGRSGRSG, which allowed us to analyze the mode of binding of RI to the RNase A tandem enzymes. Modeling studies with the crystal structures of the RI-RNase A complex and the SGRSGRSG-RNase A tandem enzyme as templates suggested a 1 : 1 binding stoichiometry for the RI-RNase A tandem enzyme complex, with binding of the RI molecule to the N-terminal RNase A entity. These results were experimentally verified by analytical ultracentrifugation, quantitative electrophoresis, and proteolysis studies with trypsin. As other dimeric RNases, which are comparably cytotoxic, either evade RI binding or potentially even bind two RI molecules, inactivation by RI cannot be the crucial limitation to the cytotoxicity of dimeric RNases., (© 2010 The Authors Journal compilation © 2010 FEBS.)

The hexokinase isoenzyme 2 of Saccharomyces cerevisiae (ScHxk2) represents an archetype of a two-domain protein with the active site located in a cleft between the two domains. Binding of the substrate glucose results in a rigid body movement of the two domains leading to a cleft closure of the active site. Both domains of this enzyme are composed of discontinuous peptide sequences. This structural feature is reflected in the stability and folding of the ScHxk2 protein. Structural transitions induced by urea treatment resulted in the population of a thermodynamically stable folding intermediate, which, however, does not correspond to a molecule with one domain folded and the other unfolded. As demonstrated by different spectroscopic techniques, both domains are structurally affected by the partial denaturation. The intermediate possesses only 40% of the native secondary structural content and a substantial increase in the Stokes radius as judged by circular dichroism and dynamic light scattering analyses. One-dimensional ¹H NMR data prove that all tryptophan residues are in a non-native environment in the intermediate, indicating substantial changes in the tertiary structure. Still, the intermediate possesses quite a high stability for a transition intermediate of about ΔG = -22 kJ mol⁻¹.

L-arginine hydrochloride (L-ArgHCl) was found to be an effective enhancer for in vitro protein refolding more than two decades ago. A detailed understanding of the mechanism of action, by which L-ArgHCl as co-solvent is capable to effectively suppress protein aggregation, while protein stability is preserved, has remained elusive. Concepts for the effects of co-solvents, which have been established over the last decades, were found to be insufficient to completely explain the effects of L-ArgHCl on protein refolding. In this article, we present data, which clearly establish that L-ArgHCl acts on the equilibrium solubility of the native model protein recombinant plasminogen activator (rPA), while for S-carboxymethylated rPA (IAA-rPA) that served as a model protein for denatured protein states, equilibrium solubilities could not be obtained. Solid to solute free transfer energies for native rPA were lowered by up to 14 kJ mol(-1) under the tested conditions. This finding is in marked contrast to a previously proposed model in which L-ArgHCl acts as a neutral crowder which exclusively has an influence on the stability of the transition state of aggregation. The effects on the apparent solubility of IAA-rPA, as well as on the aggregation kinetics of all studied protein species, that were observed in the present work could tentatively be explained within the framework of a nucleation-aggregation scheme, in which L-ArgHCl exerts a strong effect on the pre-equilibria leading to formation of the aggregation seed., (Copyright © 2010 The Protein Society.)

The RNA-dependent RNA polymerase NS5B is a key enzyme of the replication of hepatitis C virus (HCV) and a major therapeutic target. Applying a novel continuous assay with highly purified protein and a fluorescent RNA-template we provide for the first time a comprehensive mechanistic description of the enzymatic reaction. Using fluorescence spectroscopy, the kinetics of NS5B was confirmed to consist of two half-reactions, namely substrate binding and turnover. Determining the binding constants of the substrates and the rate constants of individual reaction steps, NS5B was shown to bind the template single-stranded RNA with high affinity (nanomolar range) and in a stepwise process that reflects the substrate positioning. As demonstrated by CD, NTP(s) binding caused a tertiary structural change of the enzyme into an active conformation. The second half-reaction was dissected into a sequential polymerization and a subsequent, rate-limiting product release reaction. Taking advantage of these tools, we analyzed the mechanism of action of the NS5B inhibitor HCV-796, which was shown to interfere with the formation of double-stranded RNA by blocking the second half-reaction.

FocA is a predicted formate channel with a deduced mass of 31 kDa that catalyzes the bidirectional movement of formate across the cytoplasmic membrane of Escherichia coli and is the archetype of the formate-nitrite transporter (FNT) family. Overproduced FocA variants with either an N- or a C-terminal Strep-tag increased formate import into anaerobic E. coli cells as determined by the enhanced activity of a single-copy formate-dependent fdhF::lacZ fusion. Using anti-FocA antibodies, we could show that both FocA variants were integrated into the cytoplasmic membrane. Circular dichroism spectroscopy of purified FocA(Strep-N) revealed a high alpha-helical content of 56% consistent with the predicted six transmembrane helices present in the protein. Analysis of the oligomeric state by blue-native polyacrylamide gel electrophoresis revealed FocA to have an unexpected pentameric quaternary structure. This study reports the first isolation of an FNT family member.

The incidence of herpes zoster in the elderly (50 years and older) 2004 in Germany was determined by retrospectively analysing representative treatment data of the statutory health insurance sample of AOK Hesse/KV Hesse. The overall observed incidence rate of herpes zoster was 9.4 cases per 1,000 person-years (PY). 10.1% of herpes-zoster-patients suffered at least 1 month from pain, the so called postherpetic neuralgia (PHN1), 6.9% had at least 3 months pain (PHN3). Incidence rate of herpes zoster rose markedly with age: from 6.8 per 1,000 PY in 50 to 54 year-olds to 12.4 PY in persons 80 years and older. Incidence rate in the immunocompromised was higher (11.6 per 1,000 PY) than in the immunocompetent (9.1 per 1,000 PY). According to a standardized extrapolation of the sample to the German population, about 300,000 persons 50 years and older suffered from acute herpes zoster on the year 2004 in Germany.

In previous work, a strongly stabilized variant of the beta1 domain of streptococcal protein G (Gbeta1) was obtained by an in vitro selection method. This variant, termed Gbeta1-M2, contains the four substitutions E15V, T16L, T18I, and N37L. Here we elucidated the molecular basis of the observed strong stabilizations. The contributions of these four residues were analyzed individually and in various combinations, additional selections with focused Gbeta1 gene libraries were performed, and the crystal structure of Gbeta1-M2 was determined. All single substitutions (E15V, T16L, T18I, and N37L) stabilize wild-type Gbeta1 by contributions of between 1.6 and 6.0 kJ mol(-1) (at 70 degrees C). Hydrophobic residues at positions 16 and 37 provide the major contribution to stabilization by enlarging the hydrophobic core of Gbeta1. They also increase the tendency to form dimers, as shown by dependence on the concentration of apparent molecular mass in analytical ultracentrifugation, by concentration-dependent stability, and by a strongly increased van't Hoff enthalpy of unfolding. The 0.88-A crystal structure of Gbeta1-M2 and NMR measurements in solution provide the explanation for the observed dimer formation. It involves a head-to-head arrangement of two Gbeta1-M2 molecules via six intermolecular hydrogen bonds between the two beta strands 2 and 2' and an adjacent self-complementary hydrophobic surface area, which is created by the T16L and N37L substitutions and a large 120 degrees rotation of the Tyr33 side chain. This removal of hydrophilic groups and the malleability of the created hydrophobic surface provide the basis for the dimer formation of stabilized Gbeta1 variants.

Monoclonal antibodies (mAbs) have been developed over the past years as promising anticancer therapeutics. The conjugation of tumor specific mAbs with cytotoxic molecules has been shown to improve their efficacy dramatically. These bifunctional immunotoxins, consisting of covalently linked antibodies and protein toxins, possess considerable potential in cancer therapy. Many of them are under investigation in clinical trials. As a result of general interest in new toxic components, we describe here the suitability of the bacterial protein Listeriolysin O (LLO) as cytotoxic component of an immunotoxin. Unique characteristics of LLO, such as its acidic pH optimum and the possibility to regulate the cytolytic activity by cysteine-oxidation, make LLO an interesting toxophore. Oxidized LLO shows a substantially decreased cytolytic activity when compared with the reduced protein as analyzed by hemolysis. Both oxidized and reduced LLO exhibit a cell-type-unspecific toxicity in cell culture with a significantly higher toxicity of reduced LLO. For cell-type-specific targeting of LLO to tumor cells, LLO was coupled to the dsFv fragment of the monoclonal antibody B3, which recognizes the tumor-antigen Lewis Y. The coupling of LLO to dsFv-B3 was performed via cysteine-containing polyionic fusion peptides that act as a specific heterodimerization motif. The novel immunotoxin B3-LLO could be shown to specifically eliminate antigen positive MCF7 cells with an EC(50) value of 2.3 nM, whereas antigen negative cell lines were 80- to 250-fold less sensitive towards B3-LLO.

A novel class of antibiotic acyldepsipeptides (designated ADEPs) exerts its unique antibacterial activity by targeting the peptidase caseinolytic protease P (ClpP). ClpP forms proteolytic complexes with heat shock proteins (Hsp100) that select and process substrate proteins for ClpP-mediated degradation. Here, we analyse the molecular mechanism of ADEP action and demonstrate that ADEPs abrogate ClpP interaction with cooperating Hsp100 adenosine triphosphatases (ATPases). Consequently, ADEP treated bacteria are affected in ClpP-dependent general and regulatory proteolysis. At the same time, ADEPs also activate ClpP by converting it from a tightly regulated peptidase, which can only degrade short peptides, into a proteolytic machinery that recognizes and degrades unfolded polypeptides. In vivo nascent polypeptide chains represent the putative primary target of ADEP-activated ClpP, providing a rationale for the antibacterial activity of the ADEPs. Thus, ADEPs cause a complete functional reprogramming of the Clp-protease complex.

Endonuclease G (EndoG) is a mitochondrial enzyme believed to be released during apoptosis to participate in the degradation of nuclear DNA. This paper describes a Drosophila protein, EndoGI, which inhibits EndoG specifically. EndoG and EndoGI associate with subpicomolar affinity, forming a 2:1 complex in which dimeric EndoG is bound by two tandemly repeated homologous domains of monomeric EndoGI. Binding appears to involve the active site of EndoG. EndoGI is present in the cell nucleus at micromolar concentrations. Upon induction of apoptosis, levels of the inhibitor appear to be reduced, and it is relocalized to the cytoplasm. EndoGI, encoded by the predicted open reading frame cg4930, is expressed throughout Drosophila development. Flies homozygous for a hypomorphic EndoGI mutation have a strongly reduced viability, which is modulated by genetic background and diet. We propose that EndoGI protects the cell against low levels of EndoG outside mitochondria.

Nonproteinogenic amino acids that either occur naturally or are synthesized chemically are becoming important tools in modern drug discovery. In this context, fluorinated amino acids have great potential in the development of novel pharmaceuticals and drugs. To assess whether different fluorinated aromatic amino acid analogues of phenylalanine, tyrosine, and tryptophan are potentially interesting as therapeutic drugs, we examined their cytostatic and cytotoxic effects on the growth of the human breast cancer cell line MCF-7. Of all the tested analogues L-4-fluorotryptophan, L-6-fluorotryptophan and L-p-fluorophenylalanine effectively and irreversibly inhibited cell growth with IC(50) values in the low micromolar range (3-15 microM). Additionally, using L-4-[14C]fluorotryptophan, and L-6-[14C]fluorotryptophan, we discovered that the cellular uptake of these fluorinated amino acids occurs through active transport with a 70-fold excess of intracellular over extracellular concentrations. We identified system L as the responsible amino acid transporter. Our findings fully support the idea that fluorinated aromatic amino acid analogues are promising chemotherapeutics with the potential for use in combination with classical cancer therapy, and as new cytotoxic drugs for certain tumor types such as melanoma.

Repeatedly expressed worries that doctors are at risk for malpractice suits and for ending up in prison do not really reflect reality. An empirical study on public prosecutors' offices demonstrated that malpractice proceedings where doctors were blamed for treatment errors had predominantly been closed. If a long time passes between the opening and the closing proceedings this is often due to the colleagues who take more than average time to state their expert opinion as requested. In most cases these expert medical opinions are decisive in the outcome of these proceedings.

Computational protein design has progressed rapidly over the last years. A number of design methods have been proposed and tested. In this paper, we report the successful application of a fragment-based method for protein design. The method uses statistical information on tetrapeptide backbone conformations. The previously published artificial fold of TOP 7 (Kuhlman et al., Science, 2003; 302:1364-1368) was chosen as template. A series of polypeptide sequences were created that were predicted to fold into this target structure. Two of the designed proteins, M5 and M7, were expressed and characterized by fluorescence spectroscopy, circular dichroism and NMR. They showed the hallmarks of well-ordered tertiary structure as well as cooperative folding/unfolding transitions. Furthermore, the two novel proteins were found to be highly stable against temperature and denaturant-induced unfolding., (2007 Wiley-Liss, Inc.)

The redox-regulated chaperone Hsp33 is specifically activated upon exposure of cells to peroxide stress at elevated temperatures. Here we show that Hsp33 harbors two interdependent stress-sensing regions located in the C-terminal redox-switch domain of Hsp33: a zinc center sensing peroxide stress conditions and an adjacent linker region responding to unfolding conditions. Neither of these sensors works sufficiently in the absence of the other, making the simultaneous presence of both stress conditions a necessary requirement for Hsp33's full activation. Upon activation, Hsp33's redox-switch domain adopts a natively unfolded conformation, thereby exposing hydrophobic surfaces in its N-terminal substrate-binding domain. The specific activation of Hsp33 by the oxidative unfolding of its redox-switch domain makes this chaperone optimally suited to quickly respond to oxidative stress conditions that lead to protein unfolding.

The pro-peptide of human nerve growth factor (NGF) functions as an intramolecular chaperone during oxidative renaturation of proNGF in vitro and interacts intramolecularly with the mature part of native proNGF. Here, we analyzed the structure formation and stability of the pro-peptide in the context of proNGF and its intramolecular interaction with the native mature part. Folding and unfolding of the NGF-coupled pro-peptide, as analyzed by fluorescence, were biphasic reactions with both phases depending on the interaction with the mature part. This interaction was characterized by an overall stability of DeltaG = 20.9 kJ/mol that was subdivided into two reactions, native <--> intermediate state (14.8 kJ/mol) and intermediate <--> unfolded state (6.1 kJ/mol). An additional very fast unfolding reaction was observed using circular dichroism (CD), indicating the presence of at least two kinetically populated intermediates in the unfolding of proNGF. The part of the pro-peptide involved in the intramolecular association with mature NGF comprised the peptide Trp(-83)-Ala(-63) as determined by H/D exchange experiments. Spectroscopic analyses revealed that on the NGF side, a surface area around Trp(21) interacted with the pro-peptide. Trp(21) also participates in binding to TrkA and p75 receptors. These overlapping binding sites of the pro-peptide and the NGF receptors might explain the previously observed lower affinity of proNGF to its receptors as compared to NGF.

In cytosolic Src-type tyrosine kinases the Src-type homology 3 (SH3) domain binds to an internal proline-rich motif and the presence or the absence of this interaction modulates the kinase enzymatic activity. The Src-type kinase Lck plays an important role during T-cell activation and development, since it phosphorylates the T-cell antigen receptor in an early step of the activation pathway. We have determined the crystal structure of the SH3 domain from Lck kinase at a near-atomic resolution of 1.0 A. Unexpectedly, the Lck-SH3 domain forms a symmetrical homodimer in the crystal and the dimer comprises two identical zinc-binding sites in the interface. The atomic interactions formed across the dimer interface resemble strikingly those observed between SH3 domains and their canonical proline-rich ligands, since almost identical residues participate in both contacts. Ultracentrifugation experiments confirm that in the presence of zinc ions, the Lck-SH3 domain also forms dimers in solution. The Zn(2+) dissociation constant from the Lck-SH3 dimer is estimated to be lower than 100 nM. Moreover, upon addition of a proline-rich peptide with a sequence corresponding to the recognition segment of the herpesviral regulatory protein Tip, competition between zinc-induced homodimerization and binding of the peptide can be detected by both fluorescence spectroscopy and analytical ultracentrifugation. These results suggest that in vivo, too, competition between Lck-SH3 homodimerization and binding of regulatory proline-rich sequence motifs possibly represents a novel mechanism by which kinase activity is modulated. Because the residues that form the zinc-binding site are highly conserved among Lck orthologues but not in other Src-type kinases, the mechanism might be peculiar to Lck and to its role in the initial steps of T-cell activation.

Recent kinetic and structural studies on various thiamin-dependent enzymes, including the bacterial E1 component of the pyruvate dehydrogenase complex (PDHc), suggested an active center communication between the cofactors in these multimeric enzymes. This regulatory mode has been inferred from the dissymmetry of active sites in proteolytic patterns and X-ray structures and from a complex macroscopic kinetic behavior not being consistent with independently working active sites. Here, direct microscopic kinetic evidence for this hypothesis is presented for the alpha2beta2-type E1 component of the human pyruvate dehydrogenase complex. Only one of the two thiamin molecules bound to the two active sites is in a chemically activated state exhibiting an apparent C2 ionization rate constant of approximately 50 s(-1) at pH 7.6 and 30 degrees C, whereas the thiamin in the "inactive site" ionizes with a rate that is at least 3 orders of magnitude smaller. The chemical nonequivalence is also exhibited in the ability to bind the substrate analogue methyl acetylphosphonate and in the catalytic turnover of the substrate pyruvate in the E1-only reaction. In the activated active site, pyruvate is rapidly bound and decarboxylated with apparent forward rate constants of covalent pyruvate binding of 2 s(-1) and decarboxylation of the formed 2-lactyl-thiamin intermediate of 5 s(-1). In the dormant site, these steps are as slow as 0.03 s(-1). Under the conditions that were used, only the heterotetramer can be detected by analytical ultracentrifugation, thus ruling out the possibility that multiple oligomeric species with different reactivities cause the observed kinetic effects. The results are consistent with the recently suggested model of an active site synchronization in PDHc-E1 via a proton wire that keeps the two active sites in an alternating activation state [Frank, R. A., et al. (2004) Science 306, 872]. Kinetic studies on the related thiamin enzymes transketolase, pyruvate oxidase, and bacterial pyruvate decarboxylase are not consistent with a chemical and/or functional nonequivalence of the active sites as observed in the E1 component of hsPDHc. We hypothesize that the alternating sites reaction in PDHc-E1 aids in the synchronized acyl transfer to the E2 component in the highly organized multienzyme complex.

The Gal4 protein represents a universally functional transcription activator, which in yeast is regulated by protein-protein interaction of its transcription activation domain with the inhibitor Gal80. Gal80 inhibition is relieved via galactose-mediated Gal80-Gal1-Gal3 interaction. The Gal4-Gal80-Gal1/3 regulatory module is conserved between Saccharomyces cerevisiae and Kluyveromyces lactis. Here we demonstrate that K. lactis Gal80 (KlGal80) is a nuclear protein independent of the Gal4 activity status, whereas KlGal1 is detected throughout the entire cell, which implies that KlGal80 and KlGal1 interact in the nucleus. Consistently KlGal1 accumulates in the nucleus upon KlGAL80 overexpression. Furthermore, we show that the KlGal80-KlGal1 interaction blocks the galactokinase activity of KlGal1 and is incompatible with KlGal80-KlGal4-AD interaction. Thus, we propose that dissociation of KlGal80 from the AD forms the basis of KlGal4 activation in K. lactis. Quantitation of the dissociation constants for the KlGal80 complexes gives a much lower affinity for KlGal1 as compared with Gal4. Mathematical modeling shows that with these affinities a switch based on competition between Gal1 and Gal4 for Gal80 binding is nevertheless efficient provided two monomeric Gal1 molecules interact with dimeric Gal80. Consistent with such a mechanism, analysis of the sedimentation behavior by analytical ultracentrifugation demonstrates the formation of a heterotetrameric KlGal80-KlGal1 complex of 2:2 stoichiometry.

12-Oxophytodienoate reductase (OPR) 3, a homologue of old yellow enzyme (OYE), catalyzes the reduction of 9S,13S-12-oxophytodienoate to the corresponding cyclopentanone, which is subsequently converted to the plant hormone jasmonic acid (JA). JA and JA derivatives, as well as 12-oxophytodienoate and related cyclopentenones, are known to regulate gene expression in plant development and defense. Together with other oxygenated fatty acid derivatives, they form the oxylipin signature in plants, which resembles the pool of prostaglandins in animals. Here, we report the crystal structure of OPR3 from tomato and of two OPR3 mutants. Although the catalytic residues of OPR3 and related OYEs are highly conserved, several characteristic differences can be discerned in the substrate-binding regions, explaining the remarkable substrate stereoselectivity of OPR isozymes. Interestingly, OPR3 crystallized as an extraordinary self-inhibited dimer. Mutagenesis studies and biochemical analysis confirmed a weak dimerization of OPR3 in vitro, which correlated with a loss of enzymatic activity. Based on structural data of OPR3, a putative mechanism for a strong and reversible dimerization of OPR3 in vivo that involves phosphorylation of OPR3 is suggested. This mechanism could contribute to the shaping of the oxylipin signature, which is critical for fine-tuning gene expression in plants.

The Tat (twin-arginine translocation) system of Escherichia coli serves to translocate folded proteins across the cytoplasmic membrane. The reasons established so far for the Tat dependence are cytoplasmic cofactor assembly and/or heterodimerization of the respective proteins. We were interested in the reasons for the Tat dependence of novel Tat substrates and focused on two uncharacterized proteins, YcdO and YcdB. Both proteins contain predicted Tat signal sequences. However, we found that only YcdB was indeed Tat-dependently translocated, whereas YcdO was equally well translocated in a Tat-deficient strain. YcdB is a dimeric protein and contains a heme cofactor that was identified to be a high-spin Fe(III)-protoporphyrin IX complex. In contrast to all other periplasmic hemoproteins analyzed so far, heme was assembled into YcdB in the cytoplasm, suggesting that heme assembly could take place prior to translocation. The function of YcdB in the periplasm may be related to a detoxification reaction under specific conditions because YcdB had peroxidase activity at acidic pH, which coincides well with the known acid-induced expression of the gene. The data demonstrate the existence of a class of heme-containing Tat substrates, the first member of which is YcdB.

The AAA+ protein ClpC is not only involved in the removal of misfolded and aggregated proteins but also controls, through regulated proteolysis, key steps of several developmental processes in the Gram-positive bacterium Bacillus subtilis. In contrast to other AAA+ proteins, ClpC is unable to mediate these processes without an adaptor protein like MecA. Here, we demonstrate that the general activation of ClpC is based upon the ability of MecA to participate in the assembly of an active and substrate-recognizing higher oligomer consisting of ClpC and the adaptor protein, which is a prerequisite for all activities of this AAA+ protein. Using hybrid proteins of ClpA and ClpC, we identified the N-terminal and the Linker domain of the first AAA+ domain of ClpC as the essential MecA interaction sites. This new adaptor-mediated mechanism adds another layer of control to the regulation of the biological activity of AAA+ proteins.

At present nine FDA-approved HIV protease inhibitors have been launched to market, however rapid drug resistance arising under antiviral therapy calls upon novel concepts. Possible strategies are the development of ligands with less peptide-like character or the stabilization of a new and unexpected binding-competent conformation of the protein through a novel ligand-binding mode. Our rational design of pyrrolidinedimethylene diamines was inspired by the idea to incorporate key structural elements from classical peptidomimetics with a non-peptidic heterocyclic core comprising an endocyclic amino function to address the catalytic aspartic acid side chains of Asp 25 and 25'. The basic scaffolds were decorated by side chains already optimized for the recognition pockets of HIV protease or cathepsin D. A multistep synthesis has been established to produce the central heterocycle and to give flexible access to side chain decorations. Depending on the substitution pattern of the pyrrolidine moiety, single-digit micromolar inhibition of HIV-1 protease and cathepsin D has been achieved. Successful design is suggested in agreement with our modelling concepts. The subsequently determined crystal structure with HIV protease shows that the pyrrolidine moiety binds as expected to the pivotal position between both aspartic acid side chains. However, even though the inhibitors have been equipped symmetrically by polar acceptor groups to address the flap water molecule, it is repelled from the complex, and only one direct hydrogen bond is formed to the flap. A strong distortion of the flap region is detected, leading to a novel hydrogen bond which cross-links the flap loops. Furthermore, the inhibitor addresses only three of the four available recognition pockets. It achieves only an incomplete desolvation compared with the similarly decorated amprenavir. Taking these considerations into account it is surprising that the produced pyrrolidine derivatives achieve micromolar inhibition and it suggests extraordinary potency of the new compound class. Most likely, the protonated pyrrolidine moiety experiences strong enthalpic interactions with the enzyme through the formation of two salt bridges to the aspartic acid side chains. This might provide challenging opportunities to combat resistance of the rapidly mutating virus.

The author discusses problems of the "Rule of 3" (Dreierregel) meaning that only three pre-embryos are allowed to be produced within one fertilisation process. This rule is based on the Embryo Protection Act and restricts methods of reproductive medicine. These limits have provoked a debate among the legal community on the question whether certain methods still conform to the law or if the law has to be adapted. This article both outlines and criticizes the arguments put forward in the debate and contrasts them to a proposal made by the German Medical Association.

Poly(A)-specific ribonuclease (PARN) is a processive, poly(A)-specific 3' exoribonuclease. The crystal structure of C-terminal truncated human PARN determined in two states (free and RNA-bound forms) reveals that PARNn is folded into two domains, an R3H domain and a nuclease domain similar to those of Pop2p and epsilon186. The high similarity of the active site structures of PARNn and epsilon186 suggests that they may have a similar catalytic mechanism. PARNn forms a tight homodimer, with the R3H domain of one subunit partially enclosing the active site of the other subunit and poly(A) bound in a deep cavity of its nuclease domain in a sequence-nonspecific manner. The R3H domain and, possibly, the cap-binding domain are involved in poly(A) binding but these domains alone do not appear to contribute to poly(A) specificity. Mutations disrupting dimerization abolish both the enzymatic and RNA-binding activities, suggesting that the PARN dimer is a structural and functional unit. The cap-binding domain may act in concert with the R3H domain to amplify the processivity of PARN.

The lateral mobility of individual murine polyoma virus-like particles (VLPs) bound to live cells and artificial lipid bilayers was studied by single fluorescent particle tracking using total internal reflection fluorescence microscopy. The particle trajectories were analyzed in terms of diffusion rates and modes of motion as described by the moment scaling spectrum. Although VLPs bound to their ganglioside receptor in lipid bilayers exhibited only free diffusion, analysis of trajectories on live 3T6 mouse fibroblasts revealed three distinct modes of mobility: rapid random motion, confined movement in small zones (30-60 nm in diameter), and confined movement in zones with a slow drift. After binding to the cell surface, particles typically underwent free diffusion for 5-10 s, and then they were confined in an actin filament-dependent manner without involvement of clathrin-coated pits or caveolae. Depletion of cholesterol dramatically reduced mobility of VLPs independently of actin, whereas inhibition of tyrosine kinases had no effect on confinement. The results suggested that clustering of ganglioside molecules by the multivalent VLPs induced transmembrane coupling that led to confinement of the virus/receptor complex by cortical actin filaments.

The matrix protein M of Borna disease virus (BDV) is a constituent of the viral envelope covering the inner leaflet of the lipid bilayer. BDV-M was expressed as recombinant protein in Escherichia coli, purified to homogeneity and structurally analyzed. Recombinant M (i) forms non-covalently bound multimers with a Stoke's radius of 35 Angstroms estimated by size exclusion chromatography, (ii) consists of tetramers detected by analytical ultracentrifugation, and (iii) appears by electron microscopy studies as tetramers with the tendency to assemble into high molecular mass lattice-like complexes. The structural features suggest that BDV-M possesses a dominant driving force for virus particle formation.

L-Arginine (L-Arg) has been widely used as an enhancer of protein renaturation. The mechanism behind its action is still not fully understood. Using hen egg white lysozyme as a model protein, we present data that clearly demonstrate the suppression of the aggregation of denatured protein by L-Arg. By chemical modification of free cysteines, a series of unfolded lysozyme species were obtained that served as models for unfolded and intermediate states during the process of oxidative refolding. An increased equilibrium solubility of unfolded species and intermediates in the presence of L-Arg seems to be its major mechanism of action.

The guanylate-binding proteins (GBPs) form a group of interferon-gamma inducible GTP-binding proteins which belong to the family of dynamin-related proteins. Like other members of this family, human guanylate-binding protein 1 (hGBP1) shows nucleotide-dependent oligomerisation that stimulates the GTPase activity of the protein. A unique feature of the GBPs is their ability to hydrolyse GTP to GDP and GMP. In order to elucidate the relationship between these findings, we designed point mutants in the phosphate-binding loop (P-loop) as well as in the switch I and switch II regions of the protein based on the crystal structure of hGBP1. These mutant proteins were analysed for their interaction with guanine nucleotides labeled with a fluorescence dye and for their ability to hydrolyse GTP in a cooperative manner. We identified mutations of amino acid residues that decrease GTPase activity by orders of magnitude a part of which are conserved in GTP-binding proteins. In addition, mutants in the P-loop were characterized that strongly impair binding of nucleotide. In consequence, together with altered GTPase activity and given cellular nucleotide concentrations this results in hGBP1 mutants prevailingly resting in the nucleotide-free (K51A and S52N) or the GTP bound form (R48A), respectively. Using size-exclusion chromatography and analytical ultracentrifugation we addressed the impact on protein oligomerisation. In summary, mutants of hGBP1 were identified and biochemically characterized providing hGBP1 locked in defined states in order to investigate their functional role in future cell biology studies.

Triosephophate isomerase (TIM) is a dimeric enzyme in eucarya, bacteria and mesophilic archaea. In hyperthermophilic archaea, however, TIM exists as a tetramer composed of monomers that are about 10% shorter than other eucaryal and bacterial TIM monomers. We report here the crystal structure of TIM from Thermoproteus tenax, a hyperthermophilic archaeon that has an optimum growth temperature of 86 degrees C. The structure was determined from both a hexagonal and an orthorhombic crystal form to resolutions of 2.5A and 2.3A, and refined to R-factors of 19.7% and 21.5%, respectively. In both crystal forms, T.tenax TIM exists as a tetramer of the familiar (betaalpha)(8)-barrel. In solution, however, and unlike other hyperthermophilic TIMs, the T.tenax enzyme exhibits an equilibrium between inactive dimers and active tetramers, which is shifted to the tetramer state through a specific interaction with glycerol-1-phosphate dehydrogenase of T.tenax. This observation is interpreted in physiological terms as a need to reduce the build-up of thermolabile metabolic intermediates that would be susceptible to destruction by heat. A detailed structural comparison with TIMs from organisms with growth optima ranging from 15 degrees C to 100 degrees C emphasizes the importance in hyperthermophilic proteins of the specific location of ionic interactions for thermal stability rather than their numbers, and shows a clear correlation between the reduction of heat-labile, surface-exposed Asn and Gln residues with thermoadaptation. The comparison confirms the increase in charged surface-exposed residues at the expense of polar residues.

Human nerve growth factor (NGF) belongs to the structural family of cystine knot proteins, characterized by a disulfide pattern in which one disulfide bond threads through a ring formed by a pair of two other disulfides connecting two adjacent beta-strands. Oxidative folding of NGF revealed that the pro-peptide of NGF stimulates in vitro structure formation. In order to learn more about this folding assisting protein fragment, a biophysical analysis of the pro-peptide structure has been performed. While proNGF is a non-covalent homodimer, the isolated pro-peptide is monomeric. No tertiary contacts stabilize the pro-peptide in its isolated form. In contrast, the pro-peptide appears to be structured when bound to the mature part. The results presented here demonstrate that the mature part stabilizes the structure in the pro-peptide region. This is the first report that provides a biophysical analysis of a pro-peptide of the cystine knot protein family.

The molecular chaperone Hsp33 in Escherichia coli responds to oxidative stress conditions with the rapid activation of its chaperone function. On its activation pathway, Hsp33 progresses through three major conformations, starting as a reduced, zinc-bound inactive monomer, proceeding through an oxidized zinc-free monomer, and ending as a fully active oxidized dimer. While it is known that Hsp33 senses oxidative stress through its C-terminal four-cysteine zinc center, the nature of the conformational changes in Hsp33 that must take place to accommodate this activation process is largely unknown. To investigate these conformational rearrangements, we constructed constitutively monomeric Hsp33 variants as well as fragments consisting of the redox regulatory C-terminal domain of Hsp33. These proteins were studied by a combination of biochemical and NMR spectroscopic techniques. We found that in the reduced, monomeric conformation, zinc binding stabilizes the C terminus of Hsp33 in a highly compact, alpha-helical structure. This appears to conceal both the substrate-binding site as well as the dimerization interface. Zinc release without formation of the two native disulfide bonds causes the partial unfolding of the C terminus of Hsp33. This is sufficient to unmask the substrate-binding site, but not the dimerization interface, rendering reduced zinc-free Hsp33 partially active yet monomeric. Critical for the dimerization is disulfide bond formation, which causes the further unfolding of the C terminus of Hsp3 and allows the association of two oxidized Hsp33 monomers. This then leads to the formation of active Hsp33 dimers, which are capable of protecting cells against the severe consequences of oxidative heat stress.

Granzyme B (GzmB) is a potent apoptosis-inducing serine protease of cytotoxic lymphocytes. Following receptor-mediated endocytosis, GzmB is supposed to enter the cytosol through perforin-mediated membrane disruption. We investigated whether retargeting of GzmB to Lewis Y positive surface receptors could lead to perforin-independent target cell death. We coupled recombinant GzmB to the Lewis Y-binding antibody dsFv-B3. Targeting of GzmB to Lewis Y positive cells triggered cell death with similar efficacy as dsFv-B3 targeted Pseudomonas exotoxin fragment 38 (PE38). Since GzmB was only weakly inhibited by plasma proteins, GzmB-based immunoconjugates should be useful as a new class of immunotoxins with low immunogenicity utilizing programmed cell death for therapeutic purposes.

ORF56 is a plasmid-encoded protein from Sulfolobus islandicus, which probably controls the copy number of the pRN1 plasmid by binding to its own promotor. The protein showed an extremely high stability in denaturant, heat, and pH-induced unfolding transitions, which can be well described by a two-state reaction between native dimers and unfolded monomers. The homodimeric character of native ORF56 was confirmed by analytical ultracentrifugation. Far-UV circular dichroism and fluorescence spectroscopy gave superimposable denaturant-induced unfolding transitions and the midpoints of both heat as well as denaturant-induced unfolding depend on the protein concentration supporting the two-state model. This model was confirmed by GdmSCN-induced unfolding monitored by heteronuclear 2D NMR spectroscopy. Chemical denaturation was accomplished by GdmCl and GdmSCN, revealing a Gibbs free energy of stabilization of -85.1 kJ/mol at 25 degrees C. Thermal unfolding was possible only above 1 M GdmCl, which shifted the melting temperature (t(m)) below the boiling point of water. Linear extrapolation of t(m) to 0 M GdmCl yielded a t(m) of 107.5 degrees C (5 microM monomer concentration). Additionally, ORF56 remains natively structured over a remarkable pH range from pH 2 to pH 12. Folding kinetics were followed by far-UV CD and fluorescence after either stopped-flow or manual mixing. All kinetic traces showed only a single phase and the two probes revealed coincident folding rates (k(f), k(u)), indicating the absence of intermediates. Apparent first-order refolding rates depend linearly on the protein concentration, whereas the unfolding rates do not. Both lnk(f) and lnk(u) depend linearly on the GdmCl concentration. Together, folding and association of homodimeric ORF56 are concurrent events. In the absence of denaturant ORF56 refolds fast (7.0 x 10(7)M(-1)s(-1)) and unfolds extremely slowly (5.7 year(-1)). Therefore, high stability is coupled to a slow unfolding rate, which is often observed for proteins of extremophilic organisms.

We have identified and reconstituted a multicomponent redox-chaperone network that appears to be designed to protect proteins against stress-induced unfolding and to refold proteins when conditions return to normal. The central player is Hsp33, a redox-regulated molecular chaperone. Hsp33, which is activated by disulfide bond formation and subsequent dimerization, works as an efficient chaperone holdase that binds to unfolding protein intermediates and maintains them in a folding competent conformation. Reduction of Hsp33 is catalyzed by the glutaredoxin and thioredoxin systems in vivo, and leads to the formation of highly active, reduced Hsp33 dimers. Reduction of Hsp33 is necessary but not sufficient for substrate protein release. Substrate dissociation from Hsp33 is linked to the presence of the DnaK/DnaJ/GrpE foldase system, which alone, or in concert with the GroEL/GroES system, then supports the refolding of the substrate proteins. Upon substrate release, reduced Hsp33 dimers dissociate into inactive monomers. This regulated substrate transfer ultimately links substrate release and Hsp33 inactivation to the presence of available DnaK/DnaJ/GrpE, and, therefore, to the return of cells to non-stress conditions.