Karl-Heinz Gührs

Production of spider silk proteins in tobacco and potato

Spider dragline silk is a proteinaceous fiber with remarkable mechanical properties that make it attractive for technical applications. Unfortunately, the material cannot be obtained in large quantities from spiders. We have therefore generated transgenic tobacco and potato plants that express remarkable amounts of recombinant Nephila clavipes dragline proteins. Using a gene synthesis approach, the recombinant proteins exhibit homologies of >90% compared to their native models. Here, we demonstrate the accumulation of recombinant silk proteins, which are encoded by synthetic genes of 420–3,600 base pairs, up to a level of at least 2% of total soluble protein in the endoplasmic reticulum (ER) of tobacco and potato leaves and potato tubers, respectively. Using the present expression system, spider silk proteins up to 100 kDa could be detected in plant tissues. When produced in plants, the recombinant spidroins exhibit extreme heat stability—a property that is used to purify the spidroins by a simple and efficient procedure.

4-fluorophenylglycine as a label for 19F NMR structure analysis of membrane-associated peptides

The non‐natural amino acid 4‐fluorophenylglycine (4F‐Phg) was incorporated into several representative membrane‐associated peptides for dual purpose. The 19F‐substituted ring is directly attached to the peptide backbone, so it not only provides a well‐defined label for highly sensitive 19F NMR studies but, in addition, the D and L enantiomers of the stiff side chain may serve as reporter groups on the transient peptide conformation during the biological function. Besides peptide synthesis, which is accompanied by racemisation of 4F‐Phg, we also describe separation of the epimers by HPLC and removal of trifluoroacetic acid. As a first example, 18 different analogues of the fusogenic peptide “B18” were prepared and tested for induction of vesicle fusion; the results confirmed that hydrophobic sites tolerated 4F‐Phg labelling. Similar fusion activities within each pair of epimers suggest that the peptide is less structured in the fusogenic transition state than in the helical ground state. In a second example, five doubly labelled analogues of the antimicrobial peptide gramicidin S were compared by using bacterial growth inhibition assays. This cyclic β‐sheet peptide could accommodate both L and D substituents on its hydrophobic face. As a third example, we tested six analogues of the antimicrobial peptide PGLa. The presence of d‐4F‐Phg reduced the biological activity of the peptide by interfering with its amphiphilic α‐helical fold. Finally, to illustrate the numerous uses of l‐4F‐Phg in 19F NMR spectroscopy, we characterised the interaction of labelled PGLa with uncharged and negatively charged membranes. Observing the signal of the free peptide in an aqueous suspension of unilamellar vesicles, we found a linear saturation behaviour that was dominated by electrostatic attraction of the cationic PGLa. Once the peptide is bound to the membrane, however, solid‐state 19F NMR spectroscopy of macroscopically oriented samples revealed that the charge density has virtually no further influence on the structure, alignment or mobility of the peptide.

Metal Binding Dictates Conformation and Function of the Amyloid Precursor Protein (APP) E2 Domain.

The amyloid precursor protein (APP) and its neurotoxic cleavage product Aβ are key players in the development of Alzheimers disease and appear essential for neuronal development and cell homeostasis in mammals. Proteolytic processing of APP is influenced by metal ions, protein ligands and its oligomerization state. However, the structural basis and functional mechanism of APP regulation are hitherto largely unknown. Here we identified a metal-dependent molecular switch located within the E2 domain of APP containing four evolutionary highly conserved histidine residues. Three X-ray structures of the metal-bound molecule were solved at 2.6-2.0 Å resolution. Using protein crystallographic and biochemical methods, we characterized this novel high-affinity binding site within the E2 domain that binds competitively to copper and zinc at physiological concentrations. Metal-specific coordination spheres induce large conformational changes and enforce distinct structural states, most likely regulating the physiological function of APP and its processing in Alzheimers disease.

Structure and regulatory role of the C-terminal winged helix domain of the archaeal minichromosome maintenance complex

The minichromosome maintenance complex (MCM) represents the replicative DNA helicase both in eukaryotes and archaea. Here, we describe the solution structure of the C-terminal domains of the archaeal MCMs of Sulfolobus solfataricus (Sso) and Methanothermobacter thermautotrophicus (Mth). Those domains consist of a structurally conserved truncated winged helix (WH) domain lacking the two typical ‘wings’ of canonical WH domains. A less conserved N-terminal extension links this WH module to the MCM AAA+ domain forming the ATPase center. In the Sso MCM this linker contains a short α-helical element. Using Sso MCM mutants, including chimeric constructs containing Mth C-terminal domain elements, we show that the ATPase and helicase activity of the Sso MCM is significantly modulated by the short α-helical linker element and by N-terminal residues of the first α-helix of the truncated WH module. Finally, based on our structural and functional data, we present a docking-derived model of the Sso MCM, which implies an allosteric control of the ATPase center by the C-terminal domain.

Analysis of the Overall Structure of the Multi-Domain Amyloid Precursor Protein (APP)

The amyloid precursor protein (APP) and its processing by the α-, β- and γ-secretases is widely believed to play a central role during the development of Alzheimer´s disease. The three-dimensional structure of the entire protein, its physiologic function and the regulation of its proteolytic processing remain, however, largely unclear to date. To gain a deeper understanding of the structure of APP that underlies all of its functions, we first cloned and recombinantly expressed different constructs in E. coli. Using limited proteolysis followed by mass spectrometry and Edman degradation as well as analytical gel permeation chromatography coupled static light scattering, we experimentally analyzed the structural domain boundaries and determined that the large ectodomain of APP consists of exactly two rigidly folded domains – the E1-domain (Leu18-Ala190) and the E2-domain (Ser295-Asp500). Both, the acidic domain (AcD) connecting E1 and E2 as well as the juxtamembrane region (JMR) connecting E2 to the single transmembrane helix are highly flexible and extended. We identified in-between the E1-domain and the AcD an additional domain of conservation and partial flexibility that we termed extension domain (ED, Glu191-Glu227). Using Bio-layer interferometry, pull-down assays and analytical gel filtration experiments we demonstrated that the E1-domain does not tightly interact with the E2-domain, both in the presence and in the absence of heparin. APP hence forms an extended molecule that is flexibly tethered to the membrane. Its multi-domain architecture enables together with the many known functionalities the concomitant performance of several, independent functions, which might be regulated by cellular, compartment specific pH-changes.

Surface reactive polymers for special applications in nucleic acid synthesis

Abstract This publication reviews work done in the authors laboratories on the functionalization of surface-reactive polymers and their application as a basis for the preparation of oligonucleotides of extended length, larger number or quantity. In addition to nonporous particulate materials (silicagel microbeads; polystyrene-grafted polytetrafluoroethylene particles) special focus is on recent work describing the introduction of spacered amino groups into polypropylene films and the preparation of oligonucleotides on polypropylene surface. A prototype apparatus is shown, for use with a four-column DNA synthesizer, that permits the semi-mechanized preparation of a one-dimensional array of oligonucleotides of variant sequence.

Anti-amyloid compounds protect from silica nanoparticle-induced neurotoxicity in the nematode C. elegans

Abstract Identifying nanomaterial-bio-interactions are imperative due to the broad introduction of nanoparticle (NP) applications and their distribution. Here, we demonstrate that silica NPs effect widespread protein aggregation in the soil nematode Caenorhabditis elegans ranging from induction of amyloid in nucleoli of intestinal cells to facilitation of protein aggregation in body wall muscles and axons of neural cells. Proteomic screening revealed that exposure of adult C. elegans with silica NPs promotes segregation of proteins belonging to the gene ontology (GO) group of “protein folding, proteolysis and stress response” to an SDS-resistant aggregome network. Candidate proteins in this group include chaperones, heat shock proteins and subunits of the 26S proteasome which are all decisively involved in protein homeostasis. The pathway of protein homeostasis was validated as a major target of silica NPs by behavioral phenotyping, as inhibitors of amyloid formation rescued NP-induced defects of locomotory patterns and egg laying. The analysis of a reporter worm for serotonergic neural cells revealed that silica NP-induced protein aggregation likewise occurs in axons of HSN neurons, where presynaptic accumulation of serotonin, e.g. disturbed axonal transport reduces the capacity for neurotransmission and egg laying. The results suggest that in C. elegans silica NPs promote a cascade of events including disturbance of protein homeostasis, widespread protein aggregation and inhibition of serotonergic neurotransmission which can be interrupted by compounds preventing amyloid fibrillation.

A label-free assay of exonuclease activity using a pyrosequencing technique.

Enzymes with 3-5 exonuclease activities are important in promoting the accuracy of DNA replication and DNA repair by proofreading. The alteration of the function of these enzymes by endogenous or exogenous effectors could, therefore, have a considerable impact on DNA replication and ultimately on genome integrity. We have developed a label-free high-throughput screening method for quantifying the effects of different reagents on exonuclease activity. The assay is based on a hairpin-forming biotinylated oligonucleotide substrate that contains one or more exonuclease-resistant phosphorothioate nucleotides. The activity and specificity of the selected 3-5 exonuclease is determined indirectly using a sensitive pyrosequencing reaction after cleanup of the samples. In this pyrosequencing step, the amount of nucleotides filled into each position of the exonucleolytically degraded 3 end of the substrate can be recorded quantitatively and equals the amount of the nucleotides removed by the exonuclease. This system allows the estimation of both processivity and efficiency of the exonuclease activity. We have employed compounds reported in the literature to inhibit the exonuclease activities of either exonuclease III or the large fragment of polymerase I (Klenow fragment) to evaluate the assay.

Amyloid domains in the cell nucleus controlled by nucleoskeletal protein lamin B1 reveal a new pathway of mercury neurotoxicity

Mercury (Hg) is a bioaccumulating trace metal that globally circulates the atmosphere and waters in its elemental, inorganic and organic chemical forms. While Hg represents a notorious neurotoxicant, the underlying cellular pathways are insufficiently understood. We identify amyloid protein aggregation in the cell nucleus as a novel pathway of Hg-bio-interactions. By mass spectrometry of purified protein aggregates, a subset of spliceosomal components and nucleoskeletal protein lamin B1 were detected as constituent parts of an Hg-induced nuclear aggregome network. The aggregome network was located by confocal imaging of amyloid-specific antibodies and dyes to amyloid cores within splicing-speckles that additionally recruit components of the ubiquitin-proteasome system. Hg significantly enhances global proteasomal activity in the nucleus, suggesting that formation of amyloid speckles plays a role in maintenance of protein homeostasis. RNAi knock down showed that lamin B1 for its part regulates amyloid speckle formation and thus likewise participates in nuclear protein homeostasis. As the Hg-induced cascade of interactions between the nucleoskeleton and protein homeostasis reduces neuronal signalling, amyloid fibrillation in the cell nucleus is introduced as a feature of Hg-neurotoxicity that opens new avenues of future research. Similar to protein aggregation events in the cytoplasm that are controlled by the cytoskeleton, amyloid fibrillation of nuclear proteins may be driven by the nucleoskeleton.

Recombinant production of isotope-labeled peptides and spontaneous cyclization of amino-terminal glutamine into pyroglutamic acid.

NMR is a powerful tool for the elucidation of biomolecular structures and their dynamics. Labeling with the stable isotopes N and C paved the way to determining the structures of larger systems. 2] In addition, examination of peptide–protein complexes is facilitated through the use of isotope-labeled peptides. However, the chemical synthesis of isotope-labeled peptides is expensive and recombinant production has yielded a number of peptide-specific protocols in the past. For structural analysis of the complex between the hDlg PDZ domain 2 (PDZ2) and the HPV oncoprotein E6, we generated a doubly labeled (C and N) 11-residue target peptide (QRTRQRNETQV) derived from the C terminus of HPV51 E6 by recombinant expression as C-terminal fusion to an intein. Typically, inteins are exploited to mediate ligation of protein segments. In our approach, the intein just liberates the isotopelabeled peptide through amide bond cleavage at the fusion site in the presence of thiols. Purification of the soluble intein–peptide fusion protein was carried out by affinity chromatography based on the chitinbinding domain of the intein carrier. On-column cleavage was performed in the presence of thiols (Figure S1 in the Supporting Information), followed by reversed-phase HPLC in order to purify the liberated peptide (Figure S2). All major HPLC peaks were collected and subsequently analyzed by [H,N] HSQC NMR spectroscopy and MALDI-TOF MS. The material eluting at 14 min yielded a [H,N] HSQC spectrum corresponding to the composition of the target peptide (Figure S3 A). MALDI-TOF MS (Figure S3 B, Table 1 A) detected a mass corresponding to the calculated mass of the Cand N-labeled target peptide (1493.8 Da). MS analysis also indicated the presence of two minor species that probably represent a sodium–peptide adduct and a species with a mass of 1475.9 Da (i.e. , 18 Da, detected as [M 18 Da+H] of 1476.9 Da). The target peptide was lyophilized and stored at 80 8C until further use. The average yield of the target peptide was 10.6 nmol g 1 (15.8 mg g ) of Escherichia coli wet cell mass. During the resonance assignment of the target peptide bound to the unlabeled PDZ2, we observed unexpected NMR signals in a number of through-bond correlation experiments linking the amide proton of the N-terminal residue (Gln) to its own side-chain nuclei (Figure 1), which suggested a cyclization to pGlu. Such a cyclization involves liberation of the Gln1 side chain NH2 and of one of its a-amino protons to release NH3 (Scheme 1). This explains the observation of an amide proton