[Prolonged weaning during early neurological and neurosurgical rehabilitation : S2k guideline published by the Weaning Committee of the German Neurorehabilitation Society (DGNR)].

Prolonged weaning of patients with neurological or neurosurgery disorders is associated with specific characteristics, which are taken into account by the German Society for Neurorehabilitation (DGNR) in its own guideline. The current S2k guideline of the German Society for Pneumology and Respiratory Medicine is referred to explicitly with regard to definitions (e.g.

Traceless and site-specific ubiquitination of recombinant proteins.

Protein ubiquitination is a post-translational modification that regulates almost all aspects of eukaryotic biology. Here we discover the first routes for the efficient site-specific incorporation of δ-thiol-L-lysine (7) and δ-hydroxy-L-lysine (8) into recombinant proteins, via evolution of a pyrrolysyl-tRNA synthetase/tRNA(CUA) pair. We combine the genetically directed incorporation of 7 with native chemical ligation and desulfurization to yield an entirely native isopeptide bond between substrate proteins and ubiquitin.

Early protein evolution: building domains from ligand-binding polypeptide segments.

It has been suggested that in the early evolution of proteins, segments of polypeptide, unable to fold in isolation, may have collapsed together to form folded proto-domains. We wondered whether the incorporation of segments with a pre-existing binding activity into a folded domain could, by fixing the ligand binding conformation and/or providing additional contacts, lead to large affinity improvements and provide an evolutionary advantage. As a model, we took a segment of polypeptide from hen egg lysozyme that in the native protein forms the binding interface with the monoclonal antibodies HyHEL5 and F10 (KD=60 pM).

Folding and stability of a primitive protein.

We have previously attempted to simulate domain creation in early protein evolution by recombining polypeptide segments from non-homologous proteins, and we have described the structure of one such de novo protein, 1b11, a segment-swapped tetramer with novel architecture. Here, we have analyzed the thermodynamic stability and folding kinetics of the 1b11 tetramer and its monomeric and dimeric intermediates, and of 1b11 mutants with changes at the domain interface. Denatured 1b11 polypeptides fold into transient, folded monomers with marginal stability (DeltaG<1kcalmol(-1)) which convert rapidly ( approximately 6x10(4)M(-1)s(-1)) into dimers (DeltaG=9.

A segment of cold shock protein directs the folding of a combinatorial protein.

It has been suggested that protein domains evolved by the non-homologous recombination of building blocks of subdomain size. In earlier work we attempted to recapitulate domain evolution in vitro. We took a polypeptide segment comprising three beta-strands in the monomeric, five-stranded beta-barrel cold shock protein (CspA) of Escherichia coli as a building block.

A native-like artificial protein from antisense DNA.

We describe the creation of folded chimaeric proteins by combining a designed polypeptide segment (bait) derived from a beta-sheet of a human antibody variable domain with random polypeptide segments encoded by human cDNA fragments. The repertoire of polypeptides was displayed on the surface of filamentous bacteriophage and folded polypeptides were selected by proteolysis. One of these, 2a6, was readily expressed in the Escherichia coli cytoplasm as a soluble and protease-resistant protein and could be purified after heating the bacterial lysate to 90 degrees C.

Novel folded protein domains generated by combinatorial shuffling of polypeptide segments.

It has been proposed that the architecture of protein domains has evolved by the combinatorial assembly and/or exchange of smaller polypeptide segments. To investigate this proposal, we fused DNA encoding the N-terminal half of a beta-barrel domain (from cold shock protein CspA) with fragmented genomic Escherichia coli DNA and cloned the repertoire of chimeric polypeptides for display on filamentous bacteriophage. Phage displaying folded polypeptides were selected by proteolysis; in most cases the protease-resistant chimeric polypeptides comprised genomic segments in their natural reading frames.

Single domain antibodies: comparison of camel VH and camelised human VH domains.

The antigen binding sites of conventional antibodies are formed primarily by the hypervariable loops from both the heavy and the light chain variable domains. Functional antigen binding sites can however also be formed by heavy chain variable domains (VH) alone. In vivo, such binding sites have evolved in camels and camelids as part of antibodies, which consist only of two heavy chains and lack light chains.

Interdomain interactions within the gene 3 protein of filamentous phage.

Infection of Escherichia coli by filamentous phage fd is mediated by the phage gene 3 protein (g3p). The g3p consists of three domains (g3p-D1, D2 and D3) linked by flexible glycine-rich linkers. All three domains are indispensable for phage infectivity; the g3p-D1 domain binds to the TolA receptor presumably at the inner face of the outer membrane, the g3p-D2 domain to the F-pilus and the g3p-D3 domain anchors g3p to the phage coat.

Crystal structure of the two N-terminal domains of g3p from filamentous phage fd at 1.9 A: evidence for conformational lability.

Infection of Escherichia coli by filamentous bacteriophages is mediated by the minor phage coat protein g3p and involves two distinct cellular receptors, the F' pilus and the periplasmic protein TolA. Recently we have shown that the two receptors are contacted in a sequential manner, such that binding of TolA by the N-terminal domain g3p-D1 is conditional on a primary interaction of the second g3p domain D2 with the F' pilus. In order to better understand this process, we have solved the crystal structure of the g3p-D1D2 fragment (residues 2-217) from filamentous phage fd to 1.

The C-terminal domain of TolA is the coreceptor for filamentous phage infection of E. coli.

Filamentous bacteriophages infecting gram-negative bacteria display tropism for a variety of pilus structures. However, the obligatory coreceptor of phage infection, postulated from genetic studies, has remained elusive. Here we identify the C-terminal domain of the periplasmic protein TolA as the coreceptor for infection of Escherichia coli by phage fd and the N-terminal domain of the phage minor coat protein g3p as its cognate ligand.

A conserved infection pathway for filamentous bacteriophages is suggested by the structure of the membrane penetration domain of the minor coat protein g3p from phage fd.

Background: . Gene 3 protein (g3p), a minor coat protein from bacteriophage fd mediates infection of Escherichia coli bearing an F-pilus. Its N-terminal domain (g3p-D1) is essential for infection and mediates penetration of the phage into the host cytoplasm presumbly through interaction with the Tol complex in the E.

Affinity improvement of single antibody VH domains: residues in all three hypervariable regions affect antigen binding.

Background: Through antibody engineering, immunoglobulins can be tailored for their particular application. In this respect, small recognition units are desired for the targeting of antigens in obstructed locations like solid tumors.

Objectives: To design efficient, minimum size recognition units, heavy chain variable regions (VH) had previously been modified for the use as antigen specific, single domain antibody fragments.

Rearrangement of the former VL interface in the solution structure of a camelised, single antibody VH domain.

The solution structure of the isolated antibody heavy chain variable domain (VH)-P8 was determined by NMR spectroscopy. The VH had previously been modified (camelised) at three positions in its former antibody light chain variable domain (VL) interface to reduce hydrophobicity by mimicking camelid heavy chains naturally devoid of light chains. The architecture of two pleated beta-sheets and the conformation of the H1 and H2 loops in VH-P8 are very similar to those in non-camelised, VL-associated VH domains.

Single antibody domains as small recognition units: design and in vitro antigen selection of camelized, human VH domains with improved protein stability.

Folding stabilities of camelized human antibody VH domains were studied through the determination of their melting points in thermodenaturation experiments. The melting point of a VH domain originating from a synthetic library of human VHs, which had been optimized for the use as small recognition units through the mimicking of camelid antibody heavy chains occurring naturally without light chain, was 56.6 degrees C compared with 71.

An antibody VH domain with a lox-Cre site integrated into its coding region: bacterial recombination within a single polypeptide chain.

Bacterial lox-Cre recombination within a single antibody VH domain was achieved through integration of a loxP site into its coding sequence. The 5' half of the VH gene, in which the H2 loop was replaced by a mutant loxP site, was fused to geneIII in an 'acceptor' fd-phage vector containing also a wild type loxP site. With a 'donor' plasmid vector harbouring the 3' half of the VH gene flanked by the same, differing loxP sites it recombined into a full-length VH with the loxP site-H2 loop.

Backbone assignment, secondary structure and protein A binding of an isolated, human antibody VH domain.

Antibody heavy chain variable domains (VH) lacking their light chain (VL) partner are prime candidates for the design of minimum-size immunoreagents. To obtain structural information about isolated VH domains, a human VH was labelled with 15N or doubly labelled with both 15N and 13C and was studied by heteronuclear nuclear magnetic resonance spectroscopy. Most (90%) of the 1H and 15N main-chain signals were assigned through two-dimensional TOCSY and NOESY experiments on the unlabelled VH and three-dimensional heteronuclear multiple quantum correlation TOCSY and NOESY experiments on the 15N-labelled VH.

Antibody VH domains as small recognition units.

To develop immunoglobulin based recognition units of minimum size, a human heavy chain variable domain (VH) was designed for selection of phage displayed VH. Non-specific binding of the VH through its interface for the light chain variable domain (VL) was prevented through three mutations (G44E, L45R and W47G) in this interface. These mutations were introduced to mimic camelid antibody heavy chains naturally devoid of light chain partners.

'Camelising' human antibody fragments: NMR studies on VH domains.

A human heavy chain variable domain (VH) was expressed in bacteria for structural analysis by NMR spectroscopy. NMR analysis was initially impossible due to the short transverse proton relaxation time of the VH, probably caused by aggregation through the exposed interface naturally in contact with the light chain. The relaxation time was improved to normal values when this interface was mutated to mimic heavy chains of camel antibodies naturally devoid of light chains and through the use of the detergent CHAPS.

Phage display and selection of a site-directed randomized single-chain antibody Fv fragment for its affinity improvement.

The affinity of an antibody Fv fragment was improved by semirational design involving site-directed randomization and phage display. On the basis of the predicted model of an anti-2-phenyloxazol-5-one (phOx) antibody Fv fragment, into which the ligand was inserted with the help of nuclear Overhauser enhancement (NOE) data, residues close to the hapten were identified. Seven of these residues in the third hypervariable regions of light and heavy chains were randomized in polymerase chain reactions (PCR) using degenerate oligonucleotides.

Improving the antigen affinity of an antibody Fv-fragment by protein design.

The affinity of an antibody for its ligand 2-phenyloxazolone was improved by protein design. For the design two-dimensional nuclear magnetic resonance spectroscopy, protein engineering and molecular modelling were used in an interactive scheme. Initially the binding site was localized with the help of transferred nuclear Overhauser enhancement signals from two, site specifically assigned tyrosine side-chains in the complementarity-determining regions of the antibody to the ligand 4-glycyl-2-phenyloxazolone.

Uniform labeling of a recombinant antibody Fv-fragment with 15N and 13C for heteronuclear NMR spectroscopy.

The expression of functional antibody fragments in Escherichia coli enables a detailed analysis by NMR spectroscopy. This is demonstrated with the uniform labeling of an Fv-fragment (25 kDa) comprising the antigen binding site of an antibody against 2-phenyloxazolone with 15N and 13C. The antigen-complexed Fv-fragment was analysed for a potential assignment by heteronuclear multi-dimensional NMR spectroscopy.

Use of 2D NMR, protein engineering, and molecular modeling to study the hapten-binding site of an antibody Fv fragment against 2-phenyloxazolone.

Two-dimensional (2D) 1H NMR spectroscopy was used to study the hapten-binding site of a recombinant antibody Fv fragment expressed in Escherichia coli. Point mutations of residues in the CDR loops of the Fv fragment were designed in order to investigate their influence on hapten binding and to make site-specific assignments of aromatic NMR proton signals. Two tyrosines giving NOEs to the ligand 2-phenyloxazolone were identified, residue 33 in CDR1 of the heavy chain and residue 32 in CDR1 of the light chain.

Antigen-antibody interactions: an NMR approach.

With recent advances in methodology, it now appears that NMR can be used at an unprecedented level of sophistication to obtain new insights into the solution structure and dynamics of the antibody combining site, both free and in its complex with antigen. Most promising in this regard is the Fv fragment (molecular weight approximately 25 kD) which can be produced by genetic engineering in a form suitable for NMR studies. Isotopic labeling is required to make specific resonance assignments.