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Journal Issue - Volume 17 Issue 6 (June 2008)

Abstract Leukemia‐associated Rho guanine nucleotide exchange factor (LARG) is a RhoA‐specific guanine nucleotide exchange factor (GEF) that can activate RhoA. The PDZ (PSD‐95/Disc‐large/ZO‐1 homology) domain of LARG interacts with membrane receptors, which can relay extracellular signals to RhoA signal transduction pathways. Until now there is no structural and dynamic information about these interactions. Here we report the NMR...

Abstract H‐type thioredoxins (Trxs) constitute a particularly large Trx sub‐group in higher plants. Here, the crystal structures are determined for the two barley Trx h isoforms, HvTrxh1 and HvTrxh2, in the partially radiation‐reduced state to resolutions of 1.7 Å, and for HvTrxh2 in the oxidized state to 2.0 Å. The two Trxs have a sequence identity of 51% and highly similar fold and active‐site architecture. Interestingly, the four...

Abstract Phosphatidylinositol‐4,5‐bisphosphate (PIP2) is a key player in the neurotransmitter release process. Rabphilin‐3A is a neuronal C2 domain tandem containing protein that is involved in this process. Both its C2 domains (C2A and C2B) are able to bind PIP2. The investigation of the interactions of the two C2 domains with the PIP2 headgroup IP3 (inositol‐1,4,5‐trisphosphate) by NMR showed that a well‐defined binding site can...

Abstract Conjugation or deconjugation of ubiquitin (Ub) or ubiquitin‐like proteins (UBLs) to or from cellular proteins is a multifaceted and universal means of regulating cellular physiology, controlling the lifetime, localization, and activity of many critical proteins. Deconjugation of Ub or UBL from proteins is performed by a class of proteases called isopeptidases. Herein is described a readily quantifiable novel isopeptidase...

Abstract The effect of the polypeptide environment on polyalanine‐induced fibril formation was investigated with amyloidogenic fragments from PAPBN1, a nuclear protein controlling polyadenylation. Mutation‐caused extensions of the natural 10 alanine sequence up to maximally 17 alanines result in fibril formation of PABPN1 and the development of the disease oculopharyngeal muscular dystrophy (OPMD). We explored the influence of...

Abstract The complex of barnase (bn) and barstar (bs), which has been widely studied as a model for quantitative analysis of protein–protein interactions, is significantly destabilized by a single mutation, namely, bs Asp39 → Ala, which corresponds to a change of 7.7 kcal·mol−1 in the free energy of binding. However, there has been no structural information available to explain such a drastic destabilization. In the present study,...

Abstract Alanine racemase (Alr) is an important enzyme that catalyzes the interconversion of L‐alanine and D‐alanine, an essential building block in the peptidoglycan biosynthesis. For the small size of the Alr active site, its conserved substrate entryway has been proposed as a potential choice for drug design. In this work, we fully analyzed the crystal structures of the native, the D‐cycloserine‐bound, and four mutants (P219A,...

Abstract It is assumed that the proteosome‐processing characteristics of fusion constructs can be predicted from the sum of the proteosome sensitivity of their components. In the present study, we observed that a fusion construct consisting of proteosome‐degradable proteins does not necessarily result in a proteosome‐degradable chimera. Conversely, fusion of proteosome‐resistant proteins may result in a proteosome‐degradable...

Abstract The process of self‐assembly of the triple‐helical peptide (Pro‐Hyp‐Gly)10 into higher order structure resembles the nucleation‐growth mechanism of collagen fibril formation in many features, but the irregular morphology of the self‐assembled peptide contrasts with the ordered fibers and networks formed by collagen in vivo. The amino acid sequence in the central region of the (Pro‐Hyp‐Gly)10 peptide was varied and found to affect the...

Abstract Amide‐to‐ester backbone mutagenesis enables a specific backbone–backbone hydrogen bond (H‐bond) in a protein to be eliminated in order to quantify its energetic contribution to protein folding. To extract a H‐bonding free energy from an amide‐to‐ester perturbation free energy (ΔGfolding,wt − ΔGfolding,mut), it is necessary to correct for the putative introduction of a lone pair–lone pair electrostatic repulsion, as well as for ...

Abstract The recently published microcrystal structures of amyloid fibrils from small peptides greatly enhanced our understanding of the atomic‐level structure of the amyloid fibril. However, only a few amyloid fibrils can form microcrystals. The dansyl‐tryptophan fluorescence resonance energy transfer (FRET) pair was shown to be able to detect the inter‐peptide arrangement of the Transthyretin (105–115) amyloid fibril. In this...

Abstract Amylosucrase is a transglucosidase that catalyzes amylose‐like polymer synthesis from sucrose substrate. About 60,000 amylosucrase variants from two libraries generated by the MutaGen random mutagenesis method were submitted to an in vivo selection procedure leading to the isolation of more than 7000 active variants. These clones were then screened for increased thermostability using an automated screening process. This...

Abstract The prediction of T‐cell epitopes is an essential part in virtual immunology. Apart from sequence‐based techniques, which achieve good results but fail to give insight into the binding behavior of a certain peptide binding to a major histocompatibility complex, structure‐based approaches are another important technique. An essential step is the correct placement of the side chains for a given peptide in cases where no...

Abstract Diagnosis of eukaryotic parasitic infection using antibody‐based tests such as ELISAs (enzyme‐linked immunosorbent assays) is often problematic because of the need to differentiate between homologous host and pathogen proteins and to ensure that antibodies raised against a peptide will also bind to the peptide in the context of its three‐dimensional protein structure. Filariasis caused by the nematode, Brugia malayi, is an...

Abstract When researchers build high‐quality models of protein structure from sequence homology, it is today common to use several alternative target‐template alignments. Several methods can, at least in theory, utilize information from multiple templates, and many examples of improved model quality have been reported. However, to our knowledge, thus far no study has shown that automatic inclusion of multiple alignments is...