MethodsMutagenesis and preparation of cRNA and Oocytes -Mutant 5-HT 3A receptor subunits were developed using pcDNA3.1 (Invitrogen, Abingdon, U.K.) containing the complete coding sequence for the 5-HT 3A(b) subunit from mouse neuroblastoma N1E-115 cells as previously described 1 . For nonsense suppression the proline codon at 308 was replaced by TAG as previously described 2 . Wild type and mutant receptor subunit coding sequences were then subcloned into pGEMHE. This was linearized with Nhe1 (New England Biolabs) and cRNA synthesised using the T7 mMESSAGE mMACHINE kit (Ambion). Oocytes from Xenopus laevis were prepared and maintained as described previously 2 .Synthesis of tRNA and dCA-amino acids-Unnatural amino acids were chemically synthesised as nitroveratryloxycarbonyl (NVOC) protected cyanomethyl esters and coupled to the dinucleotide dCA, which was then enzymatically ligated to 74-mer THG73 tRNA CUA as detailed previously 3 . Immediately prior to co-injection with mRNA, tRNA-aa was deprotected by photolysis. Typically 5 ng mRNA and 25 ng tRNA-aa were injected into Stage V-VI oocytes in a total volume of 50 nl. For control experiments, mRNA was injected 1) in the absence of tRNA and 2) with the THG73 74-mer tRNA.Experiments were preformed 18-36 h post injection.Characterisation of mutant receptors-5-HT-induced currents were recorded from individual oocytes using two-voltage electrode clamp with either a GeneClamp 500 amplifier or an OpusXpress system (Axon Instruments, Inc., Union City, CA). All experiments were performed at 22-25 ºC. Serotonin (creatinine sulphate complex,
Polyketides from actinomycete bacteria provide the basis for many valuable medicines, so engineering genes for their biosynthesis to produce variant molecules holds promise for drug discovery. The modular polyketide synthases are particularly amenable to this approach, because each cycle of chain extension is catalyzed by a different module of enzymes, and the modules are arranged within giant multienzyme subunits in the order in which they act. Protein-protein interactions between terminal docking domains of successive multienzymes promote their correct positioning within the assembly line, but because the overall complex is not stable in vitro, the key interactions have not been identified. We present here the NMR solution structure of a 120 residue polypeptide representing a typical pair of such domains, fused at their respective C and N termini: it adopts a stable dimeric structure which reveals the detailed role of these (predominantly helical) domains in docking and dimerization by modular polyketide synthases.
The heterochromatin protein 1 (HP1) family of proteins is involved in gene silencing via the formation of heterochromatic structures. They are composed of two related domains: an N-terminal chromo domain and a C-terminal shadow chromo domain. Present results suggest that chromo domains may function as protein interaction motifs, bringing together different proteins in multi-protein complexes and locating them in heterochromatin. We have previously determined the structure of the chromo domain from the mouse HP1β protein, MOD1. We show here that, in contrast to the chromo domain, the shadow chromo domain is a homodimer. The intact HP1β protein is also dimeric, where the interaction is mediated by the shadow chromo domain, with the chromo domains moving independently of each other at the end of flexible linkers. Mapping studies, with fragments of the CAF1 and TIF1β proteins, show that an intact, dimeric, shadow chromo domain structure is required for complex formation. Keywords: chromatin structure/chromo domain/ heterochromatin protein 1/protein complex/protein structure
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