RESUMO
We describe a method for generating moderate to high-resolution protein structures using limited NMR data combined with the ab initio protein structure prediction method Rosetta. Peptide fragments are selected from proteins of known structure based on sequence similarity and consistency with chemical shift and NOE data. Models are built from these fragments by minimizing an energy function that favors hydrophobic burial, strand pairing, and satisfaction of NOE constraints. Models generated using this procedure with approximately 1 NOE constraint per residue are in some cases closer to the corresponding X-ray structures than the published NMR solution structures. The method requires only the sparse constraints available during initial stages of NMR structure determination, and thus holds promise for increasing the speed with which protein solution structures can be determined.
Assuntos
Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Humanos , Fragmentos de Peptídeos/química , Biblioteca de Peptídeos , Estrutura Terciária de Proteína , TermodinâmicaRESUMO
The region responsible for sequence-specific DNA binding by the transcription factor ADR1 contains two Cys2-His2 zinc fingers and an additional N-terminal proximal accessory region (PAR). The N-terminal (non-finger) PAR is unstructured in the absence of DNA and undergoes a folding transition on binding the DNA transcription target site. We have used a set of HN-HN NOEs derived from a perdeuterated protein-DNA complex to describe the fold of ADR1 bound to the UAS1 binding site. The PAR forms a compact domain consisting of three antiparallel strands that contact A-T base pairs in the major groove. The three-strand domain is a novel fold among all known DNA-binding proteins. The PAR shares sequence homology with the N-terminal regions of other zinc finger proteins, suggesting that it represents a new DNA-binding module that extends the binding repertoire of zinc finger proteins.
Assuntos
Proteínas de Ligação a DNA/química , Dobramento de Proteína , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/química , Dedos de Zinco , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Sequência Conservada , Cristalização , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Elementos de Resposta/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Soluções , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
We have measured deuterium/hydrogen fractionation in three histidine-containing proteins, ecHPr, ecHPr mutant S31A, and bsHPr, and in random coil peptides using NMR and mass spectrometry. The amide protons of unstructured peptides exhibit equilibrium enrichment for deuterium, in agreement with previous studies. Enrichment for both protium and deuterium was observed in both HPrs, with fractionation factors ranging from 0.63 to 1.41. Enrichment for protium was seen in alpha-helical secondary structure. 'Strong' HBs previously identified by mutagenesis and thermodynamic measurements are significantly enriched for protium. Sites of protium enrichment are conserved in a structural context across species lines, though ecHPr and bsHPr share only 30% sequence identity, suggesting that strong HBs are conserved and may play an important role in stabilizing the folded state.
Assuntos
Proteínas de Bactérias/química , Deutério/química , Histidina/química , Hidrogênio/química , Espectroscopia de Ressonância Magnética/métodos , Bacillus subtilis/química , Proteínas de Bactérias/genética , Escherichia coli/química , Ligação de Hidrogênio , Espectrometria de Massas/métodos , Modelos Moleculares , Mutação , Peptídeos/química , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Seven subnormal spastic children aged 15--16 years, who had the additional problem of profuse dribbling, were trained to associate an auditory cue, from a small box pinned to their clothes, with swallowing. A significant reduction in dribble rate was obtained for all children within the first week. This method of dribble control has two advantages: it requires very little professional time, and the child's ability to control his dribbling is emphasized from the beginning.
