Sequence and structure space of RNA-binding peptides

Studies of RNA-binding peptides, and recent combinatorial library experiments in particular, have demonstrated that diverse peptide sequences and structures can be used to recognize specific RNA sites. The identification of large numbers of sequences capable of binding to a particular site has provided extensive phylogenetic information used to deduce basic principles of recognition. The high frequency at which RNA-binding peptides are found in large sequence libraries suggests plausible routes to evolve sequence-specific binders, facilitating the design of new binding molecules and perhaps reflecting characteristics of natural evolution.     

Fluorescence anisotropy assay for pharmacological characterization of ligand binding dynamics to melanocortin 4 receptors

Fluorescence anisotropy assay was implemented for characterization of ligand binding dynamics to melanocortin 4 (MC₄) receptors. This approach enables on-line monitoring of reactions that is essential for estimation of more correct binding parameters, understanding of ligand binding and its regulation mechanisms, and design of new drugs with desirable properties. Two different red-shifted fluorophore-labeled peptide ligands, Cy3B-NDP-α-MSH and TAMRA-NDP-α-MSH, were used and compared in assays that monitored their binding to MC₄ receptors in membranes of Sf9 insect cells. The Cy3B dye-labeled ligand exhibited improved performance in assays when compared with the TAMRA-labeled ligand, having higher photostability, insensitivity to buffer properties, and better signal/noise ratio. The binding of both ligands to membranes of Sf9 cells expressing MC₄ receptors was saturable and with high affinity. All studied MC₄ receptor-specific nonlabeled ligands displaced fluoroligands’ binding in a concentration-dependent manner with potencies in agreement with their pharmacological activities. On-line monitoring of the reactions revealed that equilibrium of peptide binding was not reached even after 3 h. Real-time monitoring of ligand binding dynamics enabled us to find optimal experimental conditions for each particular ligand and an improved estimate of their binding parameters.     

Protein domain definition should allow for conditional disorder

Abstract: Proteins are often classified in a binary fashion as either structured or disordered. However this approach has several deficits. Firstly, protein folding is always conditional on the physiochemical environment. A protein which is structured in some circumstances will be disordered in others. Secondly, it hides a fundamental asymmetry in behavior. While all structured proteins can be unfolded through a change in environment, not all disordered proteins have the capacity for folding. Failure to accommodate these complexities confuses the definition of both protein structural domains and intrinsically disordered regions. We illustrate these points with an experimental study of a family of small binding domains, drawn from the RNA polymerase of mumps virus and its closest relatives. Assessed at face value the domains fall on a structural continuum, with folded, partially folded, and near unstructured members. Yet the disorder present in the family is conditional, and these closely related polypeptides can access the same folded state under appropriate conditions. Any heuristic definition of the protein domain emphasizing conformational stability divides this domain family in two, in a way that makes no biological sense. Structural domains would be better defined by their ability to adopt a specific tertiary structure: a structure that may or may not be realized, dependent on the circumstances. This explicitly allows for the conditional nature of protein folding, and more clearly demarcates structural domains from intrinsically disordered regions that may function without folding.     

Plasma protein binding study of oxybutynin by high-performance frontal analysis

Plasma protein binding of oxybutynin (OXY) was investigated quantitatively and enantioselectively using high-performance frontal analysis (HPFA). An on-line HPLC system which consists of HPFA column, extraction column and analytical column was developed to determine the unbound concentrations of OXY enantiomers in human plasma, in human serum albumin (HSA) solutions, and in human alpha1-acid glycoprotein (AGP) solutions. OXY is bound in human plasma strongly and enantioselectively. The bound drug fraction in human plasma containing 2-10 microM (R)- or (S)-OXY was higher than 99%, and the unbound fraction of (R)-OXY was 1.56 times higher than that of (S)-isomer. AGP plays the dominant role in this strong and enantioselective plasma protein binding. The total binding affinities (nK) of (R)- and (S)-OXY to AGP were 6.86 x 10(6) and 1.53 x 10(7) M(-1), respectively, while the nK values of (R)- and (S)-OXY to HSA were 2.64 x 10(4) and 2.19 x 10(-4) M(-1), respectively. The binding affinity of OXY to AGP is much higher than that to HSA, and shows high enantioselectivity (SIR ratio of nK values is 2.2). It was found that both enantiomers are bound competitively at the same binding site on an AGP molecule. The binding property between OXY and low density lipoprotein (LDL) was investigated by using the frontal analysis method incorporated in high-performance capillary electrophoresis (HPCE/FA). It was found the binding is non-saturable and non-enantioselective.     

Zinc finger structure-function in Ikaros

The zinc finger motif was used as a vehicle for the initial discovery of Ikaros in the context of T-cell differentiation and has been central to all subsequent analyses of Ikaros function. The Ikaros gene is alternately spliced to produce several isoforms that confer diversity of function and consequently have complicated analysis of the function of Ikaros in vivo. Key features of Ikaros in vivo function are associated with six C2H2 zinc fingers; four of which are alternately incorporated in the production of the various Ikaros isoforms. Although no complete structures are available for the Ikaros protein or any of its family members, considerable evidence has accumulated about the structure of zinc fingers and the role that this structure plays in the functions of the Ikaros family of proteins. This review summarizes the structural aspects of Ikaros zinc fingers, individually, and in tandem to provide a structural context for Ikaros function and to provide a structural basis to inform the design of future experiments with Ikaros and its family members.     

Nucleic acid binding activity of pns6 encoded by genome segment 6 of rice ragged stunt oryzavirus

Rice ragged stunt disease, caused by rice ragged stuntoryzavirus (RRSV), was first discovered in 1976–1977 inIndonesia and Philippines [1]. Subsequently the diseasewas found in most rice-growing countries in south-easternand far-eastern Asia [2] and may inflict heavy loss on thecrop. RRSV is the type species of the genus Oryzavirus in thefamily Reoviridae. The virus particle is icosahedral witha diameter of about 65–70 nm and the genome consistsof 10 double stranded RNA (dsRNA) segm…     

A rapid PCR-based method for genetically mapping ESTs

A simple, semi-automatable procedure was developed for converting expressed sequence tags (ESTs) into mappable genetic markers. The polymerase chain reaction is used to amplify regions immediately 5′ or 3′ to the coding regions of genes in order to maximise sequence variability between alleles. Fragment length and nucleotide substitution polymorphisms among amplified alleles can be detected using either ethidium bromide staining or automated laser-based fluorescence. A 6% non-denaturing acrylamide gel, analysed with an ABI 377 DNA sequencer, proved capable of resolving homoduplexes and heteroduplexes formed between amplified alleles containing nucleotide substitutions as well as resolving allelic length differences. With this approach 75% of 60 ESTs from a range of Pinus species could be genetically mapped in each of three pedigrees from P. radiata and P. taeda. Furthermore, three or four alleles were detected in each pedigree for 42% of the EST markers. Received: 4 January 2000 / Accepted: 26 May 2000     

Response of SCP-2L domain of human MFE-2 to ligand removal: binding site closure and burial of peroxisomal targeting signal

In the study of the structure and function relationship of human MFE-2, we have investigated the dynamics of human MFE-2SCP-2L (hSCP-2L) and its response to ligand removal. A comparison was made with homologous rabbit SCP-2. Breathing and a closing motion are found, identifiable with an adjustment in size and a closing off of the binding pocket. Crucial residues for structural integrity have been identified. Particularly mobile areas of the protein are loop 1 that is connecting helices A and C in space, and helix D, next to the entrance of the pocket. In hSCP-2L, the binding pocket gets occupied by Phe93, which is making a tight hydrophobic contact with Trp36. In addition, it is found that the C-terminal peroxisomal targeting signal (PTS1) that is solvent exposed in the complexed structure becomes buried when no ligand is present. Moreover, an anti-correlation exists between burial of PTS1 and the size of the binding pocket. The results are in accordance with plant nsLTPs, where a similar accommodation of binding pocket size was found after ligand binding/removal. Furthermore, the calculations support the suggestion of a ligand-assisted targeting mechanism.     

De novo MECP2 duplication derived from paternal germ line result in dysmorphism and developmental delay

Xq28 duplications encompassing the methyl CpG binding protein 2 (MECP2) in males exhibit a distinct phenotype, including developmental delay, facial dysmorphism, muscular hypotonia, intellectual disability, poor or absent speech, recurrent infections and early death. The vast majority of affected males inherit the MECP2 duplication from their usually asymptomatic carrier mothers. Only a few cases with Xq28 duplication originating from de novo unbalanced X/Y translocation have been reported and the paternal origin of the aberration has only been validated in three males in the related literature. Here we present a karyotypically normal male with features characteristic of the MECP2 duplication syndrome. The genome-wide SNP genotyping shows a de novo 2.26-Mb duplication from Xq28 to the terminus. The genotypes of the SNPs within the duplicated region indicated a paternal origin. Furthermore, the results of fluorescence in situ hybridization (FISH) indicated a novel Xq:Yp translocation, characterized as der(Y)t(Y;X)(p11.32;q28), which suggests an aberrant that occurred during spermatogenesis. The phenotype is compared to the previously reported cases with Xq28 duplication originated from an unbalanced X/Y translocation, and there was no specific part of the phenotype that could be contributed to the origin of parental imbalances. This report further highlights the capacity of high-molecular cytogenetic methods, such as SNP array and FISH, in the identification of submicroscopic rearrangement, structural configuration and parental origin of aberrant while in the evaluation of children with idiopathic developmental delay and intellectual disability.