Search

The uses of dipolar shifts due to cobalt(II) substituted for zinc(II) in a consensus zinc finger peptide for refining the NMR-determined structure were examined. Substantial differences between the calculated and observed chemical shift differences between the cobalt(II) and zinc(II) complexes were observed when these dipolar shifts were not used as constraints in the structure refinement. However, inclusion of these constraints resulted in excellent agreement with minor adjustments in the structure and a slight improvement in the precision of the structure determination. Other calculations revealed that the dipolar shifts were not adequate to determine the overall folded structure by themselves, but were useful in increasing the accuracy and precision of a structure determined based only on nuclear Overhauser effects constraints involving only backbone atoms. Substitution of cobalt(II) for zinc(II) in a zinc finger peptide induces substantial shifts in 1H NMR resonances due to interactions with the magnetic dipole induced on the cobalt(II) center. The chemical shift differences between the cobalt(II) and zinc(II) complexes provide constraints for refining the three-dimensional structure as these shifts determine a surface on which the proton must lie. [Display omitted] • Zinc finger domains have structures that consist of a beta sheet followed by an alpha helix. • The structure has a zinc(II) ion that is coordinated to 2 cysteinate and 2 histidine residues. • Cobalt(II) can replace zinc(II) to produce an isostructural domain that is paramagnetic. • The paramagnetic ion leads to shifts in the 1H NMR spectrum compared with the zinc(II) complex. • These paramagnetic shifts can be used as constraints to refine the structure. [ABSTRACT FROM AUTHOR]

Abstract: A large number of proteins contain bound zinc ions. These zinc ions are frequently coordinated by a combination of histidine and cysteine residues. In addition to atoms that coordinate directly to the zinc ions, these side chains have groups that can donate or accept hydrogen bonds from other groups. These secondary interactions can help stabilize the zinc-binding sites, can contribute to protein folding and stability, and, on occasion, can participate in interactions with other macromolecules. Five examples of these secondary interactions are discussed: carbonic anhydrase (where secondary interactions involving histidine residues stabilize the zinc-binding site thermodynamically and kinetically), retroviral nucleocapsid proteins and TRAF proteins (where cysteinate sulfur to peptide NH hydrogen bonds contribute to the structural relationships between adjacent domains), and nucleic acid binding proteins, Zif268 and TIS11 where secondary interactions participate in protein–nucleic acid interactions. [Copyright &y& Elsevier]

The solution structures of nucleocapsid (NC)-like CCHC zinc-binding domains bound to nucleic acid targets have revealed that these domains bind guanosine residues within single-stranded nucleic acids. Here, we have performed initial studies examining the potential use of NC-like CCHC zinc-binding domains as modules to construct single-stranded nucleic acid binding peptides. The affinity for guanosine-containing single-stranded deoxyribooligonucleotides increases with the number of CCHC domains in the peptide. The length of the linker between domains affects the spacing of guanosine residues in oligonucleotides that are preferentially bound. These studies provide a proof of principle that NC-like CCHC zinc-binding domains can be utilized as a basis for designing peptides that bind specific single-stranded nucleic acid sequences. [ABSTRACT FROM AUTHOR]

Most proteins are targeted to the peroxisomal matrix by virtue of a peroxisomal targeting signal-i (PTS1), a short carboxy-terminal sequence specifically recognized by the PTS1 receptor Pex5p. We had previously developed a model that allowed the estimation of the affinities of many PTS1 sequences within the human proteome for Pex5p that revealed a wide range of predicted affinities. We have now experimentally determined the affinities of the PTS1-containing peptides from 42 proteins from the human proteome for Pex5p and show that these range over 4 orders of magnitude. These affinities correlate reasonably well with the predicted values and are substantially more precise. In an attempt to provide a possible explanation for the wide range of PTS1 -Pex5p affinities, we compared these affinities with mRNA levels (as a proxy for rates of protein production) of the genes encoding these proteins in 79 human tissues and cell types. We note that high affinity PTS1 -Pex5p interactions tend to correspond to proteins encoded by genes expressed at relatively low levels, whereas lower affinity PTS1 -Pex5p interactions tend to correspond to proteins encoded by genes exhibiting higher levels and wider ranges of expression. Further analysis revealed that these relationships are consistent with the notion that a relatively uniform pool of protein-Pex5p complexes is maintained for appropriate peroxisome assembly. [ABSTRACT FROM AUTHOR]

The article focuses on the ASAPbio (Accelerating Science and Publication in biology) meeting held on February 16-17, 2016. Topics discussed include the use of preprints for the dissemination of research results and ideas in the life sciences, and the roles preprints could play in communicating biology research results and debate possible advantages and disadvantages of increased use of preprints for scientific progress. It considers the perspective of funders and challenges in using preprints.

Metal-mediated protein oligomerization is an emerging mode of protein-protein interaction. The C-terminal cytosolic domains of T-cell coreceptors CD4 and CD8α form zinc-bridged heterodimers with the N-terminal region of the kinase Lck, with each protein contributing two cysteinate ligands to the complex. Using size exclusion chromatography, 1H NMR, and UV/visible absorption spectroscopy with cobalt(II) as a spectroscopic probe, we demonstrate that small peptides derived from these regions form metal-bridged heterodimers but also homodimers, in contrast to previous reports. The Lck-CD4 and Lck-CD8α cobalt(II)- bridged heterodimer complexes are more stable than the corresponding (Lck)2cobalt(II) complex by factors of 11 ± 4 and 22 ± 9, respectively. These studies were aided by the discovery that cobalt(II) complexes with a cobalt(II)(-Cys-X-X-Cys-)(-Cys-X-Cys-) chromophore show unusual optical spectra with one component of the visible d-d (4A2-to-4T1(P)) transition red-shifted and well separated from the other components. These results provide insights into the basis of specificity of metal-bridged complex formation and on the potential biological significance of metal-bridged homodimers in T-cells. [ABSTRACT FROM AUTHOR]

Abstract: Peroxisomal biogenesis disorders (PBDs) are caused by mutations in 12 distinct genes that encode the components of the peroxisome assembly machinery. Three mutations in the gene encoding Pex5p, the peroxisomal targeting signal type-1 (PTS1) receptor, have been reported, each associated with a disorder of the Zellweger spectrum of different severity. Here, we report studies of the affinities of mutated forms of Pex5p for a series of PTS1 peptides and conclude that PTS1-affinity reductions are correlated with disease severity and cell biological phenotype. A quantitative model has been developed that allows estimation of the dissociation constants for complexes with a wide range of PTS1 sequences bound to wild-type and mutant Pex5p. In the context of this model, the binding measurements suggest that no PTS1-containing proteins are targeted by Pex5p(N489K) and only a relatively small subset of PTS1-containing proteins with the highest affinity for Pex5p are targeted to peroxisomes by Pex5p(S563W). Furthermore, the results of the analysis are consistent with an approximate dissociation constant threshold near 500 nM required for efficient protein targeting to peroxisomes. [Copyright &y& Elsevier]

Zinc(II) and cobalt(II) binding to a series of zinc finger peptides with different charged residue pairs across from one another in a β-sheet were examined. Previous studies revealed a narrow range of interaction free energies (<0.5 kcal/mol) between these residues. Here, isothermal titration calorimetry studies were performed, revealing a range of over 3 kcal/mol in relative binding enthalpies. Double mutant cycle analysis revealed a range of interaction enthalpies ranging from -3.1 to -3.4 kcal/mol for the Arg-Asp pair to -0.8 kcal/mol for the Lys-Glu pair. The large range of interaction enthalpies coupled with the small range of interaction free energies reveals substantial entropy-enthalpy compensation. The magnitudes of the effects are consistent with the formation of a structurally rigid Arg-Asp contact ion pair but less direct and more mobile interactions involving the other combinations. [ABSTRACT FROM AUTHOR]

Cys2His2 zinc finger proteins make up the largest class of transcription factors encoded in the genomes of higher eukaryotes. Recent studies of the Ikaros transcription factor demonstrated that this zinc finger protein undergoes cell cycle-dependent changes in association with DNA that seem to be due to phosphorylation of Thr or Ser residues in the linker regions connecting adjacent zinc finger domains. The high degree of conservation of this linker sequence within the Cys2His2 superfamily suggested a common mechanism for the cell cycle-dependent modulation of DNA-binding affinity throughout this large class of transcription factors. The effects of linker phosphorylation on DNA-binding affinity were investigated through a direct comparison of the DNA-binding properties of four synthetic zinc finger proteins produced by native chemical ligation. The four proteins, comprising three zinc finger domains joined by two consensus Thr-Gly-Glu-Lys-Pro linkers, correspond to all four possible combinations of linker Thr phosphorylation states. Fluorescence-based DNA-binding studies of a specific DNA-binding site revealed that phosphorylation of a single linker reduced binding affinity &ap; 40-fold, whereas phosphorylation of both linkers reduced binding affinity 130-fold. These results with purified components demonstrate that linker phosphorylation does, indeed, produce a significant reduction in DNA-binding affinity and support a model wherein a single cell cycle-dependent Ser/Thr kinase could simultaneously inactivate a large number of zinc finger transcription factors. [ABSTRACT FROM AUTHOR]

Metal response element (MRE) binding transcription factor-1 (M'l'F1) is a six Cys2His2 zinc finger-containing transcription factor required for basal and zinc-induced transcription of metallothionein genes. The cobalt(II) and zinc(II) affinities of a protein fragment comprising the six zinc finger domains have been examined to reveal apparent dissociation constants (for the six domains collectively) of 0.5 ± 0.2 μM for cobalt(II) and 31 ± 14 μM for zinc(II). Two approaches have been used to determine the metal ion affinities of the individual domains. First, the six domains have been examined as single domain peptides revealing dissociation constants ranging from 0.3 to 1.7 μM for cobalt(II). The domains fall into two sets with peptides corresponding to domains 2, 3, and 4 showing relatively high affinity (Kd(CO(II)) 0.3-0.5 μM) and peptides corresponding to domains 1, 5, and 6 showing lower affinity (Kd(CO(II)) 1.6-1.7 μM). Second, we examined the affinity of each domain in the context of the six zinc finger domain protein by individually mutating one metal-binding His residue to Cys to allow independent monitoring of the cobalt(II) occupancy of each site. The affinity of each domain was higher in this context than as a single domain peptide with affinities (corrected for the effect of the mutation) ranging from 0.02 to 0.5 μM. The increase in affinity for the individual domains ranged from factors of 1.1 to 20. The order of affinities (from higher to lowest) was observed to be 4 > 2 &ap; 5 > 6 &ap; 3 &ap; 1. These results reveal that none of the Cys2His2 zinc finger domains of MTF1 have dramatically low metal ion affinities, certainly none low enough to respond to changes in free zinc ion concentrations in the micromolar range. Nonetheless, the metal ion affinities of some domains do differ by a fadtor of 25 with domains at both the amino- and carboxyl-termini showing lower intrinsic affinities for metal ions than the central domains. [ABSTRACT FROM AUTHOR]

Analyzes the frequency of occurrence of C-terminal tripeptides sequences in representative archaeal, bacterial and eukaryotic genomes. Indication that the sequence distribution in prokaryotic genomes nearly matches that generated by the randomization of the observed tripeptide set; Role of a complete sequence of genome of an organism for the analysis of sequence patterns; Determination of which sequence can be considered overrepresented.

Examines the peroxisomal targeting signal (PTS1)-binding activity of purified, recombinant, full-length PEX5 using a fluorescence anisotropy-based assay. Binding of PTS1 through the C-terminal 40-kDa tetratricopeptide repeat domain of PTS1; Demonstration of the specificity of the interaction; Recognition of enzymes by PEX5.

The thermodynamics of metal binding by the prototypical Cys[sub 2]His[sub 2] zinc finger peptide CP-1 has been examined through the use of isothermal titration calorimetry. In cholamine buffer at pH 7.0, the binding of zinc(II) to CP-1 shows an enthalpy change of ΔH°[sub obs] = -33.7 ± 0.8 kcal/mol. Between one and two protons appear to be released accompanying the metal binding process. The heat of protonation of the cholamine buffer used is quite large (-11.5 kcal/mol), indicating that a portion of the observed metal binding enthalpy is due to buffer protonation. Structure-based thermodynamic analysis including the effect of water release from zinc(II) appears to account for the entropy associated with the coupled metal binding-protein folding process semiquantitatively. The strongest driving force for the reaction is the enthalpy associated with the four bonds from zinc(II) to cysteinate and histidine residues, compared with the bonds from zinc(II) to water. The binding of cobalt(II) to CP-1 is less enthalpically driven than the binding of zinc(II) by -7.6 kcal/mol. This value is approximately equal to, but slightly larger than, the expectation based on considerations of ligand field stabilization energy. [ABSTRACT FROM AUTHOR]

Investigates the metal binding and structural properties the zinc-binding domains of a family of proteins. Presence of five conserved potential zinc-binding residues; Synthesis of a peptide corresponding to the third putative zinc-binding domain from the transcription factor NZF-1; Indication that metal binding is tetrahedral.

Examines the metal binding and DNA binding properties of a fragment of neural zinc finger factor 1 (NZF-1) containing two adjacent zinc binding domains. DNA binding structural motifs; Methods used to isolate the gene for neural zinc finger factor1; Characterization of two-domain NZF-1 fragments.

Discusses the sequential metal binding by the RING finger domain of BRC1 familial breast and ovarian cancer susceptibility gene. Description of the interleaved metal binding sites; Characterization of the metal-binding and metal-dependent folding properties of peptides; Structural changes of BRC1 RING finger peptide after metal binding.

Identifies the free energy consequences of electrostatic interaction across a model beta-sheet using a zinc finger peptide. Isolation of energies of the electrostatic interactions via double mutant cycles performed at a series of sodium chloride concentrations; Description of favorable and unfavorable free energies.

Focuses on research that deals with zinc finger protein and DNA, RNA and DNA-RNA hybrids in relation to protein binding. Details on research; Experimental procedures; Results and discussion.

Explains research findings that indicate the presence of zinc finger proteins in the genetic structure of the nematode Caenorhabditis elegans. Sequences characteristic of zinc-binding structural domains; Distribution of the proteins offering insights in the role of zinc-binding proteins in evolution; Opportunities offered in analyzing pathways of transcriptional regulation.

Zinc-finger domains are small metal-binding modules that are found in a wide range of gene regulatory proteins. Peptides corresponding to these domains have provided valuable model systems for examining a number of biophysical parameters entirely unrelated to their nucleic acid binding properties. These include the chemical basis for metal-ion affinity and selectivity, thermodynamic properties related to hydrophobic packing and beta-sheet propensities, and constraints on the generation of ligand-binding and potential catalytic sites. These studies have laid the foundation for applications such as the generation of optically detected zinc probes and the design of metal-binding peptides and proteins with desired spectroscopic and chemical properties. [ABSTRACT FROM AUTHOR]

Describes the role of zinc as a structural component of a large number of proteins. Stabilization of folded conformations of domains through the binding of zinc; Facilitation of interactions between proteins and other macromolecules; Ability of zinc to be bound specifically within a range of tetrahedral sites that allow the evolution of zinc-stabilized domains.

Presents a thermodynamic beta-sheet propensity scale for all the commonly occurring amino acids in aqueous solution. Zinc-finger peptide as the host system with a guest position within a beta-strand; Summary of beta-sheet propensity data; Details.

Presents information on a study which addressed the fundamental properties of alkylation of metal-coordinating cysteine residues within peptide complexes in aqueous solution, using modified consensus zinc finger peptides. Experimental section; Discussion on the results.

Examines specific deoxyribonucleic acid-(DNA) ribonucleic acid (RNA) binding by zinc finger proteins of the Cys2His2 type. Structural characteristics of zinc finger protein-DNA complexes; Examination of the binding properties of ZF-QQR, a zinc finger protein with different site preferences; Examination of the behavior of the homeodomain protein Ubx.

A response by Jeremy Berg to a letter to the editor about his article "The young and the innovative" in the September 21, 2007 issue is presented.

Discusses research being done on the structure of zinc fingers. Reference to study by Lu, Searles and Klug, published in the journal "Nature"; Role of RNA in zinc fingers-TFIIIA protein interaction; Biological significance of zinc finger proteins; Interaction of zinc fingers with RNA; Recognition of RNA by TFIIIA proteins.

Studies the kinetics and thermodynamics of copper(II) binding to apoazurin. Examination of the calorimetric traces for the titration of apoazurin into copper(II) in cholamine chloride buffer; Isolation of the calorimetric traces from each injection; Simulation of the integrated calorimetric traces.

Studies the expansion of DNA-recognition capabilities for zinc finger proteins beyond 20 amino acids. Key role of designed sequence-specific nucleic acid-binding proteins in the directed regulation of gene expression; Restrictions on zinc finger design; Implications on chemistry.

The recently determined structure of a zinc binding peptide reveals that a particular sequence can adopt one stable fold as an isolated peptide but adopt an alternative structure as part of a larger protein domain. [Copyright &y& Elsevier]

Offers a look at two new methods for probing protein-DNA interactions. Details on a novel fluorescence-based approach to monitoring protein-DNA interaction; Biochemical method for mapping, at single nucleotide resolution; How the elucidation of DNA-protein interaction will help present information on gene expression.

Under the title "Biochemistry, Revised Edition," this article was published in the March 26, 2001 issue of Chemical and Engineering News, p. 130, celebrating the 125th anniversary of the American Chemical Society. Written from the viewpoint of a chemist, it looks to how "many of the most intriguing biological problems can be posed in chemical terms." It is reproduced here by permission of the author and publisher. [ABSTRACT FROM AUTHOR]

Looks into a ratioable fluorescent probe for zinc(ZN)(II) that is based on the zinc finger consensus peptide (CP). Significance of Zn finger peptides; Modulation on the affinity of metal ions; Impact of spectroscopic properties of Zn(II) on the ineffectiveness of unmodified CP; Modification of Zn finger peptide with Trp and a single fluorescent dye; Synthesis of Zn finger peptide via solid phase peptide synthesis; Confirmation on the identity of the probe using mass spectroscopy; Ineffectiveness of incorporating fluorescent dyes on the metal binding and folding properties of the Zn finger peptide.

The author discusses the policy announced by the U.S. National Institutes of Health (NIH) which will apply to the next round of R01 grants. The author says that the application from investigators receiving more than $1 million a year in funding from the NIH are subjected to extra review. He suggests that the policy will complement peer review and help determine the best possible portfolio of NIH research.

A letter to the editor is presented in response to the article "A KNOCK-out award in the medicine" in the October 12, 2007 issue.

Presents a letter to the editor about issues related to the use of sponsored research funding as a determinant of bonus pay for basic science faculty at a number of U.S. medical schools.

The metal binding properties of peptides corresponding to metal-binding sites spanning regions that normally function as linkers in tandem arrays of metal-binding domain-containing proteins were examined. For a peptide with two His residues from one TFIIIA-like zinc finger domain, a canonical IFIIIA-like linker, and two Cys residues from an adjacent zinc domain, the dissociation constant for the 1:1 peptide to cobalt(ll) was found to be 15 ± 10 µM, compared with 60 nM for the corresponding zinc finger domains themselves. Peptides overlapping two sets of metal-binding domains from human TRAF (tumor necrosis factor receptor-associated factor) proteins were examined. In one case, the affinity of the presumed metal-binding domain and that for the linker region were comparable, while in the second case, the affinity of the linker peptide was higher than that for the corresponding presumed metal-binding domain peptide. These studies revealed that cobalt(ll) affinities in the micromolar range can occur even for peptides that do not correspond to natural zinc-binding domains and that the degree of distinction between authentic metal-binding domains and the corresponding linker-spanning peptides may be modest, at least for single domain peptide models. [ABSTRACT FROM AUTHOR]

The structure of a CCHHC zinc-binding domain from neural zinc finger factor-I (NZF-1) has been determined in solution though the use of NMR methods. This domain is a member of a family of domains that have the Cys-X[Sub4]-CYS-X[Sub4]-His-X[Sub7]-His-X[Sub5]-Cys consensus sequence. The structure determination reveals a novel fold based around a zinc(II) ion coordinated to three Cys residues and the second of the two conserved His residues. The other His residue is stacked between the metal-coordinated His residue and a relatively conserved aromatic residue. Analysis of His to Gin sequence variants reveals that both His residues are required for the formation of a well-defined structure, but neither is required for high-affinity metal binding at a tetrahedral site. The structure suggests that a two-domain protein fragment and a double-stranded DNA binding site may interact with a common two-fold axis relating the two domains and the two half-sites of the DNA-inverted repeat. [ABSTRACT FROM AUTHOR]

Regulation of gene expression takes place at several different levels and involves specific domains involved in specific protein-nucleic acid interactions. The protein Nup475 (also known as Tristetraprolin and TS11) binds to AU-rich sequence dements in certain mRNA molecules and favors the degradation of these mRNAs. The nucleic acid binding domain of Nup475 consists of two CCCH zinc-binding domains. A 36-amino acid peptide corresponding to the first of these CCCH domains has been synthesized and characterized. This peptide binds metal ions such as zinc(II) and cobalt(II) with affinities comparable to those of other authenticated zinc-binding domains. The zinc(II) complex of this peptide binds the RNA oligonucleotide UUUAUUU labeled with fluorescein on the 3'-end with an affinity of approximately 5 µM and discriminates against other sequences lacking the central A or the flanking U residues. These results demonstrate for the flint time that a single CCCH domain is capable of binding single-stranded RNA with considerable affinity and selectivity. The combination of this well-behaved domain and the fluorescence-based binding assay sets the stage for more detailed structure—activity studies. [ABSTRACT FROM AUTHOR]

Nup475 (also known as tristetraprolin and TIS11) includes two zinc-binding domains of the form Cys-X[sub 8]-Cys-X[sub 5]-Cys-X[sub 3]-His. These domains are required for rapid degradation of tumor necrosis factor (TNF) and other mRNAs through the interaction with AU-rich elements in their 3'-untranslated regions. The three-dimensional solution structure of the first domain was determined by multidimensional nuclear magnetic resonance spectroscopy, revealing a novel fold around a central zinc ion. The core structure is disklike with a diameter of ∼25 Å and a width of ∼12 Å. This structure provides a basis for evaluating the role of individual residues for structural stability and for nucleic acid binding. [ABSTRACT FROM AUTHOR]

Studies the nuclear magnetic resonance (NMR) of rapidly exchanging backbone amide protons in staphylococcal nuclease and the correlation with structural and dynamic properties. Dependence of pH; Comparison of measured exchange rates with crystal structural properties; Comparison with other solution NMR dynamics; Comparison with B-factors.

Background: The Cancer Genome Atlas Project (TCGA) is a National Cancer Institute effort to profile at least 500 cases of 20 different tumor types using genomic platforms and to make these data, both raw and processed, available to all researchers. TCGA data are currently over 1.2 Petabyte in size and include whole genome sequence (WGS), whole exome sequence, methylation, RNA expression, proteomic, and clinical datasets. Publicly accessible TCGA data are released through public portals, but many challenges exist in navigating and using data obtained from these sites. We developed TCGA Expedition to support the research community focused on computational methods for cancer research. Data obtained, versioned, and archived using TCGA Expedition supports command line access at high-performance computing facilities as well as some functionality with third party tools. For a subset of TCGA data collected at University of Pittsburgh, we also re-associate TCGA data with de-identified data from the electronic health records. Here we describe the software as well as the architecture of our repository, methods for loading of TCGA data to multiple platforms, and security and regulatory controls that conform to federal best practices. Results: TCGA Expedition software consists of a set of scripts written in Bash, Python and Java that download, extract, harmonize, version and store all TCGA data and metadata. The software generates a versioned, participant- and sample-centered, local TCGA data directory with metadata structures that directly reference the local data files as well as the original data files. The software supports flexible searches of the data via a web portal, user-centric data tracking tools, and data provenance tools. Using this software, we created a collaborative repository, the Pittsburgh Genome Resource Repository (PGRR) that enabled investigators at our institution to work with all TCGA data formats, and to interrogate these data with analysis pipelines, and associated tools. WGS data are especially challenging for individual investigators to use, due to issues with downloading, storage, and processing; having locally accessible WGS BAM files has proven invaluable. Conclusion: Our open-source, freely available TCGA Expedition software can be used to create a local collaborative infrastructure for acquiring, managing, and analyzing TCGA data and other large public datasets. [ABSTRACT FROM AUTHOR]