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Jeremy M Berg

Publications and source records attributed to Jeremy M Berg.

18 recordsLinked to original sources
PubMed2004

Entropy-enthalpy compensation in ionic interactions probed in a zinc finger peptide.

Zinc(II) and cobalt(II) binding to a series of zinc finger peptides with different charged residue pairs across from one another in a beta-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.

Calorimetry↗
PubMed2004

Pex5p binding affinities for canonical and noncanonical PTS1 peptides.

The majority of proteins targeted to the peroxisomal lumen contain a C-terminal peroxisomal targeting signal-1 (PTS1) that is bound by the peroxin Pex5p. The PTS1 is generally regarded as a C-terminal tripeptide that adheres to the consensus (S/A/C)(K/R/H)(L/M). Previously, we studied the binding affinity of peptides of the form YQX(-3)X(-2)X(-1) to the peptide-binding domain of human Pex5p (referred to as Pex5p-C). Optimal affinity was found for YQSKL, which bound with an affinity of 200 +/- 40 nM. To extend this work, we investigated the properties of a peptide containing the last 9 residues of acyl-CoA oxidase (RHYLKPLQSKL) and discovered that it binds to Pex5p-C with a dissociation constant of 1.4 +/- 0.4 nM, 180 times tighter than YQSKL. Further analysis revealed that the enhanced affinity is primarily due to the presence of leucine in the (-5) position. In addition, a peptide corresponding to the luciferase C-terminus (YKGGKSKL) was found to bind Pex5p-C about 20 times tighter than YQSKL. The majority of this effect results from having lysine in position (-4). Catalase contains a noncanonical PTS1 (-AREKANL). The affinity of YQANL was found to be 3600 +/- 400 nM. This relatively weak binding is consistent with previous unsuccessful attempts to direct chloramphenicol acetyltransferase to the peroxisome by fusing -ANL to its C-terminus (-GGA-ANL). The peptides YKANL, YEKANL, YREKANL, and YAREKANL all bound Pex5p-C with higher affinities than did YQANL, but the affinities are still lower than peptides that correspond to functional targeting signals in other contexts. Because both catalase and Pex5p are tetramers (as opposed to the monomeric Pex5p-C and the peptides used in our studies), multidentate effects on binding affinity between Pex5p and other oligomeric proteins should be considered. Our study provides direct thermodynamic data revealing that peptide binding to Pex5p-C binding is favored by lysine in the (-4) position and leucine in the (-5) position. Our results suggest that peptides or proteins with optimized residues in the (-4) and/or (-5) positions can bind to Pex5p with affinities that are at least two orders of magnitude greater than that of YQSKL, and that this stabilization can compensates for otherwise weakly binding PTS1s.

Acyl-CoA Oxidase↗
PubMed2004

Metal ion affinities of the zinc finger domains of the metal responsive element-binding transcription factor-1 (MTF1).

Metal response element (MRE) binding transcription factor-1 (MTF1) is a six Cys(2)His(2) 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 microM for cobalt(II) and 31 +/- 14 pM 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 microM for cobalt(II). The domains fall into two sets with peptides corresponding to domains 2, 3, and 4 showing relatively high affinity (K(d)(Co(II)) 0.3-0.5 microM) and peptides corresponding to domains 1, 5, and 6 showing lower affinity (K(d)(Co(II)) 1.6-1.7 microM). 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 microM. 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 approximately 5 > 6 approximately 3 approximately 1. These results reveal that none of the Cys(2)His(2) 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 factor of 25 with domains at both the amino- and carboxyl-termini showing lower intrinsic affinities for metal ions than the central domains.

Amino Acid Sequence↗
PubMed2004

Reduction in DNA-binding affinity of Cys2His2 zinc finger proteins by linker phosphorylation.

Cys(2)His(2) 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 Cys(2)His(2) 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 approximately 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.

Amino Acid Sequence↗
PubMed2004

Solution structure of a CCHHC domain of neural zinc finger factor-1 and its implications for DNA binding.

The structure of a CCHHC zinc-binding domain from neural zinc finger factor-1 (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(4)-Cys-X(4)-His-X(7)-His-X(5)-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 Gln 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.

Amino Acid Motifs↗
PubMed2003

Kinetics and thermodynamics of copper(II) binding to apoazurin.

The binding of copper(II) to apoazurin has been probed by isothermal titration calorimetry in cholamine buffer at pH 7.0. The standard enthalpy change was determined to be -10.0 +/- 1.4 kcal/mol. Each calorimetric trace reveals an initial exothermic phase followed by an endothermic phase. The calorimetric data could be fit to a kinetic model involving a bimolecular combination of copper(II) and apoazurin in an exothermic process (k = 2 +/-1 x 103 M-1 s-1, DeltaH degrees = -19 +/- 3 kcal/mol) to form an intermediate that spontaneously converts to Cu(II)-azurin in an endothermic process (k = 0.024 +/- 0.01 s-1, DeltaH degrees = +9 +/- 3 kcal/mol). These data suggest that copper(II) first combines with apoazurin in an irreversible process to form an intermediate that converts to copper(II)-azurin in a process driven by the release of water. The overall standard free energy of copper(II) binding to apoazurin is estimated to be -18.8 kcal/mol.

Apoproteins↗
PubMed2003

Expanding the DNA-recognition repertoire for zinc finger proteins beyond 20 amino acids.

The design of DNA binding domains based on the Cys2His2 zinc finger motif has proven to be a successful strategy for the specific recognition of novel DNA sequences. Although considerable effort has been devoted to the generation of zinc finger proteins with widely varying DNA-binding preferences, only a limited number of potential DNA binding sites have been targeted with a high degree of specificity. These restrictions on zinc finger design appear to be a consequence of the limited repertoire of side-chain lengths and functionalities available with the 20 proteinogenic amino acids. To demonstrate that these limitations can be overcome through the use of "unnatural" amino acids, expressed protein ligation was employed to incorporate the amino acid citrulline into a single position within a three-zinc finger protein. As anticipated, the resulting semisynthetic protein specifically recognizes adenine in the appropriate position of its binding site.

Amino Acids↗
PubMed2003

Selective RNA binding by a single CCCH zinc-binding domain from Nup475 (Tristetraprolin).

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 elements 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 microM and discriminates against other sequences lacking the central A or the flanking U residues. These results demonstrate for the first 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.

Anisotropy↗
PubMed2003

Correlating structure and affinity for PEX5:PTS1 complexes.

Many proteins that are destined to reside within the lumen of the peroxisome contain the peroxisomal targeting signal-1 (PTS1), a C-terminal tripeptide approximating the consensus sequence -Ser-Lys-Leu-COO(-). The PTS1 is recognized by the tetratricopeptide repeat (TPR) domains of PEX5, a cytosolic receptor that cycles between the cytoplasm and the peroxisome. To gain insight into the energetics of PTS1 binding specificity and to correlate these with features from the recently determined structure of a PEX5:PTS1 complex, we used a fluorescence-based binding assay that enables the quantitation of the dissociation constants for PTS1-containing peptide complexes with the TPR region of human PEX5. Through application of this assay to a collection of pentapeptides containing different C-terminal tripeptide sequences, including both natural and unnatural amino acids, the thermodynamic effects of sequence variation were examined. PTS1 variants that correspond to known functional targeting signals bind to the PEX5 fragment with a change in the standard binding free energy within 1.8 kcal mol(-1) of that corresponding to the peptide ending with -Ser-Lys-Leu-COO(-). The results suggest that a binding energy threshold may determine the functionality of PTS1 sequences.

Amino Acid Substitution↗
PubMed2003

A Cys3His zinc-binding domain from Nup475/tristetraprolin: a novel fold with a disklike structure.

Nup475 (also known as tristetraprolin and TIS11) includes two zinc-binding domains of the form Cys-X8-Cys-X5-Cys-X3-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 disk-like with a diameter of approximately 25 A and a width of approximately 12 A. This structure provides a basis for evaluating the role of individual residues for structural stability and for nucleic acid binding.

Amino Acid Sequence↗
PubMed2003

Nonrandom tripeptide sequence distributions at protein carboxyl termini.

The availability of complete genome sequences enables the statistical analysis of sequence features without significant database-imposed bias. The carboxyl termini of proteins often contain regions associated with protein targeting and enhanced translational termination. We analyzed the frequency of occurrence of C-terminal tripeptides in representative archaeal, bacterial, and eukaryotic genomes. The sequence distribution in prokaryotic genomes nearly matches that generated by the randomization of the observed tripeptide set. In contrast, eukaryotic genomes contain large numbers of overrepresented sequences. Some of these correspond to highly repeated sequences from either duplicated endogenous genes or transposon open reading frames. Gratifyingly, others represent previously known targeting signals or sequences associated with an increase in translational termination efficiency. However, a number of overrepresented tripeptides have not been previously noted and may represent novel functional sequences. For example, the sequence XSS may enhance translational termination efficiency in plants, whereas FWC may be a targeting or processing signal for certain amino acid permeases in yeast.

Amino Acid Motifs↗
PubMed2002

Structure-based thermodynamic analysis of a coupled metal binding-protein folding reaction involving a zinc finger peptide.

The thermodynamics of metal binding by the prototypical Cys(2)His(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 DeltaH degrees (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.

Calorimetry↗
PubMed2002

PEX5 binds the PTS1 independently of Hsp70 and the peroxin PEX12.

Most peroxisomal enzymes are targeted to peroxisomes by virtue of a type-1 peroxisomal targeting signal (PTS1) at their extreme C terminus. PEX5 binds the PTS1 through its C-terminal 40-kDa tetratricopeptide repeat domain and is essential for import of PTS1-contining proteins into peroxisomes. Here we examined the PTS1-binding activity of purified, recombinant, full-length PEX5 using a fluorescence anisotropy-based assay. Like its C-terminal fragment, full-length tetrameric PEX5 exhibits high intrinsic affinity for the PTS1, with a K(d) of 35 nm for the peptide lissamine-Tyr-Gln-Ser-Lys-Leu-COO(-). The specificity of this interaction was demonstrated by the fact that PEX5 had no detectable affinity for a peptide in which the Lys was replaced with Glu, a substitution that inactivates PTS1 signals in vivo. Hsp70 has been found to regulate the affinity of PEX5 for a PTS1-containing protein, but we found that the kinetics of PEX5-PTS1 binding was unaffected by Hsp70, Hsp70 plus ATP, or Hsp70 plus ADP. In addition, we found that another protein known to interact with the PTS1-binding domain of PEX5, the PEX12 zinc RING domain, also had no discernable effect on PEX5-PTS1 binding kinetics. Taken together, these results suggest that the initial step in peroxisomal protein import, the recognition of enzymes by PEX5, is a relatively simple process and that Hsp70 most probably stimulates this process by catalyzing the folding of newly synthesized peroxisomal enzymes and/or enhancing the accessibility of their PTS1.

Adenosine Triphosphate↗
PubMed2002

Building a metal binding domain, one half at a time.

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.

Carrier Proteins↗