PubMed Health⌕ Search

Biomedical subjects

C O Pabo

Publications and source records attributed to C O Pabo.

At least 73 records · Page 4Linked to original sources
PubMed1988

Tat protein from human immunodeficiency virus forms a metal-linked dimer.

Tat, the transactivating protein from HIV, forms a metal-linked dimer with metal ions bridging cysteine-rich regions from each monomer. This novel arrangement is distinct from the "zinc finger" domain observed in other eukaryotic regulatory proteins. Ultraviolet absorption spectra show that Tat binds two Zn2+ or two Cd2+ ions per monomer, and electrophoresis of the Tat-metal complexes demonstrates that the protein forms metal-linked dimers. Partial proteolysis and circular dichroism spectra suggest that metal binding has its primary effects in the cysteine-rich region and relatively little effect on the folding of other regions. These results suggest new directions for biological studies and new approaches to drug design.

Cadmium↗
PubMed1988

Dimerization of the tat protein from human immunodeficiency virus: a cysteine-rich peptide mimics the normal metal-linked dimer interface.

We have synthesized an 18-amino acid peptide that contains the cysteine-rich region of the tat protein from human immunodeficiency virus. Previous experiments in vitro with the intact tat protein have shown that these cysteines serve as metal ligands, causing tat to form metal-linked dimers. Ultraviolet absorption spectra show that the synthetic peptide (tat21-38) binds two Cd2+ or two Zn2+ ions per peptide monomer, and some changes in the circular dichroism spectra are seen as the metals bind. The peptide-metal complexes are completely resistant to proteolytic digestion, and mass spectrometry demonstrates that this peptide forms metal-linked dimers. The peptide can also combine with the intact tat protein to form metal-linked heterodimers. If these heterodimers are unable to trans-activate viral transcription, tat21-38 could be a lead compound for designing drugs to treat acquired immunodeficiency syndrome.

Amino Acid Sequence↗
PubMed1987

Dimerization of the operator binding domain of phage lambda repressor.

Dimerization of lambda repressor is required for its binding to operator DNA. As part of a continuing study of the structural basis of the coupling between dimer formation and operator binding, we have undertaken 1H NMR and gel filtration studies of the dimerization of the N-terminal domain of lambda repressor. Five protein fragments have been studied: three are wild-type fragments of different length (1-102, 1-92, and 1-90), and two are fragments bearing single amino acid substitutions in residues involved in the dimer interface (1-102, Tyr-88----Cys; 1-92, Ile-84----Ser). The tertiary structure of each species is essentially the same, as monitored by the 1H NMR resonances of internal aromatic groups. However, significant differences are observed in their dimerization properties. 1H NMR resonances of aromatic residues that are involved in the dimer contact allow the monomer-dimer equilibrium to be monitored in solution. The structure of the wild-type dimer contact appears to be similar to that deduced from X-ray crystallography and involves the hydrophobic packing of symmetry-related helices (helix 5) from each monomer. Removal of two contact residues, Val-91 and Ser-92, by limited proteolysis disrupts this interaction and also prevents crystallization. The Ile-84----Ser substitution also disrupts this interaction, which accounts for the severely reduced operator affinity of this mutant protein.

Amino Acids↗
PubMed1987

Metal-dependent folding of a single zinc finger from transcription factor IIIA.

A 30-amino acid peptide, which corresponds to the second "zinc finger" domain of transcription factor IIIA, has been synthesized and purified. This peptide folds in the presence of zinc: adding Zn2+ significantly changes the circular dichroism spectrum, and Zn2+ protects the peptide from tryptic digestion. The peptide also binds Co2+, and the absorption spectrum of the Co2+ complex suggests that a tetrahedral binding site is formed by two cysteines and two histidines. Experiments at higher temperatures (60-75 degrees C) suggest that these folded metal-peptide complexes are quite thermostable. The peptide shows some sequence-specific effects in DNase and methylation protection experiments. However, it does not give a clear "footprint," and some effects are observed in the absence of added zinc.

Amino Acid Sequence↗
PubMed1986

Computer-aided model-building strategies for protein design.

Model-building strategies for protein modification and design are developed. These strategies emphasize simple geometric aspects of protein structure, use local coordinate systems defined at particular residues, and systematically consider a large number of alternative sequences and conformations. We have written a computer program, PROTEUS, to implement these search methods. PROTEUS has been used to find positions where disulfide bonds could be added to the N-terminal domain of the lambda repressor and to predict how a loop on the surface of repressor could be shortened.

Computer Simulation↗
PubMed1986

An engineered intersubunit disulfide enhances the stability and DNA binding of the N-terminal domain of lambda repressor.

Site-directed mutagenesis has been used to replace Tyr-88 at the dimer interface of the N-terminal domain of lambda repressor with cysteine. Computer model building had suggested that this substitution would allow formation of an intersubunit disulfide without disruption of the dimer structure [Pabo, C. O., & Suchanek, E. G. (1986) Biochemistry (preceding paper in this issue)]. We find that the Cys-88 protein forms a disulfide-bonded dimer that is very stable to reduction by dithiothreitol and has increased operator DNA binding activity. The covalent Cys88-Cys88' dimer is also considerably more stable than the wild-type protein to thermal denaturation or urea denaturation. As a control, Tyr-85 was replaced with cysteine. A Cys85-Cys85' disulfide cannot form without disrupting the wild-type structure, and we find that this disulfide bond reduces the DNA binding activity and stability of the N-terminal domain.

Amino Acid Sequence↗
PubMed1985

Systematic variation in DNA length yields highly ordered repressor-operator cocrystals.

Crystals have been grown that contain the operator-binding domain of the lambda repressor and the lambda operator site OL1. Crystallization conditions were tested with a set of DNA fragments, ranging in length from 17 to 23 base pairs. The best crystals were grown with a 20-base pair DNA fragment. These crystals have space-group symmetry P2I, with unit cell dimensions a = 37.1 A, b = 68.8 A, c = 56.8 A, and a beta angle of 91.5 degrees. They diffracted to at least 2.5 A resolution. High resolution data from these crystals should allow the direct determination of how a repressor recognizes its operator site.

Base Sequence↗
PubMed1985

Crystallization of the Arc repressor.

Arc, a repressor from Salmonella phage P22 has been crystallized from ammonium phosphate at pH 8.0. The crystals form in space group P212121 with a = 90.26 A, b = 52.88 A and c = 47.58 A. The crystals diffract to 2.2 A resolution.

Crystallography↗
PubMed1984

Protein-DNA recognition.

Several general principles emerge from the studies of Cro, lambda repressor, and CAP. The DNA-binding sites are recognized in a form similar to B-DNA. They do not form cruciforms or other novel DNA structures. There seem to be proteins that bind left-handed Z-DNA (87) and DNA in other conformations, but it remains to be seen how these structures are recognized or how proteins recognize specific sequences in single-stranded DNA. Cro, repressor, and CAP use symmetrically related subunits to interact with two-fold related sites in the operator sequences. Many other DNA-binding proteins are dimers or tetramers and their operator sequences have approximate two-fold symmetry. It seems likely that these proteins will, like Cro, repressor, and CAP, form symmetric complexes. However, there is no requirement for symmetry in protein-DNA interactions. Some sequence-specific DNA-binding proteins, like RNA polymerase, do not have symmetrically related subunits and do not bind to symmetric recognition sequences. Cro, repressor, and CAP use alpha-helices for many of the contacts between side chains and bases in the major groove. An adjacent alpha-helical region contacts the DNA backbone and may help to orient the "recognition" helices. This use of alpha-helical regions for DNA binding appears to be a common mode of recognition. Most of the contacts made by Cro, repressor, and CAP occur on one side of the double helix. However, lambda repressor contacts both sides of the double helix by using a flexible region of protein to wrap around the DNA. Recognition of specific base sequences involves hydrogen bonds and van der Waals interactions between side chains and the edges of base pairs. These specific interactions, together with backbone interactions and electrostatic interactions, stabilize the protein-DNA complexes. The current models for the complexes of Cro, repressor, and CAP with operator DNA are probably fundamentally correct, but it should be emphasized that model building alone, even when coupled with genetic and biochemical studies, cannot be expected to provide a completely reliable "high-resolution" view of the protein-DNA complex. For example, the use of standard B-DNA geometry for the operator is clearly an approximation.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1983

Comparison of the structures of cro and lambda repressor proteins from bacteriophage lambda.

The three-dimensional structures of cro repressor protein and of the amino-terminal domain of lambda repressor protein, both from bacteriophage lambda, are compared. The second and third alpha-helices, alpha 2 and alpha 3, are shown to have essentially identical conformations in the two proteins, confirming the significance of the amino acid sequence homology previously noted between these and other DNA binding proteins in the region corresponding to these helices. The correspondence between the two-helical units in cro and lambda repressor protein is better than the striking agreement noted previously between two-helical units in cro and catabolite gene-activator protein. Parts of the first alpha-helices of repressor and cro show a structural correspondence that suggests a revised sequence homology between the two proteins in their extreme amino-terminal regions. In particular, there is a short loop between the alpha 1 and alpha 2 helices of lambda repressor that is missing from cro. This structural difference may account for the observed differences found with different cros and repressors in the pattern of phosphates whose ethylation prevents the binding of these proteins to their specific recognition sites. Although the two proteins have strikingly similar alpha 2-alpha 3 helical units that are presumed to bind to DNA in an essentially similar manner, stereochemical restrictions prevent the alpha 2-alpha 3 units of the respective proteins aligning on the DNA in exactly the same way.

Amino Acid Sequence↗
PubMed1983

Systematic analysis of possible hydrogen bonds between amino acid side chains and B-form DNA.

The ways in which amino acid side chains could make a pair of hydrogen bonds within the major groove of B DNA are systematically analyzed. Hydrogen bond donors within the major groove are characterized by determining the idealized position of the hydrogen bond acceptors that they might bond with. It appears that an amino acid side chain could, at most, contact a pair of base pairs. The ten possible pairs of base pairs are analyzed to determine how they could be recognized by the amino acid side chains.

Amino Acids↗
PubMed1982

The N-terminal arms of lambda repressor wrap around the operator DNA.

From differences in the ability of fragments of the lambda repressor to protect operator bases from chemical modification we suggest that the first few N-terminal residues of the lambda repressor form an extended arm that reaches around the back of the DNA helix when repressor binds to the operator.

Amino Acid Sequence↗
PubMed1982

The operator-binding domain of lambda repressor: structure and DNA recognition.

The structure of the operator-binding domain of the lambda repressor has been determined at 3.2 A resolution. This domain contains an extended N-terminal arm and five alpha-helices. Model-building studies of the repressor-operator complex suggest that alpha-helices, especially the N-terminal parts of these helices, may provide a useful surface for protein-DNA interactions.

Amino Acid Sequence↗
PubMed1982

Homology among DNA-binding proteins suggests use of a conserved super-secondary structure.

The amino acid sequences of the repressor and cro proteins of phages lambda, 434 and P22 are homologous, especially in a region in which repressor and lambda cro have a similar alpha-helix-turn-alpha-helix secondary structure. Model-building studies indicate that this structure is important in DNA binding, and we suggest it may be a common feature of many DNa-binding proteins.

Amino Acid Sequence↗