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S R Holbrook

Publications and source records attributed to S R Holbrook.

36 records · Page 2Linked to original sources
PubMed1990

Predicting surface exposure of amino acids from protein sequence.

The amino acid residues on a protein surface play a key role in interaction with other molecules, determined many physical properties, and constrain the structure of the folded protein. A database of monomeric protein crystal structures was used to teach computer-simulated neural networks rules for predicting surface exposure from local sequence. These trained networks are able to correctly predict surface exposure for 72% of residues in a testing set using a binary model, (buried/exposed) and for 54% of residues using a ternary model (buried/intermediate/exposed). In the ternary model, only 11% of the exposed residues are predicted as buried and only 5% of the buried residues are predicted as exposed. Also, since the networks are able to predict exposure with a quantitative confidence estimate, it is possible to assign exposure for over half of the residues in a binary model with greater than 80% accuracy. Even more accurate predictions are obtained by making a consensus prediction of exposure for a homologous family. The effect of the local environment of an amino acid on its accessibility, though smaller than expected, is significant and accounts for the higher success rate of prediction than obtained with previously used criteria. In the absence of a three-dimensional structure, the ability to predict surface accessibility of amino acids directly from the sequence is a valuable tool in choosing sites of chemical modification or specific mutations and in studies of molecular interaction.

Amino Acid Sequence↗
PubMed1990

Prediction of the disulfide-bonding state of cysteine in proteins.

The bonding states of cysteine play important functional and structural roles in proteins. In particular, disulfide bond formation is one of the most important factors influencing the three-dimensional fold of proteins. Proteins of known structure were used to teach computer-simulated neural networks rules for predicting the disulfide-bonding state of a cysteine given only its flanking amino acid sequence. Resulting networks make accurate predictions on sequences different from those used in training, suggesting that local sequence greatly influences cysteines in disulfide bond formation. The average prediction rate after seven independent network experiments is 81.4% for disulfide-bonded and 80.0% for non-disulfide-bonded scenarios. Predictive accuracy is related to the strength of network output activities. Network weights reveal interesting position-dependent amino acid preferences and provide a physical basis for understanding the correlation between the flanking sequence and a cysteine's disulfide-bonding state. Network predictions may be used to increase or decrease the stability of existing disulfide bonds or to aid the search for potential sites to introduce new disulfide bonds.

Amino Acid Sequence↗
PubMed1989

Molecular model of the G protein alpha subunit based on the crystal structure of the HRAS protein.

A structural model of guanine nucleotide-binding regulatory protein alpha subunits (G alpha subunits) is proposed based on the crystal structure of the catalytic domain of the human HRAS protein (p21ras). Because of low overall sequence similarity, structural and functional constraints were used to align the G alpha consensus sequence with that of p21ras. The resulting G alpha model specifies the spatial relationship among the guanine nucleotide-binding site, the binding site of the beta gamma subunit complex, likely regions of effector and receptor interaction, and sites of cholera and pertussis toxin modification. The locations in the model of the experimentally determined sites of proteolytic digestion, point mutation, monoclonal antibody binding, and toxin modification are consistent with and help explain the observed biological activity. Two important findings from our model are (i) the orientation of the G alpha model with respect to the membrane and (ii) the identification of the spatial proximity of the N- and C-terminal regions. Furthermore, by analogy to p21ras, the model assigns specific residues in G alpha required for binding the guanosine (G-box) and phosphates (PO4-box) and identifies residues potentially involved in the conformational switch mechanism (S-box). Specification of these critical regions in the G alpha model suggests guidelines for construction of mutants and chimeric proteins to experimentally test structural and functional hypotheses.

Adenosine Diphosphate Ribose↗
PubMed1988

Local mobility of nucleic acids as determined from crystallographic data. III. A daunomycin-DNA complex.

The local mobility of the complex between the anti-tumor drug daunomycin and a DNA hexanucleotide duplex of sequence d(CpGpTpApCpG)2 has been determined by anisotropic refinement of single crystal X-ray diffraction data of 1.2 A resolution (1 A = 0.1 nm). The directions and amplitudes of the local motion indicate that changes in mobility of DNA due to daunomycin binding are primarily limited to the residues forming the intercalation site and do not propagate to the neighboring residues. The intercalated daunomycin ring system (aglycone) is rigidly fixed in the base stack, apparently serving as an anchor for the amino sugar segment of the drug which is one of the most mobile regions of the entire complex. The high flexibility of this amino sugar may be important for inhibition of replication and transcription not only by sterically blocking the minor groove, but also by allowing nonproductive interactions to be formed with various polymerases or other DNA-binding proteins. The crystallographic model is improved sufficiently by the rigid group anisotropic refinement to allow additional bound water molecules to be located.

Chemical Phenomena↗
PubMed1987

Mechanism of damage recognition by Escherichia coli DNA photolyase.

Escherichia coli DNA photolyase binds to DNA containing pyrimidine dimers with high affinity and then breaks the cyclobutane ring joining the two pyrimidines of the dimer in a light- (300-500 nm) dependent reaction. In order to determine the structural features important for this level of specificity, we have constructed a 43 base pair (bp) long DNA substrate that contains a thymine dimer at a unique location and studied its interaction with photolyase. We find that the enzyme protects a 12-16-bp region around the dimer from DNase I digestion and only a 6-bp region from methidium propyl-EDTA-Fe (II) digestion. Chemical footprinting experiments reveal that photolyase contacts the phosphodiester bond immediately 5' and the 3 phosphodiester bonds immediately 3' to the dimer but not the phosphodiester bond between the two thymines that make up the dimer. Methylation protection and interference experiments indicate that the enzyme makes major groove contacts with the first base 5' and the second base 3' to the dimer. These data are consistent with photolyase binding in the major groove over a 4-6-bp region. However, major groove contacts cannot be of major significance in substrate recognition as the enzyme binds equally well to a thymine dimer in a 44-base long single strand DNA and protects a 10-nucleotide long region around the dimer from DNase I digestion. It is therefore concluded that the unique configuration of the phosphodiester backbone in the strand containing the pyrimidine dimer, as well as the cyclobutane ring of the dimer itself are the important structural determinants of the substrate for recognition by photolyase.

Base Sequence↗
PubMed1986

Local mobility of nucleic acids as determined from crystallographic data. II. Z-form DNA.

Directions and magnitudes of the local mobility of the Z-DNA hexamer duplex CpGpCpGpCpG have been determined by crystallographic refinement of anisotropic displacement parameters using the observed X-ray diffraction data. The cytidine and guanosine residues demonstrate different modes of mobility, implying that a dinucleotide is the smallest repeating unit in terms of flexibility as well as structure. Directions of librational and translational mobility of the cytidine and guanosine residues of Z-DNA are similar to those observed for the same nucleotides in B-DNA. This suggests that the local mobility of DNA is primarily determined by the individual nucleotide type and by the constraints of Watson-Crick base-pairing, rather than by helical form. Differences in the magnitudes of mobility may be responsible for some of the different physical properties of B-DNA and Z-DNA. The B to Z transition is discussed in terms of the observed flexibilities of these two helical forms.

Base Sequence↗
PubMed1986

Action mechanism of ABC excision nuclease on a DNA substrate containing a psoralen crosslink at a defined position.

Many carcinogenic as well as chemotherapeutic agents cause covalent linkages between complementary strands of DNA. If unrepaired, DNA crosslinks are blocks to DNA replication and transcription and therefore represent potentially lethal lesions to the cell. Genetic studies of Escherichia coli have demonstrated that the repair enzyme ABC excision nuclease, coded for by the three unlinked genes, uvrA, uvrB, and uvrC, plays a crucial role in DNA crosslink repair. To study the molecular events of ABC excision nuclease-mediated crosslink repair, we have engineered a DNA fragment with a psoralen-DNA interstrand crosslink at a defined position, digested this substrate with pure enzyme, and analyzed the reaction products on DNA sequencing gels. We find that the excision nuclease cuts only one of the two strands involved in the crosslink, incises the crosslink by hydrolyzing the ninth phosphodiester bond 5' and the third phosphodiester bond 3' to the furan-side thymine of the crosslink, and does not produce double-strand breaks at any significant level. Based on these data, we present a model by which ABC excision nuclease initiates crosslink repair in vivo.

DNA↗
PubMed1986

Neutral reactions of haloacetaldehydes with polynucleotides: mechanisms, monomer and polymer products.

The generally accepted mechanism for the formation of etheno derivatives upon reaction of adenosine or cytidine with haloacetaldehydes involves two intermediates. The first, a primary addition to the exocyclic amino group, has not been experimentally verified. The second, a cyclic form of the first intermediate, has been described in monomers but presumed to be too unstable to exist in polynucleotides since such derivatives would be readily dehydrated to other derivatives at pHs below neutrality. We have found that the cyclic intermediates of adenosine and cytidine are the predominant products in polynucleotides, even upon extensive reaction with chloroacetaldehyde at neutrality. The hydrated compounds have half-lives at pH 7, 37 degrees C, of 1.4 h and 13 h for adenosine and cytidine, respectively. Two types of evidence are presented for the existence of the first intermediate, a (1-hydroxy-2-chloroethyl)-substituted exocyclic amino group. Firstly, poly d[A-T] cannot form etheno derivatives (except when denatured) and the observed cross-linking is therefore attributed to alkylation by the chlorinated sidechain of the adenine residue (A), acting on the N6 of A on the opposite strand. Secondly, our results show that blocking of the acceptor nitrogen, needed for cyclization, leads to the formation of relatively stable derivatives of adenosine and cytidine. Guanosine, as a monomer, is modified extensively, but in synthetic polymers no reaction was detected, possibly due to secondary structure.

Acetaldehyde↗
PubMed1985

Molecular models for DNA damaged by photoreaction.

Structural models of a DNA molecule containing a radiation-induced psoralen cross-link and of a DNA containing a thymine photodimer were constructed by applying energy-minimization techniques and model-building procedures to data from x-ray crystallographic studies. The helical axes of the models show substantial kinking and unwinding at the sites of the damage, which may have long-range as well as local effects arising from the concomitant changes in the supercoiling and overall structure of the DNA. The damaged areas may also serve as recognition sites for repair enzymes. These results should help in understanding the biologic effects of radiation-induced damage on cells.

Animals↗
PubMed1984

Local mobility of nucleic acids as determined from crystallographic data. I. RNA and B form DNA.

The local mobility of DNA and RNA can be described well by a segmented rigid body model in which the phosphates, riboses and bases are treated as independent groups. We have developed a computer program for extracting information about the translational and rotational mobility of these groups from X-ray diffraction data of single crystals of DNA and RNA fragments. We plan to extend our studies from the B DNA helix studied here to A and Z form helices for which diffraction data are available. The mobilities described here may be important in allowing the flexibility necessary for interaction of nucleic acids with proteins, ligands or other nucleic acids, and are crucial to our understanding of the factors governing both structural stability and motion of nucleic acids on a larger scale.

DNA↗
PubMed1981

Absence of correlation between base-pair sequence and RNA conformation.

A survey of all available double-stranded RNA crystal structures shows that there is a considerable range of variation in local conformation of a given base-pair doublet, but that there is no significant correlation between base-pair sequence and RNA local conformation.

Base Composition↗
PubMed1977

RNA-ligant interactions. (I) Magnesium binding sites in yeast tRNAPhe.

X-ray crystallagraphic studies studies indicate that there are at least four site-specifically bound hydrated Mg2+ ions, [Mg(H2O)n]2+, in yeast tRNAPhe. The size and the octahedral coordination geometry, rather than the charge, of [Mg(H2O)N]2+ appear to be the primary reasons for the specificity of magnesium ions in site-binding and in the stabilization of the tertiary structure of tRNA.

Binding Sites↗