Biomedical subjects

P L de Haseth

Publications and source records attributed to P L de Haseth.

8 recordsLinked to original sources

PubMed1992

Differences in the amino acid distributions of 3(10)-helices and alpha-helices.

Local determinants of 3(10)-helix stabilization have been ascertained from the analysis of the crystal structure data base. We have clustered all 5-length substructures from 51 nonhomologous proteins into classes based on the conformational similarity of their backbone dihedral angles. Several clusters, derived from 3(10)-helices and multiple-turn conformations, had strong amino acid sequence patterns not evident among alpha-helices. Aspartate occurred over twice as frequently in the N-cap position of 3(10)-helices as in the N-cap position of alpha-helices. Unlike alpha-helices, 3(10)-helices had few C-termini ending in a left-handed alpha conformation; most 3(10) C-caps adopted an extended conformation. Differences in the distribution of hydrophobic residues among 3(10)- and alpha-helices were also apparent, producing amphipathic 3(10)-helices. Local interactions that stabilize 3(10)-helices can be inferred both from the strong amino acid preferences found for these short helices, as well as from the existence of substructures in which tertiary interactions replace consensus local interactions. Because the folding and unfolding of alpha-helices have been postulated to proceed through reverse-turn and 3(10)-helix intermediates, sequence differences between 3(10)- and alpha-helices can also lend insight into factors influencing alpha-helix initiation and propagation.

Amino Acid Sequence↗

PubMed1990

A common pentapeptide conformation occurs in viral acid proteases and other proteins.

We found a pentapeptide conformation, termed a type I twist, which has a strikingly high propensity (56%) for aspartic acid in the first position. Type I twists include the active site loops from cellular and viral aspartic proteases, with the catalytic Asp in the first position. Fifteen other type I twists, from non-homologous proteins, were found among high-resolution structures in the Protein Data Bank using a comparison method based on main-chain torsion angles. We propose that the Asp affects electrostatic interactions and thus plays a major structural role in the formation of this recurring motif, in addition to its catalytic role in the aspartic proteases.

Amino Acid Sequence↗

PubMed1989

Comparing short protein substructures by a method based on backbone torsion angles.

An efficient algorithm was characterized that determines the similarity in main chain conformation between short protein substructures. The algorithm computes delta t, the root mean square difference in phi and psi torsion angles over a small number of amino acids (typically 3-5). Using this algorithm, large numbers of protein substructure comparisons were feasible. The parameter delta t was sensitive to variations in local protein conformation, and it correlates with delta r, the root mean square deviation in atomic coordinates. Values for delta t were obtained that define similarity thresholds, which determine whether two substructures are considered structurally similar. To set a lower bound on the similarity threshold, we estimated the component of delta t due to measurement noise from comparisons of independently refined coordinates of the same protein. A sample distribution of delta t from nonhomologous protein comparisons identified an upper bound on the similarity threshold, one that refrains from incorporating large numbers of nonmatching comparisons. Unlike methods based on C alpha atoms alone, delta t was sensitive to rotations in the peptide plane, shown to occur in several proteins. Comparisons of homologous proteins by delta t showed that the active site torsion angles are highly conserved. The delta t method was applied to the alpha-chain of human hemoglobin, where it readily demonstrated the local differences in the structures of different ligation states.

Algorithms↗

PubMed1984

Failure of translational repression in the phage f2 op3 mutant is not due to an altered coat protein-RNA interaction.

A secondary phenotype of the op3 mutant of RNA bacteriophage f2 is the absence of translational repression of the phage replicase gene by the phage coat protein. We have synthesized RNA fragments corresponding to the site of translational repression for both the wild type and the op3 mutant. Using a quantitative assay, we show that the affinity of the closely related R17 coat protein for the mutant and wild type RNA fragments is the same. In addition, we find that the op3 and R17 coat proteins bind to the wild type RNA fragment with essentially identical dissociation constants. Thus, the altered regulation of replicase protein synthesis in the op3 mutant does not appear to be due simply to a reduced affinity of the translational repressor for its target site.

Bacteriophages↗

PubMed1983

Sequence-specific interaction of R17 coat protein with its ribonucleic acid binding site.

The interaction between phage R17 coat protein and its RNA binding site for translational repression was studied as an example of a sequence-specific RNA--protein interaction. Nuclease protection and selection experiments define the binding site to about 20 contiguous nucleotides which form a hairpin. A nitrocellulose filter retention assay is used to show that the binding between the coat protein and a synthetic 21-nucleotide RNA fragment conforms to a simple bimolecular reaction. Unit stoichiometry and a Kd of about 1 nM are obtained at 2 degrees C in buffer containing 0.19 M salt. The interaction is highly sequence specific since a variety of RNAs failed to compete with the 21-nucleotide fragment for coat protein binding.

Amino Acid Sequence↗

PubMed1982

Enzymatic synthesis of a 21-nucleotide coat protein binding fragment of R17 ribonucleic acid.

An oligoribonucleotide with a sequence identical with the bacteriophage R17 replicase initiator region has been synthesized. The sequence also encompasses the binding domain of R17 coat protein, which is known to act as a translational repressor at this site. The 21-nucleotide fragment was synthesized entirely by enzymatic methods, T4 RNA ligase being used to join shorter oligomers. The resulting fragment has a secondary structure with the expected thermal stability. Since the synthetic fragment binds R17 coat protein with the same affinity as a 59-nucleotide fragment isolated from R17 RNA, we conclude that it has full biological activity.

Base Sequence↗

PubMed1980

Interaction of Escherichia coli host factor protein with oligoriboadenylates.

The interaction of Escherichia coli host factor 1 with oligoadenylate [oligo(A)] was studied by fluorescence and filter retention techniques. The intrinsic fluorescence of the host factor is quenched by up to 60% by the addition of oligo(A). Fluorescence titrations at high protein concentrations (6 microM) give a saturation point of 14 A residues per host factor hexamer regardless of chain length or ionic strength. Nitrocellulose filter retention experiments at much lower concentrations (1 nM) indicate equimolar complexes form between (pA)l (12 less than l less than 27) and host factor hexamers. The smallest number of contiguous A residues which allows the formation of all favorable protein--RNA contacts is 16 at both low and high salt concentrations. At 0.1 M NaCl, the molar association constants are in the range of 10(10)--10(11) M-1 (15 less than l less than 27) and decrease only slightly with ionic strength, indicating a large nonionic component in the interaction. Cyclized (pA)l was shown to have a higher affinity for host factor than its linear counterparts when l is 18 or greater but a lower relative affinity when l is 15. This suggests that the binding site on the hexamer has a circular spatial orientation.

Adenine Nucleotides↗

PubMed1980

Interaction of Escherichia coli host factor protein with Q beta ribonucleic acid.

The affinity of Escherichia coli host factor protein for a variety of ribonucleic acids (RNAs) is compared in an equilibrium competition assay with (pA)15 or (pA)27 as the common probe. Of the homopolymers tested, only polyriboadenylate [poly(rA)] binds the protein with a high affinity. At low ionic strength (0.1 M NaCl), the binding to Q beta RNA is much stronger than to the oligoadenylates, but the situation is reversed upon fragmentation of the RNA with ribonuclease T1. Increasing the ionic strength results in a drastic reduction of the affinity of host factor for Q beta RNA over a relatively narrow salt range (0.1--0.3 M NaCl). Over the same range, added salt greatly reduces the tendency of host factor hexamers to aggregate. The tight binding of host factor to Q beta RNA is proposed to result from the binding of an aggregate, which can interact with several low affinity sites on the RNA simultaneously.

Binding Sites↗