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R P Bray

Publications and source records attributed to R P Bray.

12 recordsLinked to original sources
PubMed1981

pH dependence of the high-resolution proton nuclear magnetic resonance spectrum of the lac repressor headpiece.

The pH dependence of the N-terminal 51 amino acid headpiece (HP) of the lac repressor has been followed by using 1H NMR spectroscopy to monitor the chemical shifts of resolved aromatic and methyl resonances. The NMR evidence reveals the folded HP domain to be stable from pH 1 to 10 at 23 degrees C. All observed resonances shift toward their expected random-coil positions above pH 10, which suggests that a general unfolding occurs. The four tyrosine rings reflect a combination of unfolding and titration in the order 7 greater than 17, 12 greater than 47. This pH-induced unfolding is completely reversible. In addition, strikingly similar pH behavior for selected tyrosine and methyl resonances at acid pH values suggests that clusters of various tyrosine, methyl, and carboxyl side chains exist in the native structure.

Amino Acids↗
PubMed1981

High-resolution proton nuclear magnetic resonance studies of the exchangeable resonances of the lac repressor headpiece.

The exchangeable hydrogens of the N-terminal 51 amino acid headpiece (HP) of the lac repressor have been examined with high-resolution 1H NMR. Resonances from 35 +/- 3 protons are observable in 2H2O solutions at p2H 4.2. The exchange rates for some of these have been measured over the p2H range 1.5-5. In H2O, resonances from 70 +/- 6 protons are observable at pH 3.2 and 35 +/- 5 protons at pH 7.1. The temperature dependence of the spectrum at neat neutral pH shows the existence of a group of approximately 22 protons particularly slow to exchange. The pH dependence of the spectrum in H2O solution over the range 1-11 is discussed. Results are compared with NMR studies of the nonexchanging protons and CD and IR results.

Deuterium↗
PubMed1981

High-resolution nuclear magnetic resonance studies of the Lac repressor. 1. Assignments of tyrosine resonances in the N-terminal headpiece.

The DNA binding site of the lac repressor protein has been implicated to lie within the N-terminal 51 amino acid fragment termed headpiece (HP-51 or LR-51). High-resolution NMR suggests that isolated HP-51 retains most of the secondary and tertiary structure which it has in the whole repressor. Four of the eight tyrosines of repressor are in HP-51. 1H NMR spectra (360 MHz) over the aromatic region of native HP-51 show that the four tyrosines are nonequivalent with an unusual distribution of chemical shifts. Denaturation leads to loss of these chemical shift differences. Homonuclear decoupling and a two-dimensional autocorrelated spectrum allow unequivocal pairing of resonances from Tyr A at 6.99 and 6.79 ppm, Tyr B at 6.98 and 6.39 ppm, Tyr C at 6.70 and 6.54 ppm, and Tyr D at 6.39 and 6.33 ppm. The 2,6 protons are low field of the 3,5 protons for each Tyr residue. Selective chemical modification with nitration reagents allows assignments of Tyr A to Tyr-47, Tyr B to Tyr-7, Tyr C to Tyr-12, and Tyr D to Tyr-17 in HP-51. All four tyrosines are essential for maintaining the structure of the isolated headpiece, and Tyr-7, -12, and -17 appear to be stacked.

Escherichia coli↗
PubMed1981

High-resolution nuclear magnetic resonance studies of the Lac repressor. 2. Partial analysis of the aliphatic region of the Lac repressor headpiece spectrum.

The 360-MHz 1H NMR spectrum of native lac repressor headpiece (HP-51 or HP-59) contains a large number (greater than 30%) of aliphatic side-chain methyl and backbone alpha-CH resonances and three of four aromatic tyrosine multiplet resonances shifted to high-field chemical shift positions, indicating the presence of extensive folded structure. Denaturation leads to loss of the NMR chemical shift differences. Resonance identifications of the 27 methyl-possessing amino acids in HP-59 have been made by using resolution enhancement, double-resonance, and difference spectra. There are three firmly assigned methyl resonances and 21 pairwise identifications of methyl resonances in HP-51. Comparison of HP-51 and HP-59 allows identification of four additional methyl groups in amino acid residues 52--59. The sequence HP-50--59 is not essential to maintain the structure of HP-59, but it is of interest itself as the flexible hinge portion connecting HP to the tetrameric core of whole repressor.

Escherichia coli↗
PubMed1981

High-resolution nuclear magnetic resonance studies of the Lac repressor. 3. Unfolding of the Lac repressor headpiece.

At temperatures below 20 degrees C, the lac repressor headpiece (N-terminal amino acids 1--51) has a well-defined structure which is independent of ionic strength. Its unfolding with increasing temperature proceeds gradually with a characteristic transition temperature which depends on ionic strength. Unfolding has been studied by using NMR and CD. Shifts of several methyl and all of the tyrosyl resonances can be followed, allowing a detailed analysis of the temperature denaturation. At high ionic strength (1 M), the unfolding is complete at 85 degrees C, while at low ionic strength (0.01 M), it is complete by 65 degrees C. Native and partially unfolded structures are in rapid exchange during the unfolding, and the process appears completely reversible at all ionic strengths.

Circular Dichroism↗
PubMed1980

Sequence of the low activity equine erythrocyte carbonic anhydrase and delineation of the amino acid substitutions in various polymorphic forms.

the sequence of the low activity form of equine erythrocyte carbonic anhydrase has been determined. The most common electrophoretic form, designated D, has been found to have five substitutions. Amino acid exchanges in the electrophoretic variants known as A1, A2, B, and T have been found at six other positions. The data do not permit calculation of the number of polymorphic forms of this enzyme. The equine D isozyme and the analogous human enzyme are quite homologous, 211 of their 260 residues, or 81%, being identical.

Amino Acid Sequence↗
PubMed1980

Small-angle X-ray studies of the quaternary structure of the lac repressor from Escherichia coli.

The quarternary structures of the lac repressor molecule from Escherichia coli and its tetrametic core, which can be derived from it by proteolytic cleavage, were studied in dilute solutions by small-angle X-ray scattering. The dimensions and general shape of the lac repressor and of the tetrameric core are reported. The core itself appears to be an elongated structure, and in the intact repressor the headpieces are located at its ends. The results ar derived from model calculations and from the following molecular parameters determined from the scattering curve and the pair distance distribution function: for lac repressor, radius of gyration 5.30 +/- 0.02 nm, radius of gyration of the cross section 2.20 +/- 0.03 nm, maximum diameter 18.0 +/- 0.5 nm, hydrated volume 329 +/- 20 nm3, relative molecular mass 149 000 +/- 15 000, for tetrameric core, radius of gyration 4.92 +/- 0.02 nm, radius of gyration of the cross section 2.24 +/- 0.03 nm, maximum diameter 16.0 +/- 0.5 nm, hydrated volume 278 +/- 15 nm3, relative molecular mass 120 000 +/- 10 000.

Escherichia coli↗
PubMed1975

Carbonic anhydrase isozymes in American ponies and riding horses: a new polymorphic high-activity type isozyme.

A study of the erythrocyte carbonic anhydrases of 219 American ponies and 76 riding horses has revealed the presence of five variants of the low-activity CA B isozyme and two variants of the high-activity CA C isozyme. The previously undetected variant of CA C was found only in the pony population and had an allele frequency of 8.9%. A family study of animals possessing the CA B variant A2 showed an unexpected high frequency of inheritance.

Alleles↗