Papain does not cleave operator-bound lambda repressor: structural characterization of the carboxy terminal domain and the hinge.

The circular dichroism spectra of three different purified carboxy terminal fragments 93-236, 112-236 and 132-236 of the bacteriophage lambda cI repressor have been measured and compared with those of the intact repressor and the amino terminal fragment 1-92. All three carboxy terminal fragments contain mostly beta-strands and loops, a minor helix content increasing with the size of the fragment, showing that the 93-131 region previously called a hinge is structured. Fourier transformed infrared spectra also showed that fragment 93-236 contains alpha-helices, alpha-sheets and turns but fragment 132-236 contains no detectable alpha-helix, only beta-sheets and turns. Papain is known to cleave the lambda repressor, but it is shown here that it cannot cleave the operator-bound repressor dimer. For the 132-236 fragment, both the wt and the SN228 mutant previously shown to be dimerization defective in the intact, gave similar dimerization properties as investigated by HPLC at 2 to 100 microM protein concentration, with a KD of 13.2 microM and 19.1 microM respectively. The papain cleavage for wt and SN228 proceed at equal rates for the first cleavage at 92-93; however, the subsequent cleavages are faster for SN228. The three Cys residues in the 132-236 fragment were found to be unreactive upon incubation with DTNB, indicating the thiol sulfur atoms are buried in the repressor carboxy terminal domain. Denaturation of the 132-236 fragment studied by tryptophan fluorescence shows two transitions centered at 1.5 M and 4.5 M of urea.

Model of a LexA repressor dimer bound to recA operator.

A complete three dimensional model (RCSB000408; PDB code 1qaa) for the LexA repressor dimer bound to the recA operator site consistent with relevant biochemical and biophysical data for the repressor is proposed. A model of interaction of the N-terminal operator binding domain 1-72 with the operator was available. We have modelled residues 106-202 of LexA on the basis of the crystal structure of a homologous protein, UmuD'. Residues 70-105 have been modelled by us, residues 70-77 comprising the real hinge, followed by a beta-strand and an alpha-helix, both interacting with the rest of the C-domain. The preexponential Arrhenius factor for the LexA autocleavage is shown to be approximately 10(9) s(-1) at 298K whereas the exponential factor is approximately 2 x 10(-12), demanding that the autocleavage site is quite close to the catalytic site but reaction is slow due to an activation energy barrier. We propose that in the operator bound form, Ala 84- Gly 85 is about 7-10A from the catalytic groups, but the reaction does not occur as the geometry is not suitable for a nucleophilic attack from Ser 119 Ogamma, since Pro 87 is held in the cis conformation. When pH is elevated or under the action of activated RecA, cleavage may occur following a cis --> trans isomerization at Pro 87 and/or a rotation of the region beta9-beta10 about beta7-beta8 following the disruption of two hydrogen bonds. We show that the C-C interaction comprises the approach of two negatively charged surfaces neutralized by sodium ions, the C-domains of the monomers making a new beta barrel at the interface burying 710A2 of total surface area of each monomer.

Purification and biochemical characterization of a novel thermostable lipase from Aspergillus niger.

An extracellular 1,3-specific lipase with molecular weight of 35.5 kDa and an isoelectric point of 4.4 from Aspergillus niger has been purified 50-fold by pH precipitation followed by a series of chromatographic steps with an overall yield of 10%. The enzyme was homogeneous as judged by denaturing polyacrylamide gel electrophoresis and size-exclusion fast-performance liquid chromatography. It contained 2.8% sugar which was completely removed by endoglycosidase F treatment, and the deglycosylated enzyme retained full activity. The native lipase showed optimal activity between temperatures 35 and 55 degrees C and pH 5.0 and 6.0. The amino acid composition and the N-terminal sequence were found to be different from lipases previously purified from A. niger. The enzyme was resistant to trypsin, chymotrypsin, endoprotease Glu-C, thrombin, and papain under native conditions but was susceptible to cleavage by the same proteases when heat-denatured.

Heusinger-s syndrome-a case report.

Heusinger's syndrome or Branchio-oto-renal dysplasia is a rare congenital anomaly. One such case with external ear anomalies, branchial fistula and renal abnormality is presented. The patient underwent successful excision of the branchial fistula. The case is discussed with review of relevant literature.

Calmodulin structure refined at 1.7 A resolution.

We have determined and refined the crystal structure of a recombinant calmodulin at 1.7 A resolution. The structure was determined by molecular replacement, using the 2.2 A published native bovine brain structure as the starting model. The final crystallographic R-factor, using 14,469 reflections in the 10.0 to 1.7 A range with structure factors exceeding 0.5 sigma, is 0.216. Bond lengths and bond angle distances have root-mean-square deviations from ideal values of 0.009 A and 0.032 A, respectively. The final model consists of 1279 non-hydrogen atoms, including four calcium ions, 1130 protein atoms, including three Asp118 side-chain atoms in double conformation, 139 water molecules and one ethanol molecule. The electron densities for residues 1 to 4 and 148 of calmodulin are poorly defined, and not included in our model, except for main-chain atoms of residue 4. The calmodulin structure from our crystals is very similar to the earlier 2.2 A structure described by Babu and coworkers with a root-mean-square deviation of 0.36 A. Calmodulin remains a dumb-bell-shaped molecule, with similar lobes and connected by a central alpha-helix. Each lobe contains three alpha-helices and two Ca2+ binding EF hand loops, with a short antiparallel beta-sheet between adjacent EF hand loops and one non-EF hand loop. There are some differences in the structure of the central helix. The crystal packing is extensively studied, and facile crystal growth along the z-axis of the triclinic crystals is explained. Herein, we describe hydrogen bonding in the various secondary structure elements and hydration of calmodulin.

Structure of a T4 hairpin loop on a Z-DNA stem and comparison with A-RNA and B-DNA loops.

The synthetic DNA oligomer C-G-C-G-C-G-T-T-T-T-C-G-C-G-C-G crystallizes as a Z-DNA hexamer, capped at one end by a T4 loop. The crystals are monoclinic, space group C2, with a = 57.18 A, b = 21.63 A, c = 36.40 A, beta = 95.22 degrees, and one hairpin molecule per asymmetric unit. The structure of the z-hexamer stem was determined by molecular replacement, and the T4 loop was positioned by difference map methods. The final R factor at 2.1 A resolution for hairpin plus 70 water molecules is 20% for 2 sigma data, with a root-mean-square error of 0.26 A. The (C-G)3 stem resembles the free Z-DNA hexamer with minor crystal packing effects. The T4 loop differs from that observed on a B-DNA stem in solution, or in longer loops in tRNA, in that it shows intraloop and intermolecular interactions rather than base stacking on the final base-pair of the stem. Bases T7, T8 and T9 stack with one another and with the sugar of T7. Two T10 bases from different molecules stack between the C1-G12 terminal base-pairs of a third and fourth molecule, to simulate a T.T "base-pair". Distances between thymine N and O atoms suggest that the two thymine bases are hydrogen bonded, and a keto-enol tautomer pair is favored over disordered keto-keto wobble pairs. The hairpin molecules pack in the crystal in herringbone columns in a manner that accounts well for the observed relative crystal growth rates in a, b and c directions. Hydration seems to be most extensive around the phosphate groups, with lesser hydration within the grooves.

Conformation and dynamics of a left-handed Z-DNA hairpin: studies of d(CGCGCGTTTTCGCGCG) in solution.

The physical properties of the DNA oligomer d(CGCGCGTTTTCGCGCG) in solvents containing 4 M NaClO4 and 0.1 M NaCl were investigated by proton NMR, optical melting, and circular dichroism spectroscopy. Results of these investigations are as follows: (i) The DNA hexadecamer exists as a unimolecular hairpin in either high or low salt. (ii) In high salt the stem region of the hairpin is in the left-handed Z conformation. (iii) In either high or low salt, the duplex stem of the hairpin is stabilized against melting by approximately 40 degrees C compared to the linear core duplex. The added stability of the hairpin is entropic in origin. (iv) In high salt, as the temperature is elevated, the equilibrium structure of the duplex stem of the hairpin shifts from the Z to the B conformation before melting. (v) In low salt, when the DNA duplex exists in the B conformation, attachment of a T4 single-strand loop to one end only slightly decreases (by 14%) the correlation time of the CH5-CH6 interproton vector. In high salt, when the DNA duplex exists in the Z conformation, the correlation time of the CH5-CH6 interproton vector decreases by 51%. Since these viscosity-corrected correlation times are taken to be indicators of duplex motions on the nanosecond time scale, this result directly suggests a larger amplitude of these motions is present in the duplex stem of the hairpin when it exists in the Z conformation.

Solving DNA structures by MERLOT.

MERLOT, a molecular replacement computer package, has been successfully used for the determination of initial phases for five different oligonucleotide structures. Two of these, CCGG and CGCGCGTTTTCGCGCG, with 316 and 323 non-H atoms respectively in the asymmetric unit, have been solved ab initio using the MERLOT package. The rest are re-examinations of previously solved DNA oligomer structures, ioCCGG, CGCGAATTCGCG and CGCGAATTbrCGCG, with 160, 486 and 488 non-H atoms in the asymmetric unit respectively. Problems involved in applying the molecular-replacement technique to DNA structures, and their possible solutions, are discussed.

X-ray structure of a DNA hairpin molecule.

We have solved the crystal structure of a synthetic DNA hexadecanucleotide of sequence: C-G-C-G-C-G-T-T-T-T-C-G-C-G-C-G, at 2.1 A resolution, and observed that it adopts a monomeric hairpin configuration with a Z-DNA hexamer stem. In the T4 loop the bases stack with one another and with neighbouring molecules of the crystal, and not with base pairs of their own hexamer stem. Two thymine T10 rings from different molecules stack between the C1-G16 ends of a third and a fourth hairpin helix, in a manner that suggests T-T base 'pairing' and simulates a long, 13-base-pair helix. Although such T-T interactions would not be present in solution, they illustrate a remarkable tendency of thymines for self-association. Purine-purine G-A base pairs are known to exist in the anti-anti conformation with an increase in local helix width; it may be that more serious consideration should be given to the possible existence of pyrimidine-pyrimidine C-T base pairs with decreased local helix width, particularly where several such base pairs occur sequentially.

NMR study of a synthetic DNA hairpin.

The secondary structure of the synthetic oligodeoxyribonucleotide d(CGCGCGTTTTCGCGCG) (I) has been demonstrated to be a unimolecular hairpin structure (hairpin I) over a wide range of oligonucleotide concentrations (2 X 10(-5) to 1.6 X 10(-3) M) and temperature (0-87 degrees C). The assignments of the resonances to specific protons were carried out by use of two-dimensional nuclear Overhauser effect and COSY spectra and by comparison with the spectra of the duplex formed by d(CG)3. Comparison of hairpin I and the hairpin of d(ATCCTATTTTTAGGAT) (II) reveals that the exchange of imino protons in stem base pairs with solvent is much slower in I than in II. However, the exchange of thymine imino protons in the loop region is much faster in I than in II even though both hairpins contain four unpaired thymine residues. The secondary structure of hairpin I contains only six G X C base pairs, yet it is more stable than the d(CG)8 duplex containing 16 G X C base pairs at all concentrations of duplex lower than 10(-3) M. These observations suggest that intramolecular hairpin formation may effectively compete with bimolecular duplex formations when the appropriate intramolecular base pairs can form.

The kinetics of phosphotriester condensation reactions on solid support.

A study has been made of the kinetics of condensation reactions in phosphotriester synthesis of oligonucleotides on polystyrene copolymer resin support, using monomers, dimers, and trimers as the coupling units. The condensations follow pseudo first order kinetics, with a rate constant that decreases from monomers to dimers to trimers. Arrhenius plots indicate a nearly constant activation energy of around 10.4 kcal mol-1. Extrapolation to find the Arrhenius preexponential factor, A, is inaccurate because of the limited temperature range studied. But if the assumption is made that Ea is the same for monomer, dimer, and trimer reactions, then the A values are inversely proportional to the surface areas of the coupling units, reinforcing the interpretation of A as a collision efficiency factor.