Helix formation in enzymically ligated peptides as a driving force for the synthetic reaction: example of alpha-globin semisynthetic reaction.

The alpha-globin semisynthetic reaction, namely, the ligation of the complementary fragments of alpha-globin, alpha 1-30 and alpha 31-141, in the presence of 30% l-propanol that is catalyzed by V8 protease is distinct as compared with the previously studied protease-catalyzed splicing of the discontinuity sites of the fragment complementing systems [Sahni et al. (1989) Biochemistry 28, 5456]. The complementary fragments of alpha-globin do not exhibit noncovalent interaction between them even in the presence of l-propanol, the organic cosolvent used to facilitate the alpha-globin semisynthetic reaction. Besides, a significant portion of the fragment alpha 31-141 does not contribute to the protease-catalyzed splicing reaction. Alpha 1-30 and alpha 31-40 are ligated by V8 protease to yield alpha 1-40 in much the same way as the splicing of alpha 1-30 with either alpha 31-141 or alpha 31-47 to yield alpha-globin or alpha 1-47, respectively. An equimolar mixture of alpha 1-30 and alpha 31-40 does not show any 'complexation' in the presence of 30% l-propanol, the medium used for the synthetic reaction. The splicing junction, i.e., Glu30-Arg31 peptide bond, is located in the middle of the B-helix (residues 20-35) of the parent protein. Most of the residues from the A-helix of the protein could also be deleted from segment alpha 1-30 without influencing the V8 protease-catalyzed splicing reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pyridoxal phosphate site in glycogen phosphorylase b: structure in native enzyme and in three derivatives with modified cofactors

The detailed environment of the essential cofactor pyridoxal 5'-phosphate in glycogen phosphorylase b, resulting from crystallographic refinement at 1.9-A resolution, is described. The pyridoxal ring is buried in a nonpolar site containing three aromatic rings while the 5'-phosphate group is highly solvated and makes only three direct contacts to the protein. The pyridine nitrogen interacts via a water with protein atoms [main chain carbonyl oxygen (Asn-133) and OH of tyrosine (Tyr-90)]. The crystal structures of three active derivatives of phosphorylase reconstituted with 5'-deoxypyridoxal 5'-methylenephosphonate (PDMP), 6-fluoropyridoxal 5'-phosphate (6-FPLP), and pyridoxal (PL) in place of the natural cofactor have been determined at 2.5-A resolution. The results for PDMP-phosphorylase show a closer proximity of the phosphonate group to the NZ atom of a lysine (Lys-574) than that observed in the native enzyme, consistent with 31P NMR studies that have shown a change in ionization state of the phosphonate group compared to the native cofactor phosphate. The replacement of the polar 5'-ester linkage by a CH2 group results in a small shift of a water and its hydrogen-bonded tyrosine (Tyr-648). In 6-FPLP-phosphorylase the fluorine is accommodated with no significant change in structure. It is suggested that substitution of the electronegative fluorine at the 6-position may result in lower activity of 6-FPLP-phosphorylase through a strengthening of hydrogen-bonded interactions to the pyridine nitrogen N1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Refined structure of baboon alpha-lactalbumin at 1.7 A resolution. Comparison with C-type lysozyme

The solution of the structure of alpha-lactalbumin from baboon milk (Papio cynocephalus) at 4.5 A resolution using the isomorphous replacement method has been reported previously. Initial refinement on the basis of these low-resolution studies was not successful because of the poor isomorphism of the best heavy-atom derivative. Because of the striking similarity between the structure of lysozyme and alpha-lactalbumin, a more cautious molecular replacement approach was tried to refine the model. Using hen egg-white lysozyme as the starting model, preliminary refinement was performed using heavily constrained least-squares minimization in reciprocal space. The model was further refined using stereochemical restraints at 1.7 A resolution to a conventional crystallographic residual of 0.22 for 1141 protein atoms. In the final model, the root-mean-square deviation from ideality for bond distances is 0.015 A, and for angle distances it is 0.027 A. The refinement was carried out using the human alpha-lactalbumin sequence and "omit maps" calculated during the course of refinement indicated eight possible sequence changes in the baboon alpha-lactalbumin X-ray sequence. During the refinement, a tightly bound calcium ion and 150 water molecules, of which four are internal, have been located. Some of the water molecules were modelled for disordered side-chains. The co-ordination around the calcium is a slightly distorted pentagonal bipyramid. The Ca-O distances vary from 2.2 A to 2.6 A, representing a tight calcium-binding loop in the structure. The calcium-binding fold only superficially resembles the "EF-hand" and presumably has no evolutionary relationship with other EF-hand structures. The overall structure of alpha-lactalbumin is very similar to that of lysozyme. All large deviations occur in the loops where all sequence deletions and insertions are found. The C terminus appears to be rather flexible in alpha-lactalbumin compared to lysozyme. The experimental evidence supports the earlier predictions for the alpha-lactalbumin structure that were based upon the assumption that alpha-lactalbumin and lysozyme have similar three-dimensional structures, with minimal deletions and insertions. A detailed comparison of the two structures shows striking features as well as throwing some light on the evolution of these two proteins from a common precursor.     

Short Communication: Leaching of Chromite overburden with various native bacterial strains

Four heterotrophic bacterial strains were isolated from soil and water samples from a chromite mine in Sukinda valley in Orissa. They were identified as Lactobacillus acidophilus, Staphylococcus lactis, Lactobacillus sp. and Propionibacterium acnes. Each strain was used for leaching of metals from chromite overburden of Sukinda. Comparatively, Lactobacillus sp. showed maximum capability for metal solubilization. It leached a maximum of 5.98% of nickel in 35 days of contact time.     

Complete genome sequence of Pseudomonas frederiksbergensis ERDD5:01 revealed genetic bases for survivability at high altitude ecosystem and bioprospection potential

Pseudomonas frederiksbergensis ERDD5:01 is a psychrotrophic bacteria isolated from the glacial stream flowing from East Rathong glacier in Sikkim Himalaya. The strain showed survivability at high altitude stress conditions like freezing, frequent freeze-thaw cycles, and UV-C radiations. The complete genome of 5,746,824 bp circular chromosome and a plasmid of 371,027 bp was sequenced to understand the genetic basis of its survival strategy. Multiple copies of cold-associated genes encoding cold active chaperons, general stress response, osmotic stress, oxidative stress, membrane/cell wall alteration, carbon storage/starvation and, DNA repair mechanisms supported its survivability at extreme cold and radiations corroborating with the bacterial physiological findings. The molecular cold adaptation analysis in comparison with the genome of 15 mesophilic Pseudomonas species revealed functional insight into the strategies of cold adaptation. The genomic data also revealed the presence of industrially important enzymes.     

Dihydroxypropylation of amino groups of proteins: use of glyceraldehyde as a reversible agent for reductive alkylation

The mode of derivatization of amino groups of proteins by glyceraldehyde, an aldotriose, depends on the presence or absence of reducing agent. In the presence of sodium cyanoborohydride, the Schiff base adducts of the aldehyde with the amino groups are reduced, and dihydroxypropylation of amino groups takes place (reductive mode). The reductively glycated lysine residue, N epsilon-(2,3-dihydroxypropyl)lysine, is a substituted alpha-amino alcohol. This alpha-amino alcoholic function of the derivatized lysine should be susceptible to periodate oxidation, and this oxidation is anticipated to result in the regeneration of the lysine residue. This aspect has been now investigated. Indeed, on mild periodate oxidation (15 mM periodate, 15 min at room temperature) of dihydroxypropylated ribonuclease A, nearly 95% of its N epsilon-(2,3-dihydroxypropyl)lysine residues were regenerated to lysine residues. The removal of the dihydroxypropyl groups by periodate oxidation could be accomplished within a wide pH range with little variation in the recovery of lysines. The possible usefulness of this reversible chemical modification procedure in the primary structural studies of proteins was investigated with a tryptic peptide of dihydroxypropylated streptococcal M5 protein, namely, DHP-T4. This 12-residue tryptic peptide contains one internal N epsilon-(dihydroxypropyl)lysine. The dihydroxypropylated peptide released most of its dihydroxypropyl groups on mild periodate oxidation. Redigestion of the periodate-treated peptide with trypsin generated the two expected peptides, demonstrating the generation of a trypsin-susceptible site. Reductive dihydroxypropylation of amino groups of RNase A resulted in the loss of its enzyme activity, the extent of inactivation increasing with the concentration of the glyceraldehyde used.(ABSTRACT TRUNCATED AT 250 WORDS)