Intramolecular Folding in Human ILPR Fragment with Three C-Rich Repeats

Enrichment of four tandem repeats of guanine (G) rich and cytosine (C) rich sequences in functionally important regions of human genome forebodes the biological implications of four-stranded DNA structures, such as G-quadruplex and i-motif, that can form in these sequences. However, there have been few reports on the intramolecular formation of non-B DNA structures in less than four tandem repeats of G or C rich sequences. Here, using mechanical unfolding at the single-molecule level, electrophoretic mobility shift assay (EMSA), circular dichroism (CD), and ultraviolet (UV) spectroscopy, we report an intramolecularly folded non-B DNA structure in three tandem cytosine rich repeats, 5'-TGTC4ACAC4TGTC4ACA (ILPR-I3), in the human insulin linked polymorphic region (ILPR). The thermal denaturation analyses of the sequences with systematic C to T mutations have suggested that the structure is linchpinned by a stack of hemiprotonated cytosine pairs between two terminal C4 tracts. Mechanical unfolding and Br2 footprinting experiments on a mixture of the ILPR-I3 and a 5′-C4TGT fragment have further indicated that the structure serves as a building block for intermolecular i-motif formation. The existence of such a conformation under acidic or neutral pH complies with the strand-by-strand folding pathway of ILPR i-motif structures

Intrinsic halotolerance of the psychrophilic alpha-amylase from Pseudoalteromonas haloplanktis

The halotolerance of a cold adapted alpha-amylase from the psychrophilic bacterium Pseudoalteromonas haloplanktis (AHA) was investigated. AHA exhibited hydrolytic activity over a broad range of NaCl concentrations (0.01-4.5 M). AHA showed 28% increased activity in 0.5-2.0 M NaCl compared to that in 0.01 M NaCl. In contrast, the corresponding mesophilic (Bacillus amyloliquefaciens) and thermostable (B. licheniformis) alpha-amylases showed a 39 and 46% decrease in activity respectively. Even at 4.5 M NaCl, 80% of the initial activity was detected for AHA, whereas the mesophilic and thermostable enzymes were inactive. Besides an unaltered fluorescence emission and secondary structure, a 10 degrees C positive shift in the temperature optimum, a stabilization factor of > 5 for thermal inactivation and a Delta T-m of 8.3 degrees C for the secondary structure melting were estimated in 2.7 M NaCl. The higher activation energy, half-life time and T-m indicated reduced conformational dynamics and increased rigidity in the presence of higher NaCl concentrations. A comparison with the sequences of other halophilic alpha-amylases revealed that AHA also contains higher proportion of small hydrophobic residues and acidic residues resulting in a higher negative surface potential. Thus, with some compromise in cold activity, psychrophilic adaptation has also manifested halotolerance to AHA that is comparable to the halophilic enzymes