Towards characterization of DNA structure under physiological conditions in vivo at the single-molecule level using single-pair FRET

Fluorescence resonance energy transfer (FRET) under in vivo conditions is a well-established technique for the evaluation of populations of protein bound/unbound nucleic acid (NA) molecules or NA hybridization kinetics. However, in vivo FRET has not been applied to in vivo quantitative conformational analysis of NA thus far. Here we explored parameters critical for characterization of NA structure using single-pair (sp)FRET in the complex cellular environment of a living Escherichia coli cell. Our measurements showed that the fluorophore properties in the cellular environment differed from those acquired under in vitro conditions. The precision for the interprobe distance determination from FRET efficiency values acquired in vivo was found lower (～31%) compared to that acquired in diluted buffers (13%). Our numerical simulations suggest that despite its low precision, the in-cell FRET measurements can be successfully applied to discriminate among various structural models. The main advantage of the in-cell spFRET setup presented here over other established techniques allowing conformational analysis in vivo is that it allows investigation of NA structure in various cell types and in a native cellular environment, which is not disturbed by either introduced bulk NA or by the use of chemical transfectants.     

Effect of local sugar and base geometry on <Superscript>13</Superscript>C and <Superscript>15</Superscript>N magnetic shielding anisotropy in DNA nucleosides

Density functional theory was employed to study the dependence of 13C and 15N magnetic shielding tensors on the glycosidic torsion angle (chi) and conformation of the sugar ring in 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxycytidine, and 2'-deoxythymidine. In general, the magnetic shielding of the glycosidic nitrogens and the sugar carbons was found to depend on both the conformation of the sugar ring and chi. Our calculations indicate that the magnetic shielding anisotropy of the C6 atom in pyrimidine and the C8 atom in purine bases depends strongly on chi. The remaining base carbons were found to be insensitive to both sugar pucker and chi re-orientation. These results call into question the underlying assumptions of currently established methods for interpreting residual chemical shift anisotropies and 13C and 15N auto- and cross-correlated relaxation rates and highlight possible limitations of DNA applications of these methods.     

Structurally diverse disaccharide analogs of antifreeze glycoproteins and their ability to inhibit ice recrystallization

The β-d-galactosyl-(1,3)-α-N-acetyl-d-galactosamine disaccharide is present in antifreeze glycoproteins (AFGPs). Analogs of this disaccharide including the β-linked (1,3)-, (1,4)-, and (1,6)-galactosyl-N-acetyl galactosamine and the β-(1,3)-galactosyl-galactoside were synthesized and evaluated for ice recrystallization inhibition (IRI) activity. The results from this study demonstrate that the β-linked-(1,4) disaccharide exhibits more potent IRI activity than the native β-linked-(1,3) disaccharide. The C2 N-acetyl group of the disaccharide does not affect IRI activity but in monosaccharides, the presence of the C2 N-acetyl group decreases IRI activity. The current study will facilitate the design of potent small-molecule ice recrystallization inhibitors.     

Construction and expression of human/bovine P45017 alpha chimeric proteins: evidence for distinct tertiary structures in the same P450 from two different species

In the human and bovine adrenal cortex, 17 alpha-hydroxylase (P45017 alpha) catalyzes reactions involved in the production of C21-glucocorticoids (17 alpha-hydroxylation) and C19-androgens (17,20-lyase). The bovine and human forms of P45017 alpha share 71% primary sequence identity. Using naturally occurring restriction sites common to cDNAs encoding both human and bovine P45017 alpha, we have constructed bovine/human (bovine amino terminus and human carboxy terminus) and human/bovine (human amino terminus and bovine carboxy terminus) cDNAs that have been expressed in COS 1 cells, and the enzymatic properties of the resultant chimeric proteins have been examined. The three bovine/human chimeras studied have 17 alpha-hydroxylase activities intermediate between those of the wild-type bovine and wild-type human enzymes, although the 17,20-lyase activity of these chimeras is significantly lower than that of either of the wild-type enzymes. Surprisingly, the opposite chimeras (those containing a human amino-terminal sequene and a bovine carboxy-terminal sequence) are all virtually inactive, even though they appear to be expressed at normal levels. These results indicate that the folding of P45017 alpha initiated by the bovine amino terminus can accommodate human P45017 alpha sequences of various lengths to produce a relatively normal 17 alpha-hydroxylase having decreased 17,20-lyase activity. On the other hand, folding initiated by the human P45017 alpha amino terminus does not easily accommodate bovine carboxy-terminal sequences to produce a functional enzyme. Presumably this difference arises from the fact that the tertiary structures of the bovine and human forms of P45017 alpha are sufficiently different so that interchanging sequences will not lead to functional enzymes in a predictable fashion.     

A-like guanine-guanine stacking in the aqueous DNA duplex of d(GGGGCCCC)

We have used CD spectroscopy, NMR spectroscopy and unrestrained molecular dynamics to study conformational properties of a DNA duplex formed by the self-complementary octamer d(GGGGCCCC). Its unusual CD spectrum contains features indicating A-like stacking of half of the bases, whereas the other half stack in a B-like fashion. Unrestrained molecular dynamics simulations converged to a stable B-like double-helix of d(GGGGCCCC). However, the double-helix contained a central hole whose size was half of that occurring in structure A. In the canonical structure B, the hole does not exist at all because the base-pairs cross the double-helix centre. The cytosine bases were stacked in the duplex of d(GGGGCCCC) as in structure B, while stacking of the guanine bases displayed features characteristic for structure A. NMR spectroscopy revealed that the A-like guanine-guanine stacking was accompanied by an increased tendency of the deoxyribose rings attached to the guanine bases to be puckered in an A-like fashion. Otherwise, the duplex of d(GGGGCCCC) showed no clash, no bend and no other significant deviation from structure B. The present analysis demonstrates a remarkable propensity of the guanine runs to stack in an A-like fashion even within the B-DNA framework. This property explains why the oligo(dG). oligo(dC) tracts switch into structure A so easily. Secondly, this property may influence replication, because structure A is replicated more faithfully than structure B. Thirdly, the oligo(dG) runs might have played an important role in early evolution, when DNA took on functions that originally evolved on RNA. Fourthly, the present study extends the vocabulary of DNA secondary structures by the heteronomous duplex of d(GGGGCCCC) in which the B-like strand of oligo(dC) is bound to the A-like strand of oligo(dG).     

Recombinant perciform GnRH-R activates different signaling pathways in fish and mammalian heterologous cell lines

Perciforms have three forms of gonadotropin-releasing hormone (GnRH) in their brain. All three GnRHs are potent secretogogues for luteinizing hormone (LH) from the pituitary. The pivotal role of GnRH-R-GnRH interactions in reproductive homeostasis is well established; however, there is a paucity of information on how a GnRH-R responds to the three endogenous GnRH forms in a perciform species. In this study, a recombinant pituitary GnRH-R from striped bass (stb) was expressed in a mammalian cell line (COS-7) and a fish cell line (CHSE-214). Activation of the signaling pathways was monitored by reporter gene (luciferase) based assays, which were specific for cAMP-PKA or Ca 2+/calmodulin kinase (activated via c-fos promoter) signaling pathways. The stbGnRH-R expressed in two different cell lines triggered different downstream signaling in response to the treatments with chicken (c) GnRH II. Interestingly, when endogenous GnRHs were used in combinations, the luciferase activity was significantly attenuated in transfected CHSE-214 cells.