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Pulse dipolar ESR of doubly labeled mini TAR DNA and its annealing to mini TAR RNA.

ABSTRACT: Pulse dipolar electron-spin resonance in the form of double electron electron resonance was applied to strategically placed, site-specifically attached pairs of nitroxide spin labels to monitor changes in the mini TAR DNA stem-loop structure brought on by the HIV-1 nucleocapsid protein NCp7. The biophysical structural evidence was at Ångstrom-level resolution under solution conditions not amenable to crystallography or NMR. In the absence of complementary TAR RNA, double labels located in both the upper and the lower stem of mini TAR DNA showed in the presence of NCp7 a broadened distance distribution between the points of attachment, and there was evidence for several conformers. Next, when equimolar amounts of mini TAR DNA and complementary mini TAR RNA were present, NCp7 enhanced the annealing of their stem-loop structures to form duplex DNA-RNA. When duplex TAR DNA-TAR RNA formed, double labels initially located 27.5 Å apart at the 3'- and 5'-termini of the 27-base mini TAR DNA relocated to opposite ends of a 27 bp RNA-DNA duplex with 76.5 Å between labels, a distance which was consistent with the distance between the two labels in a thermally annealed 27-bp TAR DNA-TAR RNA duplex. Different sets of double labels initially located 26-27 Å apart in the mini TAR DNA upper stem, appropriately altered their interlabel distance to ~35 Å when a 27 bp TAR DNA-TAR RNA duplex formed, where the formation was caused either through NCp7-induced annealing or by thermal annealing. In summary, clear structural evidence was obtained for the fraying and destabilization brought on by NCp7 in its biochemical function as an annealing agent and for the detailed structural change from stem-loop to duplex RNA-DNA when complementary RNA was present.

SUBMITTER: Sun Y

PROVIDER: S-EPMC4336369 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Pulse dipolar ESR of doubly labeled mini TAR DNA and its annealing to mini TAR RNA.

Sun Yan Y Borbat Peter P PP Grigoryants Vladimir M VM Myers William K WK Freed Jack H JH Scholes Charles P CP

Biophysical journal 20150201 4

Pulse dipolar electron-spin resonance in the form of double electron electron resonance was applied to strategically placed, site-specifically attached pairs of nitroxide spin labels to monitor changes in the mini TAR DNA stem-loop structure brought on by the HIV-1 nucleocapsid protein NCp7. The biophysical structural evidence was at Ångstrom-level resolution under solution conditions not amenable to crystallography or NMR. In the absence of complementary TAR RNA, double labels located in both t ...[more]

PMID: 25692594

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Project description:The molecular dynamics of spin-labeled compounds included into the solid phase of cyclodextrins (CDs) has been studied using conventional (X-band) ESR at 9 GHz and high-field high-frequency (HFHF) ESR at 240 and 170 GHz. The patterns of axial rotation at these higher frequencies are clear just by inspection of the spectrum, unlike the case for 9 GHz spectra. That is HFHF ESR is sensitive to molecular motion about the diffusion axis collinear with the X, Y or Z-direction of the magnetic g- and A-tensors of the nitroxide moiety (referred to, respectively, as X, Y or Z-rotation). For doxyl stearic acids (Z-rotation) and TEMPOyl caprylate (X-rotation) included in beta- and gamma-CDs we were able to determine the rate of molecular motion and the corresponding potential barriers. We emphasize that determining the rate of Z-rotation by ESR is feasible only using HFHF ESR. For the X-rotation case we suggest that the motion of the nitroxide moiety consists of fast small-angle librations about the magnetic X-axis superimposed by rotational diffusion about the same axis. The potential barrier of 1.7 Kcal mol(-1) for this rotational diffusion is unusually low. A fascinating feature of TEMPO derivatives included in beta-CD is the detectable molecular motion at temperatures below 77 K. For the other CD-spin probe systems, we used multifrequency analysis to assign the conformations of spin-labeled molecules. A dramatic spectral change for 16-sasl in beta- and gamma-CDs at approximately 260 K corresponds to a tilting of the position of the nitroxide moiety on the rotating molecule relative to the long diffusion axis, while for TEMPO derivatives in gamma-cyclodextrin below 200 K, we observe a rapid transition from fast to very slow rotational motion. More complex features are best studied by means of multifrequency ESR experiments. The visual clarity and the simplicity of analysis of the ESR spectra shown in this work should provide a benchmark for future studies of molecular motion by HFHF ESR.

Dataset Information

Pulse dipolar ESR of doubly labeled mini TAR DNA and its annealing to mini TAR RNA.

Publications

Pulse dipolar ESR of doubly labeled mini TAR DNA and its annealing to mini TAR RNA.

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