Binding studies between CUGBP1ab and (GTCT) oligonucleotide repeats Ravi Ramenani(1), Qi Zhu(2), Prahatees Eswaramoorthy(1), Jinchun Wang(2), Youlin Xia(2), Kyu-Yeon Jun(2), Xiaolian Gao(1) and Dar-Chone Chow(2). (1) Department of Biology and Biochemistry and (2) Department of Chemistry, University of Houston, Houston, TX, 77004 Isothermal Titration Calorimetry Fluorescence Analysis Size Exclusion Chromatography Analysis Introduction Isothermal Titration Calorimetry was used to investigate the nucleotide binding thermodynamics of CUGBP1ab. All experiments were done by titrating protein with DNA in 25 mM NaH2PO4, 50 mM NaCl, 0.25 mM NaN3, at pH 5.8 buffer in VP-ITC titration calorimeter (Microcal Inc., Northampton, MA, USA). Sizing chromatography has been an amazing tool in reflecting the solution, protein-DNA complex model. Fixed amount of protein was mixed with various concentrations of DNA and were run on a superdex-200 column. CUGBP1 is a CUG trinucleotide repeat binding protein known of playing a role in the Myotonic Dystrophy type 1 (DM1), a genetic disease affecting about 1 in 8000 births (Alwazzan et al., 1999). It was found to be binding to the CUG-repeat in the 3’ untranslated region of mutated DMPK (myotonic dystrophy protein kinase) transcript in diseased conditions. CUGBP1 was previously shown to bind to several different RNA repeats besides the CUG repeats (Timchenko et. al., 1996). The microarray and NMR experiments from Dr.Gao’s lab have revealed that CUGBP1ab also binds to single-stranded GTCT-repeats [(GTCT)n] with micromolar affinity. To understand the binding mechanism better, we have measured the binding thermodynamics between CUGBP1ab and various (GTCT)n (n: number of repeats) using Isothermal Titration Calorimetry (ITC). Our results demonstrate that CUGBP1ab-(GTCT)n bindings have an average unit binding enthalpy of –11kcal/mol (favorable), –25 cal/mol/K of entropy (unfavorable), –8 kcal/mol of free energy and heat capacity changes close to zero to slightly positive. Stoichiometry was shown to be about one protein per GTCT repeat for n<5 and with a limited accuracy for n>5. We have also performed fluorescence experiments to show the DNA binding induced quenching of intrinsic fluorescence of CUGBP1ab, which allowed us to validate the binding stoichiometry between the [(GTCT)n] and CUGBP1ab. We further carried out size exclusion chromatography analysis to show distinct protein/DNA ratio dependent complexes. Based on these results on the CUGBP1ab-oligonucleotide complexes we were able to derive a binding model of the interaction which suggests that there is a hierarchy order of binding of CUGBP1ab to the sites on a (GTCT)n. This model suggests that the trinucleotide-repeat-containing-polynucleotide CUGBP1 interaction is optimized for maximal binding of CUGBP1 in vivo. Fluorescence quenching of CUGBP1ab by oligonucleotide repeats was done by titrating 500nM CUGBP1ab with increasing amounts of different (GTCT)n oligonucleotides in 25 mM NaH2PO4, 50 mM NaCl, 0.25 mM NaN3, at pH 5.8 buffer . Baseline adjustments for the titrations were done by linearly moving the curve to adjust with the original fluorescence curve. (GTCT)4, 15°C (GTCT)2, 6°C 0.00 0.25 0.50 0.75 -32 -28 -24 -20 -16 -12 -8 -4 -0.4 -0.2 0.0 100 200 300 Time (min) µcal/sec Molar Ratio kcal/mole of injectant (GTCT)3, 15°C Baseline adjusted (GTCT)6, 20°C (GTCT)7, 15°C (GTCT)10, 20°C Stoichiometric Reaction Curves 0.36 0.32 Previous Results Elution pattern of CUGBP1ab complexed with (GTCT)7 in Superdex-200 column. Peaks Right to left: 30 µM CUGBP1ab, 3 µM (GTCT)7, 3 µM (GTCT)7 and 5 µM CUGBP1ab, 3 µM (GTCT)7 and 10 µM CUGBP1ab, 3 µM (GTCT)7 and 15 µM CUGBP1ab, 3 µM (GTCT)7 and 30 µM CUGBP1ab, 3 µM (GTCT)7 and 55 µM CUGBP1ab. INSET: Identical plot with the peaks being normalized. CUGBP1 is a 482 amino acid long protein containing three RNA Binding Domains (RBD 1, 2 and 3). Previous studies using a truncated mutant, CUGBP1ab (RBD 1 and RBD 2), showed that RBD1 and RBD 2 are necessary for specific binding to the CUG repeats (Timchenko et al., 1999). The microarray experiments revealed this novel ssDNA binding property of CUGBP1ab and with higher affinity to GTCT repeats. Also the NMR titrations of (GTCT) repeats with CUGBP1ab showed it is only RRM1, which is involved in nucleotide repeats binding. Hierarchical binding model: DNA Oligomer (GTCT)n ITC Titration Results n = [DNA] (µM) [Protein] (µM) Temp N 1/N Kb (M-1) ΔH (cal/mol) ΔS (cal/mol/K) (ºC) 2 80 20 6 0.47 2.12 2.84 x 106 -2.2 x 104 -50 12 0.55 1.81 1.66 x 106 -2.1 x 104 -46 24 0.48 2.08 0.62 x 106 -2.0 x 104 -90 30 0.46 2.17 0.40 x 106 -1.8 x 104 -36 3 36 15 0.24 4.16 2.56 x 106 -2.69 x 104 -64 4 60 0.15 6.66 2.59 x 106 -4.53 x 104 -128 50 40 0.10 .10.00 3.21 x 106 -7.50 x 104 -226 7 27 0.08 12.50 6.49 x 106 -8.61 x 104 -262 10 28 0.09 11.11 5.55 x 106 -13.5 x 104 -329 3.71 x 106 -14.1 x 104  -401 18 3.11 x 106 -17.1 x 104  -466 0.21 0.18 CUGBP1 with all three domains RRM1, RRM2 and RRM3 As there is an increase in the protein concentration, CUGBP1ab binds sequentially to all the available DNA instead of cooperatively sequestering to one DNA molecule. Stoichiometric binding reactions with 500nM CUGBP1ab was titrated with increasing amounts of different (GTCT) oligonucleotide repeats. Data points from steeply increasing and saturated regions were independently fit and the resulting lines were shown. Conclusion We report here the DNA binding characteristics of the RNA binding protein CUGBP1ab determined from ITC, Fluorescence and Size-exclusion chromatography methods. ITC experiments gave us an in-depth thermodynamic information about the complex. Fluorescence analysis supported the binding model. Size exclusion chromatography revealed the hierarchical binding pathway. Proposed Binding Model ITC Data Analysis CUGBP1ab Scanned image of DNA oligonucleotide aptamer chip, where the intensity of each spot represents the extent of binding of CUGBP1ab to DNA oligonucleotides. ΔCp calculation Stoichiometry Intensity Vs number of GTCX repeats from the oligonucleotide chip data References (GTCT) oligonucleotide repeat 1. Timchenko, L.T., et al. Nucleic Acids Res. 24, 4407-4414 (1996) . 2. Timchenko, L.T., et al. Hum. Mol. Genet. 5, 115-121 (1996). 3. Kim, C., et al. Biochemistry 34, 3028-2064 (1995). 4. Kowalczykowski, S.C., et sl. Biochemistry 25, 1226-1240 (1986). 5. Jun, K.Y. et al. J Biomol. NMR. 30: 371-372 (2004). 6. Timchenko, N.A., et al. Nucleic Acids Res. 27, 4517-4525 (1999). 7. Kino, Y., et al. Hum. Mol. Genet. 13, 495-507 (2004). 8. Alwazzan, m., et al. Hum. Mol. Genet. 8, 1491-1497 (1999). 9. Michalowski, S., et al. Nucl. Acids Res. 27, 3534-3542 (1999). ΔCp for (GTCT)2/CUGBP1ab binding was found to be very low positive value at about 150cal/mol/oK The stoichiometry of DNA binding by CUGBP1ab obtained by DNA repeats plotting against 1/N Enthalpy Enthalpy per Repeat Acknowledgement American heart Association NIH (GM49957) Robert-A-Welch Foundation(E-1027) Jihong Wang Han Fang Toan.D.Nguyen Superimposition of the 2D 15N-1H HSQC spectra of the uniformly 15N-labled CUGBP1ab (red) with those in the presence of (GTCT)3 (blue) at the protein : DNA molar ratios of 1:0.70. The peaks that were shifted or disappeared are marked with their residue name and residue number. The binding enthalpy (kcal/mol) plotted against the GTCT repeats, also the enthalpy change (kcal/mol) per GT region. It is evident that the binding reactions are enthalpy driven and there is about a constant 10Kcal increase in enthalpy change with the increase in each repeats. The proposed model like beads on string, suggesting that about one CUGBP1ab binding to one GT region of the GTCT repeats.