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Structural Biology (LSBS5648) Biophysical Structural Program

Structural Biology (LSBS5648) Biophysical Structural Program The UV and CD spectroscopic studies on nucleic acids and proteins Lou-sing Kan, Ph. D. Institute of Chemistry, Academia Sinica May 27, 2009. Absorption and Emission Spectroscopy.

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Structural Biology (LSBS5648) Biophysical Structural Program

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  1. Structural Biology (LSBS5648) Biophysical Structural Program The UV and CD spectroscopic studies on nucleic acids and proteins Lou-sing Kan, Ph. D. Institute of Chemistry, Academia Sinica May 27, 2009

  2. Absorption and Emission Spectroscopy

  3. Advantages of Spectroscipic studies on Biomolecules High sensitivity Easy to use Multiple parameters Absorption: ultraviolet, visible, infrared Emission: fluorescence

  4. Ultraviolet (UV) spectroscopy of nucleotides

  5. L L max/ min L l max L min

  6. Hypochromicity Concentration determination Extinction coefficient (a) AAGCUU = AA + AG + GC + CU + UU – A – G – C – U = 27300 + 25580 + 18380 + 25580 + 27300 – 15340 – 12160 – 7600 – 8700 = 80340 UUCGAA = UU + UC + CG + GA + AA – U – C – G – A = 27300 + 25840 + 18780 + 25840 + 27300 –8700 – 7600 – 12160 – 15340= 81260 A = abC

  7. Stoichiometry

  8. Melting temperature Temperature °C Chemistry (Taipei), A54, 24-32 (1996).

  9. 3. Triplex is a unique molecular. It is not a complex of a single strand and a double strand DNA’s.

  10. a = a/b

  11. The study of The nearest neighbor stacking effect Single strand Double strand AA AC AG AT CA CC CG CT GA GC GG GT TA TC TG TT in RNA, U=T AA AC AG AT CA CC CG CT GA GC GG GT TA TC TG TT in RNA, U=T DH of AAGCUU = AA + AG + GC + CU + UU = 2 AA + 2 AG + GC

  12. Nucleic acids, structures, properties, and function, V. A. Bloomfield, D. M. Crothers, I. Tinoco, Jr. p. 276 University Science Book, Sausalito, CA

  13. Self-complementary 2A ------ A2 K = [A2]/[A]2 = a/[2(1- a)2CT] At Tm, a = ½, K = 1/CT -RTmlnK = DG = DH – TmDS = RTmlnCT Nonself-complementary A + B ---- AB K = [AB]/[A][B] = 2 a /[(1- a)2CT] At Tm, a = ½, K = 4/CT -RTmlnK = DG = DH – TmDS = RTmlnCT/4

  14. a = a/b

  15. DH DS AG -7.8 -0.021 GA -8.2 -0.0222 TA -7.2 -0.0213 Total -86.8 -0.236 CT RlnCT RlnCT + DS Tm (K) Tm (C) 0.002 -12.4 -0.249 349 76 0.0002 -16.9 -0.253 243 70 0.00002 -21.5 -0.258 336 63 2 x 10-6 -26.1 -0.262 331 58 CTCTCTAGAGAG ---- (CTCTCTAGAGAG)| CT + TC + CT + TC + CT + TA + AG + GA + AG + GA + AG = 6AG + 4GA + TA Tm = (DH/(RlnCT + DS)

  16. Circular Dichroism Circular Dichroism (CD) is a spectroscopic method which depends on the fact that certain molecules interact differently with reight and left circularly polarized light.

  17. 5’- CTCTCTCTCTCTCTCT 5’-AGAGAGAGAGAGAGAG 3’-TCTCTCTCTCTCTCTC 5’- CTCTCTCTCTCTCTCT 5’-AGAGAGAGAGAGAGAG 3’-TCTCTCTCTCTCTCTC + + + + + + + + + + + + + + + + Academia Sinica中央研究院Founded 1928 5’-AGAGAGAGAGAGAGAG 3’-TCTCTCTCTCTCTCTC Neutral pH 5’-AGAGAGAGAGAGAGAG 3’-TCTCTCTCTCTCTCTC Acidic pH J. Biomol. Str. Dyn. 12, 1235-1245 (1995) Synchrotron Radiation CD 2006 11 22

  18. TCTCTCTC AGAGAGAG

  19. (TC)3 (CT)3(AG)3 4℃ 4℃ 4℃ 15℃ 15℃ 15℃ 35℃ 25℃ 25℃ 45℃ 45℃ 35℃ 45℃ 55℃ 55℃ 75℃ 55℃ 75℃ (CT)3(AG)3(CT)3 (AG)3 (CT)3 (TC)3 Figure4. Plot of the CD spectra intensity at 217nm and 240nm againse temperature for TCA,CAC and ACT at pH= 6.5

  20. (TC)3 (CT)3(AG)3 (CT)3(AG)3(CT)3 4℃ 4℃ 4℃ 35℃ 15℃ 15℃ 25℃ 35℃ 45℃ 45℃ 45℃ 55℃ 55℃ 65℃ 55℃ 75℃ 75℃ 75℃ (AG)3 (CT)3 (TC)3 Figure5. Plot of the CD spectra intensity at 217nm and 240nm againse temperature for TCA,CAC and ACT at pH= 7.5

  21. 217nm/240nm pH=4 pH=5 pH=6 pH=(6.5) pH=7 pH=(7.5) pH=8 (TC)3(CT)3(AG)3 66.9 64.6 48.3 28.5 50.2 56.5 48.8 (CT)3(AG)3(CT)3 54.9 55.6 45.8 56.9 50.6 54.9 53.7 (AG)3(CT)3(TC)3 57.9 68.5 51.3 28.6 15.5 59.7 14.260.3 56.8 (CT)3(TC)3(GA)3 62.8 69.4 57.1 38.6 25.2 49.3 13.950.8 45.8 (TC)3(GA)3(TC)3 47.1 49.2 54.9 51.8 49.5 50.2 48.5 (GA)3(TC)3(CT)3 71.7 60.2 51.1 29.9 13.4 55.9 51.9 49.8 Table3. The melting temperture (ºC) of the six oligonulectides with CD spectra at 217nm and 240nm as a function of pH

  22. CD on proteins

  23. The study of protein folding. Phys. Rev., E., 70, 011904 (1-8) (2004).

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