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Non-cooperative thermal denaturation of acyl-CoA binding protein

10 th Group Meeting July 8, 2009. Non-cooperative thermal denaturation of acyl-CoA binding protein. Tetsu KIMURA. California Institute of Technology. -Purpose- Molecular Mechanism of Protein Folding. U. I. N. Bryngelson, Onuchic & Wolynes, Proteins (1995) 21 , 167.

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Non-cooperative thermal denaturation of acyl-CoA binding protein

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  1. 10th Group Meeting July 8, 2009 Non-cooperative thermal denaturation of acyl-CoA binding protein Tetsu KIMURA California Institute of Technology

  2. -Purpose-Molecular Mechanism of Protein Folding U I N Bryngelson, Onuchic & Wolynes, Proteins (1995) 21, 167 The determination of the factors that control the assembly of structural components. Quantitative estimates of the changes in energies Characterization of the populated structures along the folding pathway

  3. -Introduction & Purpose-Denatured State Ensemble U Bryngelson, Onuchic & Wolynes, Proteins (1995) 21, 167 The determination of the structures of the denatured state ensemble is required as the starting point of the reaction to understand the protein folding mechanism.

  4. -Previous Studies-Single-molecule FRET measurements Hoffmann A. et.al. PNAS;2007;104:105-110

  5. r0 6 1k(r) = 1k0 1 + r S I(t) = P(k) exp(–kt) k -Result-Static FET Kinetics U Acceptor N r hn Donor 26.8 Å Native State 10 15 20 25 30 35 40 45 50 55 r (Å) Unfolded State (3M GuHCl) 34.7 Å FET kinetics is the best method to monitor the distance distribution experimentally. 10 15 20 25 30 35 40 45 50 55 r (Å)

  6. O CH2 C CH2NH NH N O N NO2 -Pair of Donor and Acceptor- IAEDANS and QSY®35 O NHCH2CH2NH CH2 Cys C R0 = ~35 Å 1,5-IAEDANS (DNS) (10.5 < r < 52.5 Å) SO3H Cys QSY®35 e475 = 24,000 cm-1 M-1 No mission DNS-QSY pair is a sensitive probe for conformational changes.

  7. 16 86 50 21 2 38 62 66 -Target Protein-Acyl-CoA Binding Protein (ACBP) Four-helix bundle 86 residues 10 kDa Double-Cys Mutants D(Cg-Cg), Å <RCDrms>, Å Helix-1 Helix-2 Helix-3 Helix-4 Helix-1 /Helix-4 Q2-K16 D21-D38 K50-K62 K66-I86 Q2-K66 K16-I86 25.7 25.1 22.8 25.6 11.0 5.7 42 46 39 49 87 91 Kyte & Doolittle (1982) JMB 157, 105 Muñoz & Serrano (1995) JMB 245, 275 ACBP is a good model for both experiment and simulation.

  8. -Method-How to label the proteins DTT Incubate 30 min Desalting column to remove DTT & change the buffer (3 M GuHCl, pH 8.0) Stock solution of unlabeled ACBP (0.4 – 1mM) Desalting column to refold (20 mM Tris-HCl (pH 8.0) + x mM NaCl) monoQ column to purify (20 mM Tris-HCl (pH 8.0) with NaCl gradient) Dyes TCEP

  9. -Previous Study-Purification and Static Properties of Double-labeled ACBP UV-vis & Fluorescence spectra monoQ purification of DNS-labeled ACBP N U in 3.0 M GuHCl 2-16 2-16(DNS) 2(DNS)-16 280 nm 2(DNS)-16(DNS) 355 nm 490 nm monoW purification of DNS & QSY-labeled ACBP 2-66 2(QSY)-16(DNS) 490 nm 280 nm 355 nm DNS fluorescence is highly quenched by QSY®35 in the native state.

  10. -Result-Temperature Melting Followed by CD 2(QSY)-66(DNS) WT WT* (W55F) 66 2-66 66-86 66(DNS)-86(QSY) Double-labeled ACBPs are as stable as single-labeled ACBP.

  11. -Result-Thermal Melting by Fluorescence Spectra 2(QSY)-66(DNS) 11.0 Å 515 nm 80 ℃ 10 ℃ DNS is partially buried from the solvent in the native state.

  12. -Result-Thermal Melting of Labeled ACBPs 2(QSY)-66(DNS) 66(DNS)-86(QSY) 11.0 Å (~87 Å) 25.6 Å (~49 Å) Non-cooperative melting of helix-4

  13. 10 15 20 25 30 35 40 45 50 55 10 15 20 25 30 35 40 45 50 55 r (Å) r (Å) -Result-Distance Distributions of Native and Unfolded States 2(QSY)-66(DNS) 66(DNS)-86(QSY) 20 ℃ 20 ℃ 45 ℃ 49 ℃ 75 ℃ 75 ℃ A bimodal distritbuion of native and denatured states for 2-66. 66-86 does not show the cooperative folding.

  14. -Discussion-Cooperative or Non-cooperative Folding? U U N N N U Sadqi, Fushman, Muñoz Nature 442, 317-321 (2006)

  15. 10 15 20 25 30 35 40 45 50 55 10 15 20 25 30 35 40 45 50 55 r (Å) r (Å) -Result-Residual Structures in the Unfolded States 66(DNS)-86(QSY) 25.6 Å (~49 Å) Native State in 0.25 M GuHCl 20 ℃ Unfolded State in 2.1 M GuHCl 45 ℃ Unfolded State in 6.0 M GuHCl 75 ℃

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