Determination of the protein-ligand binding volume by high-pressure spectrofluorimetry

Transcription

1 Determination of the protein-ligand binding volume by high-pressure spectrofluorimetry Vytautas Petrauskas Department of Biothermodynamics and Drug Design Institute of Biotechnology, Vilnius University February 15-17, 2016 Vytautas Petrauskas (Vilnius University) IMBP 8, Dortmund February 15-17, / 18

2 Introductory remarks Protein-ligand binding (reaction) volume Definition: the change in protein volume observed upon protein-ligand binding; Motivation: fundamental knowledge about protein-ligand interaction and pressure-induced protein denaturation. ( ) Gb Protein-ligand binding volume: V b = P ; T Vytautas Petrauskas (Vilnius University) IMBP 8, Dortmund February 15-17, / 18

3 Introductory remarks Heat shock protein 90 and carbonic anhydrases N-terminal domain of human Hsp90 (229 a.a.), PDB ID: 2YI6 Human carbonic anhydrase (CA) II (260 a.a.), PDB ID: 3HS4 Vytautas Petrauskas (Vilnius University) IMBP 8, Dortmund February 15-17, / 18

4 Experimental set-up of high-pressure spectrofluorimetry Pressure range: ( ) MPa; Temperature range: (10 95) ; Fluorescence probe: intrinsic tryptophan (exited at 295 nm); Emission spectra range: ( ) nm. Vytautas Petrauskas (Vilnius University) IMBP 8, Dortmund February 15-17, / 18

5 Description of pressure-induced unfolding profiles Equations Unfolding profiles: f (p) = f N + f U f N 1 + exp( G(p)/RT ). The Gibbs energy of unfolding: G = G 0 + V 0 p + β 2 ( p)2. The center of spectral mass: λ CSM = i f iλ i. i f i Vytautas Petrauskas (Vilnius University) IMBP 8, Dortmund February 15-17, / 18

6 Pressure-induced unfolding profiles of CA II and CA I Intrinsic tryptophan fluorescence spectra at various pressures Vytautas Petrauskas (Vilnius University) IMBP 8, Dortmund February 15-17, / 18

7 The shift in P m and dosing curves Fluorescent Pressure Shift Assay (FPSA) Vytautas Petrauskas (Vilnius University) IMBP 8, Dortmund February 15-17, / 18

8 Description of dosing curves Mathematical background P t = N f + N b + U, L t = L f + N b, K U = U N f, K b = N b N f L f. The final form of dosing curve equation: ( 1 L t = (K U 1) K b ( = exp G ) b = RT P t and L t the bulk concentrations of protein and ligand; N f and N b concentrations of native ligand-free and bound protein; U concentration of unfolded protein; K U and K b equilibrium constants of protein unfolding and protein-ligand binding reaction; At melting pressure p m, U = P t /2. K b + P t 2K U ) G 0b + V 0b (p m p 0 ) + β b 2 (p m p 0 ) 2 RT Vytautas Petrauskas (Vilnius University) IMBP 8, Dortmund February 15-17, / 18

9 Hsp90N protein stability diagram in P T coordinates With added ligand Petrauskas et al. Eur Biophys J 42 (2013) Vytautas Petrauskas (Vilnius University) IMBP 8, Dortmund February 15-17, / 18

10 Hsp90N protein stability diagram The Gibbs energy dependence on pressure and temperature Vytautas Petrauskas (Vilnius University) IMBP 8, Dortmund February 15-17, / 18

11 Binding volume correlation with affinity Affinity was determined by ITC and Fluorescent Thermal Shift Assay Vytautas Petrauskas (Vilnius University) IMBP 8, Dortmund February 15-17, / 18

12 NMR experiments Preliminary results of Hsp90N interaction with two ligands by NMR; ( ) Gb V b = p ; T Vytautas Petrauskas (Vilnius University) IMBP 8, Dortmund February 15-17, / 18

13 NMR data 1 H- 1 5N HSQC spectra of Hsp90N (red) and Hsp90N+ICPD9 (blue) Vytautas Petrauskas (Vilnius University) IMBP 8, Dortmund February 15-17, / 18

14 NMR data analysis Analysis of dosing curves ( ) Gb V b = p G b = RT ln(k b ) = RT ln(k d ) T Experimental changes in chemical shifts: ( ) γ δ = (δ0 H N 2 δh ) 2 + (δ0 N δn ) 2 γ H δ 0 chemical shift without ligand. δ = δ max (L t + P t + K d ) (L t + P t + K d ) 2 4P t L t 2P t Chemical shift equation reference: Morton et al. Structure 4 (1996) Vytautas Petrauskas (Vilnius University) IMBP 8, Dortmund February 15-17, / 18

15 Gibbs energy as a function of pressure G b = G b0 + V b0 p + β b 2 ( p)2. G b0 = 20.7 kj/mol V b0 = 13 cm 3 /mol β b = 0 cm 6 /(J mol) Vytautas Petrauskas (Vilnius University) IMBP 8, Dortmund February 15-17, / 18

16 Gibbs energy as a function of pressure G b = G b0 + V b0 p + β b 2 ( p)2. G b0 = 20.7 kj/mol V b0 = 13 cm 3 /mol β b = 0 cm 6 /(J mol) G b0 = 20.8 kj/mol V b0 = 20 cm 3 /mol β b = 0.05 cm 6 /(J mol) Vytautas Petrauskas (Vilnius University) IMBP 8, Dortmund February 15-17, / 18

17 Binding volume correlation with affinity Including NMR data (blue points) Vytautas Petrauskas (Vilnius University) IMBP 8, Dortmund February 15-17, / 18

18 Concluding remarks Our results suggest that weak ligands of Hsp90N protein induce smaller changes in volume than tight-binding ligands. Both techniques FPSA and NMR provide similar values of binding volume. Vytautas Petrauskas (Vilnius University) IMBP 8, Dortmund February 15-17, / 18

19 Acknowledgments High pressure team at the department of Biothermodynamics and Drug Design: Prof. Daumantas Matulis, Gediminas Skvarnavičius, Dr. Zigmas Toleikis, Joana Smirnovienė, Dr. Piotras Cimmperman, Martynas Grigaliūnas, Povilas Norvaišas NMR data: Prof. Christian Roumestand (CBS Montpellier) Funding: Research Council of Lithuania (grant No. MIP-004/2014) High-pressure team Thank you for your attention! Vytautas Petrauskas (Vilnius University) IMBP 8, Dortmund February 15-17, / 18

20 Supplementary material Vytautas Petrauskas (Vilnius University) IMBP 8, Dortmund February 15-17, / 7

21 FTSA and FPSA methods Examples of Hsp90N protein stabilization by ligand against T and P denaturation Vytautas Petrauskas (Vilnius University) IMBP 8, Dortmund February 15-17, / 7

22 FTSA and FPSA methods Expressions for the Gibbs energy f = f N + f U f N 1 + exp( U G/RT ) U G as a function of temperature at a constant pressure: ( U G T = U G 0 U C p [T ln T ) ] ( ) T 1 + T 0 ( U H 0 U G 0 ) 1 T 0 T 0 U G as a function of pressure at a constant temperature: U G P = U G 0 + U V 0 (p p 0 ) + Uβ 2 (p p 0 ) 2 Vytautas Petrauskas (Vilnius University) IMBP 8, Dortmund February 15-17, / 7

23 The Gibbs energy change The full differential of the Gibbs energy change G = G U G N d( G) = SdT + V dp as a function of temperature T and pressure P. G = β 2 (p p 0) 2 + V 0 (p p 0 ) + α(p p 0 )(T T 0 ) ( C p [T ln T ) ] ( ) T 1 + T 0 ( H 0 G 0 ) 1 + G 0 T 0 T 0 Vytautas Petrauskas (Vilnius University) IMBP 8, Dortmund February 15-17, / 7

24 Hsp90N protein stability diagram in P T coordinates With added ligand Vytautas Petrauskas (Vilnius University) IMBP 8, Dortmund February 15-17, / 7

25 Hsp90N stabilization by ligands The use of guanidine hydrochloride a protein destabilizing agent Vytautas Petrauskas (Vilnius University) IMBP 8, Dortmund February 15-17, / 7

26 Protein stabilization by ligands Hsp90N + radicicol Hsp90N melting temperatures T m [Hsp90N] = 14 µm [Radicicol] µm T m ( ) Hsp90N + radicicol PDB ID: 4EGK Zubriene et al. Int J Mol Sci 10 (2009) Vytautas Petrauskas (Vilnius University) IMBP 8, Dortmund February 15-17, / 7