Rapid Kinetics with IR Protein folding examples

Rapid Kinetics with IR Protein folding examples

Time dependent data with FTIR Stop-flow methods - msec limits so far Continuous, micro-flow methods - < 100 µsec Rapid scan FT-IR - msec Multichannel laser Raman, faster - µsec T-jump and Flash photolysis -nsec time scales using step scan methods

Tyr92 Tyr97 H1 Tyr115 Tyr73 Ribonuclease A combined uv-cd and H2 Tyr25 H3 Tyr76 FTIR study Simona Stelea, Prot Sci 2001 124 amino acid residues, 1 domain, MW= 13.7 KDa 3 α-helices 6 β-strands in an AP β-sheet 6 β β sheet 6 Tyr residues (no Trp), 4 Pro residues, 2 (2 cis, ) 2 trans)

Scheme of Stop Flow Syringe drive system Backplate Cell and mixer blowout Gasket Cell Window Spacer Cell Window Front Plate Reagent Protein Cell nest Mixer To Cell Luer Plug Mix protein and perturbant rapidly to get new state, follow spectra

Refolding of Ribonuclease A by FTIR Inverse T-jump: Refolding initiated by injecting Ribo A stored in syringe at 80 C into IR cell at 25 C 0.02 1660 cm -1 loss of random coil 0.01 1660 cm -1 (random coil) k = 0.342 s -1 0.01 log (S i /S f ) 0.00-0.01 Peak Intensity 0.00-0.01 1632 cm -1 (sheet) k = 0.156 s -1-0.02-0.03 1750 1700 1650 Wavenumber (cm -1 ) 1630 cm -1 gain of sheet 1600 1550-0.02-0.03 Sheet refolding 2x slower than loss of coil 0 5 10 15 20 time (s) One single beam spectrum (IF scan) is collected for each time point. Time resolution = 50 ms. IR resolution separate coil decrease sheet fold.

%TFE Austin, Gerwert PNAS 98 2001, 6646 Continuous flow mixer Top view: green: inlet channels, red: 8µm deep outlet channel Side view: 2D Fluid dynamics simulation

Lifetimes of intermediates in the β-sheet to α helix transition of β-lactoglobulin using diffusional IR mixer E. Kauffmann, N. C. Darntont, R. H. Austin, C. Batts, and K. Gerwert PNAS 2001 98 6646-6649 a) Spectra along channel: 1.1,3.4,5.7,10.2,21.6,103 ms b) 2 nd deriv. & 3-state fit c) 3 basic spectra derived d) Time course of 3 states

Lipid-induced Conformational Transition of β-lactoglobulin: Equilibrium and Kinetic Studies Globular protein with 9-stranded sheet (flattened β-barrel) and one helical segment Terminal segments have high helical propensity Good model for β-to-α conversion Binding to lipid vesicle acts as perturbation cell model Too complex to waste on a single technique! Xiuqi Zhang, Ning Ge,TAK Biochemistry 2006/2007

Lipid-induced Conformational Transition of β-lactoglobulin Introduction β α transition Driving force Membrane insertion β-lactoglobulin: Native state: β-sheet dominant, but high helical propensity. Model: intramolecular β α transition pathway as opposed to folding pathways from a denatured state.

Lipid-induced Conformational Transition of β-lactoglobulin Introduction β α transition Driving force Membrane insertion 1. DMPG-dependent β α transition at ph 6.8 [θ] 10-3 /deg.cm 2.dmol -1 25 20 15 10 5 0-5 -10-15 [θ] 222nm 10-3 /deg.cm 2.dmol -1-4 -6-8 -10-12 -10 0 2 4 DMPG/mM 190 200 210 220 230 240 250 Wavelength/nm -5-6 -7-8 -9 A -11 0.0 0.1 0.2 0.3 0.4 Fractional secondary structure 0.5 0.4 0.3 0.2 0.1 Unordered α-helix β-sheet 0 1 2 3 4 5 DMPG / mm

Lipid-induced Conformational Transition of β-lactoglobulin Introduction β α transition Driving force Membrane insertion 1. DMPG-dependent β α transition at ph 6.8 2. Tertiary structure change Fluorescence 10 8 6 4 2 Relative fluorescence 1.4 1.3 1.2 1.1 1.0 0.0 0.5 1.0 1.5 2.0 DMPG/mM 0 300 330 360 390 420 450 Wavelength/nm [θ]/deg.cm 2.dmol -1-20 -40-60 -80 0 Near-UV CD 284nm 0mM DMPG 1mM DMPG 3mM DMPG 9mM DMPG 6M GndCl 292nm 260 280 300 320 340 Wavelength/nm

Dynamics--Scheme of Stopped-flow -add dynamics to experiment System protein solution Lipid vesicle solution

Kinetics for βlg in Membrane Fluorescence Circular Dichroism -10 2.0 2mM DOPG 1.9 1mM DOPG N Ellipticity(mdeg) -30 0.5mM DMPG -40 1mM DMPG 1.8 Relative Intensity 0.15mM DMPG 0.5mM DMPG -20 0.5mM DOPG 1.7 0.25mM DOPG 1.6 1.5 1.4 0.15mM DOPG 2mM DMPG 5mM DMPG 1.3 1.2-50 0 5 10 Time/s 15 20 0 5 10 15 20 Time/s CD fits single exponential, fluorescence (Fl) fits two. Rate constant for CD is slower than fast Fl kinetic

Summary N w Binding N s Unfolding U s Insertion U m

Laser induced Temperature jump IR pulse heats the solvent ( Raman shifted YAG to 1.9 µ for D 2 O) Probe heated spot with tunable IR laser (Pb-salt diode, FTIR experiments proposed) Fast MCT needed for ns response Repetition rate limited by cooling back initial state Analysis is relaxation kinetics, k rel = k f + k r Signal average thousands of shots, single frequency (diode laser) normal method

Callender/Dyer general T-jump setup Fluorescence use cavity doubled, lots cw power Fast MCT Diode laser 180 o back scatter geom. H 2 gives 1.9 µ for D 2 O, CH 4 ~1.5 µ H 2 O Generic design for T-jump, IR diode laser detection transmit to MCT

Character of Temperature jump--timing Helix example D 2 O - - - Sample Difference: a-1655 cm -1 b-1644 cm -1 c-1637 cm -1 d-1632 cm -1 Fit to biexpon. <10 ns 160+/-60 ns 10ns D 2 O Pump T at 2µm focus to 300µm, 110 µm path use split cell fast (50MHz) MCT detector, avg. 9000 shots, 10 Hz T-jump calibrated by change of D 2 O absorption with temperature 3.0x10-5 to 4.0x10-5 (OD)/ C.µm for 1700 and 1632 cm -1

Apo-Mb kinetics, T-jump Fluorescence & IR Fluorescence Follow different processes, µs response IR Fluorescence tertiary structure unfold IR secondary structure - helices

Kinetic IR response to T-jump (45-60 C) - apo Mb Solvated helix (1632 cm -1 ) lost very fast, ~100 ns, as is 1664 (turns?) protected helices (1655 cm -1 ) slower. Laser pulse heat water in 10 s ns Gilmanshin, et al. PNAS 1997

Vilin head-piece very fast folder

A57 13 C labeled Vilin Head-piece Results (IR/T- Jump) 1573 cm -1 1644 cm -1 Dyer