Chem 344 slides for Kinetics Methods extras!

Transcription

1 Chem 344 slides for Kinetics Methods extras! These are slides from a talk to my research group on dynamic protein spectra. It was meant ot be an example of the ideas we were talking about with Methods of Fast Kinetics. I tried to show some of them in class Wed, 9/13, but the printing failed. The idea is we can (a) mix things fast and do millisec kinetics or (b) do temperature jumps and get to nanosec time scales We detect change with fluorescence, absorbance, CD or IR With IR we can look at helix and sheet separately due to frequency discrimination (helix ~1650 and sheet ~1630 cm -1 ) With CD we can do this for helix, monitor 222 nm (negative) Fluorescence is mainly tertiary structure information Absorbance in UV is good for DNA, RNA changes

2 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 Most T-jump single ν with tunable IR laser for S/N, filtering and..

3 Scheme of Stop Flow initialize by rapid mixing Syringe drive system Backplate Cell and mixer blowout Gasket Cell Window Spacer Cell Window Front Plate Reagent Protein Cell nest Time restriction from flow To Cell between windows and size Mixer Luer Plug Mix protein and perturbant rapidly to get new state, follow spectra

4 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 cm -1 loss of random coil cm -1 (random coil) k = s log (S i /S f ) Peak Intensity cm -1 (sheet) k = s Wavenumber (cm -1 ) 1630 cm -1 gain of sheet Sheet refolding 2x slower than loss of coil time (s) One single beam spectrum (IF scan) is collected for each time point. Time resolution = 50 ms, but could be faster, if modify. IR resolution 8 cm -1 sufficient to separate increase in sheet, decrease in coil as folds.

5 Bacteriorhodopsin - flash photolysis: initiate cis-trans in retinal follow time resolved step-scan FTIR 700 ns 8 µs Terrific sensitivity from measuring the baseline for each pulse by recording the signal just before the strobe no drift Difference yields spectrum of L intermediate Systems that can be photo initiated to new state (like BR) and relax back reversibly offer possibility of fast kinetics, specific sites Weidlich, Siebert, Appl. Spect. 1993

6 Callender/Dyer fluorescence T-jump setup Cavity doubled, lots cw power 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, similar for IR (except transmit to MCT)

7 Apo-Mb kinetics, T-jump Fluorescence & IR Fluorescence IR

8 T-dependence of rates IR-two phases Fluorescence (+) match fast IR (x) Slow phase IR

9 Searle design (Nottingham) concept like Dyer IR T-jump Diode laser probe sample Fast MCT detector YAG pump Raman shifter Minimal schematic transient recorder

10 Apo-Mb thermal unfolding, amide I Very modest spectral change highly helical native state helix 2 nd derivative shows mostly helical contribution Difference spectra show loss of helix (1648) gain at 1673 cm -1 Gilmanshin, et al. PNAS 1997

11 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

12 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 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

13 Helix T-jump, setup data suc-fs 21-peptide: Suc-AAAAA-(AAARA) 3 A-NH 2 (Suc - succinyl, A - alanine, and R -arginine). Williams...Dyer, Biochemistry, 35, 691, 1996

14 Analysis--partial sigmoid fit Stern-Volmer

15 Unlabeled helix 1658 cm -1 colder 1632 cm -1 Gai, et al. J. Am. Chem. Soc., 123, 9235, 2001

16 Slow unfolding phase - Arrhenius behavior unlabeled helix Fast component, <20 ns is this instrumental rise (artifact?)

17 Isotopically labeled helix unfolding Gai, Degrado, PNAS 99, , 2002 Stretched exponential best fit OD(t)= A[1-Bexp(t /τ) β ] β B