Fluorescence Scanning and Kinetics of Lysozyme

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This technical note is a case study into a lysozyme folding pathway using the Scanning emission Monochromator (SX/SM). The accessory comprises a second programmable monochromator and a light guide to connect the cell-block to the second monochromator and detector. The SX/SM is designed to extend the experimental capabilities of the standard fl uorescence configuration.


SINGLE WAVELENGTH KINETIC STUDIES

Protein folding, followed using intrinsic tryptophan fluorescence to probe structural change,demonstrates the extended fluorescence capability of the SX20 stopped-flow instrument. Unfolded lysozyme 2.2mg/mL lysozyme in 6M guanidine hydrochloride was mixed in a ratio of 1:10 with pH 7.0 phosphate buffer to obtain a final lysozyme concentration of 200μg/mL. 280nm was used for fluorescence excitation. Emission kinetic records were acquired at wavelengths between 305 and 405nm (1000 data points per second, 25°C).


GLOBAL ANALYSIS OF MULTIPLE WAVELENGTH KINETIC STUDIES

The SX/SM also enables multiple wavelength fluorescence kinetic studies (i.e. collection of a series of single wavelength fluorescence traces recorded over a range of selected wavelengths). This allows complex reactions to be reliably measured. The very large data sets generated in multiple wavelength studies are easily analysed using our ProK-IV global analysis software. This technique simultaneously fits across a complete wavelength range and time course, producing kinetic parameters which satisfy the data at all points.

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  • Biologic stopped-flows are based on the design shown here.
  • This presentation is based exactly upon the SX series technical note, Fluorescence Scanning and Kinetics Using Option SX/SM and follows the document step by step. The technical note should be handed out at the end of the presentation. This section tells a very nice story of how the correct conditions can be calculated for a folding study (excitation/emission ranges) and then characteristics of the kinetics calculated from firstly single wavelength, then multi-wavelength experiments.
  • These results show us the excitation maxima (280nm) to be used and the emission range (300-405nm) to be studied in the next two experiments discussed in this presentation and the accompanying technical note. Note - overall sensitivity is less since narrower emission bandwidths are selected and optical coupling between the emission viewport and the monochromator is via an optical light guide rather than direct coupling. However, much of this sensitivity can be restored by operating the emission detector at a higher applied voltage.
  • The next part of our story. The unfolded lysozyme is at 2.2mg/mL in 6M guanidine hydrochloride,mixed 1:10 with pH 7.0 phosphate buffer (final lysozyme concentration, 200μg/mL). Using data from the steady-state fluorescence scanning experiment, 280nm is used for fluorescence excitation and the known emission range of 305-405nm is studied, in this case 305nm and 340nm spectra are chosen, 1000 data points over 1.6 seconds, experiment ran at 25°C.
  • The data obtained at 340nm shows a biphasic change which fits well using the two exponential equation present in the equation library of the instrument control software (rate 1 = 64.3 s-1; rate 2 = 4.32 s-1).
  • The data at 305nm shows a monophasic change only and a good fit is obtained (rate = 5.53 s-1) using the single exponential equation.
  • This slide continues the story that much more data can be obtained by using the SX/SM, but now also using Pro-K IV global analysis software
  • The very large data sets generated in multiple wavelength studies are easily analysed using Pro-K IV global analysis software. This technique simultaneously fits across a complete wavelength range and time course, producing kinetic parameters which satisfy the data at all points. In the case of lysozyme refolding, fluorescence kinetic traces were automatically recorded every 5nm between 305 and 405nm; a 3-D representation of this data generated by Pro-K IV is shown
  • SVD results calculated in Pro-K IV provide information on the number of reaction components and hence the minimum reaction complexity. In this case, three species are indicated. Analysis employing a two-step model A>B>C yields rate constants of k1 = 67.581s-1 and k2 = 4.90s-1. The model describes the transition of the unfolded lysozyme through a stable refolding intermediate prior to reaching the refolded state.
  • This, and the next slide show the power of the Pro-K IV ‘display review’ capability. This slide gives the overlaid data (actual data overlaid with ‘best fit’ data) and concentration profiles of the lysozyme species
  • This, and the previous slide show the power of the Pro-K IV ‘display review’ capability. Shown here are calculated spectra of the lysozyme species and residuals plot of the kinetics being studied – these are the differences between original data set and best fit data set.
  • C1 = Biologic, C2 = TgK Scientific (or Hi-Tech Scientific), C3 = Kintek, C4 = Olis (the Olis figure is an overestimate because on some stopped-flow papers, the use of Olis software is cited but an Olis stopped-flow is not being used).
  • Fluorescence Scanning and Kinetics of Lysozyme

    1. 1. PRÄSENTATIONSTITEL, ARIAL 14 2013, LEATHERHEAD APPLIED PHOTOPHYSICS LTD Protein Folding Kinetics; A Study using Stopped Flow Spectrometry
    2. 2. 2013, LEATHERHEAD  A spectroscopic technique used for studying fast reactions in solution over timescales in the region 1ms up to 100’s seconds.  Two reagents are rapidly mixed together and the flow is ‘stopped’ in an observation cell.  The product of the reaction must have different optical properties from the reagents so that the changes in the optical signal over time (usually a fluorescence or an absorbance change) is recorded as the reaction is proceeding in the observation cell. What is Stopped-Flow? The figure shows a fluorescence reaction (protein refolding). Note that in this example two kinetic events occur before the reaction is complete.
    3. 3. 2013, LEATHERHEAD  Kinetic analysis of the resulting trace can determine the reaction rate or rates, information on complexity of the reaction mechanism, information on short-lived reaction intermediates etc. k1 = 68.96 0.23 s-1 k2 = 4.10 0.01 s-1 The figure shows the same trace after kinetic analysis – curve fitting to a two phase reaction model i.e. k1 k2 A B C The fitted curve is shown overlaid on the data trace. The calculated reaction rates are shown What is Stopped-Flow?
    4. 4. 2013, LEATHERHEAD Typical research areas for stopped-flow include reaction mechanisms, drug-binding processes, determination of protein structure.  More specifically:  Protein-protein interactions  Ligand binding  Electron transfer  Fluorescence resonance energy transfer (FRET):  Protein folding  Enzyme reactions  Chemical reactions  Coordination reactions  A series of stopped-flow experiments are also used to show the effect of parameters such as temperature, pH and reagent concentration on the kinetics of the reaction.  Most universities in Europe and the USA have at least one stopped-flow spectrometer. What is Stopped-Flow?
    5. 5. 2013, LEATHERHEAD Understanding Stopped-Flow Drive syringes Stop syringe Mixer Light source Fluorescence detector Longpass filter Observation cell Absorbance detector Hard stop Trigger leaf Drive ram Typical stopped-flow design.  Reagents contained in two drive syringes
    6. 6. 2013, LEATHERHEAD Light source Absorbance detector Fluorescence detector Drive ram Hard stop Trigger leaf Stop syringeDrive syringes Observation cell Mixer Longpass filter Understanding Stopped-Flow Typical stopped-flow design.  Reagents contained in two drive syringes  Drive ram pushes the syringe-pistons: Reagents pass through mixer to the observation cell.  ‘Old’ cell contents goes to the stop-syringe, filling until the piston hits a trigger- switch and hard stop.  This simultaneously stops the flow and starts data acquisition.
    7. 7. 2013, LEATHERHEAD Light source Absorbance detector Fluorescence detector Drive ram Hard stop Trigger leaf Stop syringeDrive syringes Observation cell Mixer Longpass filter Understanding Stopped-Flow Typical stopped-flow design.  Flow is stopped.  The Dead Time (reaction time of the newly mixed reagents in the observation cell is approx 1ms.  Dead Time is dependent upon the design of the observation cell and the stopped-flow sample handling unit.  Observation cell now irradiated with light; detector connected to the trigger leaf so data acquisition begins as flow is stopped.
    8. 8. 2013, LEATHERHEAD light source absorbance detector Fluorescence detector Flow circuit of the SX20 stopped-flow.  Mixer - Integral part of the (quartz) observation cell.  Stop valve - Located above the stop- syringe. Enables contents of the stop- syringe to be emptied after each experiment, ready for the next stopped- flow drive. Automated to allow multiple experiments to be performed without user intervention Stop-syringe empty (to a waste bottle) Understanding Stopped-Flow
    9. 9. 2013, LEATHERHEAD Light source Fluorescence detector ‘Stopping valve’ Drive syringes Observation cell Mixer Longpass filter waste Absorbance detector Understanding Stopped-Flow  In some designs, flow is ended by the drive ram(s) stopping. This requires coordination with the closing of a ‘stopping-valve’ in the flow line, an added level of complication.  This design causes issues with very fast kinetics, the valve taking 1-2ms to close.
    10. 10. 2012, LEATHERHEAD  The SX20 system, in its basic fluorescence configuration has exceptional sensitivity, but can be further optimised  In this mode, the fluorescence detector is mounted directly to the emission viewport  A cut-off filter is used to eliminate scattered excitation light  A second, programmable monochromator with light guide fitted between the cell and detector greatly enhances the experimental capabilities of SX20  Single, or multi-wavelength fluorescence data can be achieved by automatically scanning each emission spectra with the second scanning monochromator  Steady-state emission and excitation scanning is also supported SX/SM Second Programmable Scanning Emission Monochromator Application – Lysozyme Folding Kinetics using SX/SM Typical component layout for the SX/SM option
    11. 11. 2012, LEATHERHEAD  Option SX/SM allows emission wavelengths to be selected using a second monochromator  Enabling excitation/emission spectra and wavelength dependent kinetic traces to be obtained  Green – Excitation spectrum of folded lysozyme (maxima 280nm)  Red – Emission spectrum of folded lysozyme (300nm to 405nm) SX/SM Steady-state Fluorescence Scanning of Lysozyme Application – Lysozyme Folding Kinetics using SX/SM
    12. 12. 2012, LEATHERHEAD  This shows the folding of lysozyme using option SX/SM demonstrating the extended fluorescence capability that a second scanning monochromator offers  The experiment follows folding using intrinsic tryptophan fluorescence to probe structural change  Unfolded lysozyme in GuHCl is mixed with Phosphate buffer at pH 7.0  1:10 mixing ratio  280nm was chosen as the excitation wavelength  Traces were acquired at 305 (red) and 340nm (green) SX/SM Single wavelength Kinetic Study – Lysozyme Folding Application – Lysozyme Folding Kinetics using SX/SM
    13. 13. 2012, LEATHERHEAD  Data obtained at 340nm shows a biphasic change  Fits well with a two exponential equation  Rate 1 = 64.3 s-1  Rate 2 = 4.32 s-1 SX/SM Single wavelength Kinetic Study – Lysozyme Folding Spectral Biphasic kinetic trace observed at 340nm Application – Lysozyme Folding Kinetics using SX/SM
    14. 14. 2012, LEATHERHEAD  Whereas data obtained at 305nm shows a monophasic change only  Fitting well with a single exponential equation  Rate = 5.53 s-1 SX/SM Single wavelength Kinetic Study – Lysozyme Folding Single phase kinetic trace observed at 305nm Application – Lysozyme Folding Kinetics using SX/SM
    15. 15. 2012, LEATHERHEAD  As can be seen in the previous example, a reaction can give different exponential fits, obtained at different wavelengths  It is also possible to perform multiple wavelength fluorescence kinetics  A series of single wavelength traces recorded over a selected range  Complex reactions can be reliably analysed  A large data set is generated  Easily analysed by global analysis software SX/SM Multiple wavelength Kinetic Studies and Global Analysis Application – Lysozyme Folding Kinetics using SX/SM
    16. 16. 2012, LEATHERHEAD  Looking again at Lysozyme refolding –  Fluorescence kinetic traces recorded between 305nm and 405nm at 5nm steps  Giving this 3 dimensional representation of the recorded data SX/SM Multiple wavelength Kinetic Studies and Global Analysis Application – Lysozyme Folding Kinetics using SX/SM
    17. 17. 2012, LEATHERHEAD  Looking again at Lysozyme refolding –  Singular value decomposition (SVD) results can be calculated  Giving information on the number of reaction components  This also indicates minimum reaction complexity  Rows 4 to 8 show only noise  Therefore three species are indicated SX/SM Multiple wavelength Kinetic Studies and Global Analysis Application – Lysozyme Folding Kinetics using SX/SM
    18. 18. 2012, LEATHERHEAD  Looking again at Lysozyme refolding –  Overall information obtained from global analysis:  Actual data overlaid with ‘best fit’ data  Concentration profiles of the lysozyme species SX/SM Multiple wavelength Kinetic Studies and Global Analysis Application – Lysozyme Folding Kinetics using SX/SM
    19. 19. 2012, LEATHERHEAD  Looking again at Lysozyme refolding –  Overall information obtained from global analysis:  Calculated spectra of the lysozyme species  Residuals of the fit SX/SM Multiple wavelength Kinetic Studies and Global Analysis Application – Lysozyme Folding Kinetics using SX/SM
    20. 20. 2012, LEATHERHEAD 0 1000 2000 3000 4000 5000 6000 7000 Google Scholar Publications APL C1 C2 C3 C4 This table shows the number of stopped-flow publications on Google Scholar for each stopped-flow manufacturer. Applied Photophysics stopped-flows contribute more that the combined total of the two main competitors Applied Photophysics is the Market Leader
    21. 21. 2012, LEATHERHEAD  Over 40 years supplying spectrometry to the global scientific community.  Unrivalled technical and applications support in  Circular Dichroism Stopped-Flow  UV-VIS – Fluorescence Stopped-Flow  Laser Flash Photolysis Stopped-Flow  Large number of installed systems and reference sites worldwide.  Reliable and responsive customer care throughout the world.  Ongoing commitment to cutting edge technological advancement and education through webinars, symposia and support materials. Applied Photophysics Ltd
    22. 22. 2012, LEATHERHEAD Applied Photophysics Ltd. 21, Mole Business Park Leatherhead Surrey, KT22 7BA, UK Tel (UK): +44 1372 386 537 Tel (USA): 1-800 543 4130 Fax: +44 1372 386 477 www.photophysics.com Thank you.

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