Embed Size (px)
DESCRIPTION
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.
Citation preview
PRÄSENTATIONSTITEL, ARIAL 14
2013, LEATHERHEAD
APPLIED PHOTOPHYSICS LTD
Protein Folding Kinetics; A Study using Stopped Flow Spectrometry
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.
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?
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?
2013, LEATHERHEAD
Understanding Stopped-Flow
Drive syringes Stop syringe
Mixer
Light source
Fluorescence detector
Longpass filter
Observationcell
Absorbance detector
Hard stop
Trigger leaf
Drive ram
Typical stopped-flow design.
Reagents contained in two drive syringes
2013, LEATHERHEAD
Light source
Absorbance detector
Fluorescence detector
Drive ramHard stop
Trigger leaf
Stop syringeDrive syringes
Observationcell
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.
2013, LEATHERHEAD
Light source
Absorbance detector
Fluorescence detector
Drive ramHard stop
Trigger leaf
Stop syringeDrive syringes
Observationcell
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.
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
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.
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
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
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
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
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
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
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
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
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
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
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
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
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 4130Fax: +44 1372 386 477
www.photophysics.com
Thank you.
