timetable of course 09.08.07 · outline of the experiments during the course: 1. modelling i...

Timetable of Course „Modelling and Spectroscopy of Proteins“

09.00-12.00 13.30-16.30 Monday September 10th Lecture

(K. Gerwert) Introduction into structure and function of proteins

Experimental Course (C. Kötting, C. Burisch, W. Stacklies) Introduction into Pymol Software for protein structure

Tuesday September 11th

Lecture (K. Gerwert) Structure and Mechanism of proteins on examples

• bacteriorhodopsin • Ras

Introduction into MD simulations (Frauke Gräter)

Experimental Course (C. Kötting, C. Burisch, W. Stacklies) Modelling of proteins

Wednesday September 12th

Lecture (K. Gerwert) Introduction into Spectroscopy: UV/VIS, fluorescence, IR, Raman

Experimental Course (C. Kötting, C. Burisch, W. Stacklies) UV/VIS Spectroscopy on proteins

Thursday September 13th Experimental Course

(C. Kötting, C. Burisch, W. Stacklies) time-resolved Spectroscopy

Experimental Course (C. Kötting, C. Burisch, W. Stacklies) Analysis of time-resolved data

Friday September 14th Experimental Course

(C. Kötting, C. Burisch, W. Stacklies) Fluorescence Spectroscopy

Symposium of participants 5 minutes talk of each participant on selected experiment

Literature for preparation:

1. Information on the protein bR, which is used throughout the course:

http://www.bph.rub.de/br_en.htm

2. Information on time-resolved spectroscopy of proteins (short):

Kötting, C., Gerwert, K. Protein Reactions: Resolved with tr-FTIR Spectroscopy europe (19) Vol.3, 19-23 (2007) (PDF-File - 828kB)

3. Information on time-resolved spectroscopy of proteins (long):

Kötting, C., Gerwert, K. Monitoring Protein-Protein Interactions by Time-resolved FTIR Difference Spectroscopy In: Protein-Protein Interactions; A molecular cloning Manual, 2nd edition, Editor: Golemis, E., Adams, P. Cold Spring Harbor, NY, USA, 279-299 (2005) (PDF-File - 11,4MB)

4. Information on data treatment:

Heßling, B., Souvignier, G., Gerwert, K. A Model-Independent Approach to Assigning Bacteriorhodopsin's Intramolecular Reactions to Photocycle Intermediates. Biophysical J. 65, 1929-1941 (1993). (PDF-File - 6,9 MB!) - (Abstract)

http://www.bph.rub.de/br_en.htm

http://www.bph.ruhr-uni-bochum.de/%7Egerwert/113.pdf





http://www.biophysj.org/cgi/content/abstract/65/5/1929?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&searchid=1039704352210_1238&stored_search=&FIRSTINDEX=0&volume=65&firstpage=1929&journalcode=biophysj

Outline of the Experiments during the course:

1. Modelling I (Pymol)

1.1 Selection of atoms (example bR)

1.2 Display types (example bR)

1.3 Measuring distances of functional groups in bR

1.5 Measuring dihedral angles in bR

1.6 Displaying the temperature factor of bR

1.7 Comparison of temperature factors of bR and myoglobin (graph in Origin)

2. Modelling II and MD simulation (Pymol and Yasara)

2.1 Analysis and display of the active site of Ras

2.2 Structural comparison of inactive and active Ras (superposition)

2.3 Dihedral angle difference plot of inactive and active Ras

2.4 Short introduction to force fields and MD simulation of bR

2.5 Structural comparison of X-ray and MD structure

3. Static UV/Vis Spectroscopy on proteins

3.1 Determination of the absorption coefficient of bR

Determine the concentration of the bR suspension. Now measure the absorption at 568 nm with the spectrometer and calculate ε568 according to Lambert Beer.

3.2 Measurement of the UV/Vis spectrum of

A Whole cells

B destroyed cells

C isolated purple membrane

3.3 Calculation of the number of cells in A (Z=1.5*109 OD [cells/ml]

3.4 Calculation of the concentration of bR in A, B and C

Iscatter~λ-4

3.5 Calculation of the bR molecules per cell

3.6 Calculation of the percentage of the coverage of the cell membrane of bR

cell: cylinder with 5µm height and 1 µm diameter

bR: cylinder with a diameter of 40 Å.

3.7 Use sample C and dilute, so that E568=0.4 and take a spectrum

Then change the pH to ~2-3, take a spectrum, change to pH 6-7 and take another spectrum

3.8 Add 20 µl 20% SDS and mix. Take spectra until no further change is observed

3.9 Add some crystals of hydroxylamine until a clear change in the spectrum is observed

4 Time resolved spectroscopy

4.1 Measurement of the bR-photocycle at pH=7

Export kinetics at 410, 530, 570 and 640 nm

4.2 Repeat measurements at pH=5 and pH=8

4.3 Repeat the measurement of the D96N mutant (PH=7)

5 Analysis of time resolved data

5.1 Introduction into curve fitting with Origin

5.2 Analysis of the data obtained in 4

6 Fluorescence

6.1 Measure the Absorption and the Fluorescence Spectra of Resorufin and Tryptophan

6.2 Measure the Emission spectrum of the ceiling lamp http://en.wikipedia.org/wiki/Fluorescent_lamp

6.3 Measure the Absorption spectrum of Pyranin (50µm, 1M KCl) at pH 2, 7 and 10.

6.4 Measure the bR Photocycle (10µm bR, 50 µm Pyranin, pH=7, 1M KCl)

Do a control measurement as in 5.4 but without bR!

http://en.wikipedia.org/wiki/Fluorescent_lamp

Manual

UV-VIS 190-720nm

UV-VIS 350-1050nm

Temperature-controlled sample holder TCL 50FTM

Diode array spectrometer 24.08.2007

Table of contents

Table of contents...................................................................................................... 2 1 Diode array spectrometer UV-VIS 190-720nm ............................................. 3 1.1 ..................................................................... 3 Setting up and starting operation1.2 .............................................................................. 5 Switching on the apparatus2 Diode array spectrometer UV-VIS 350-1050nm ........................................... 6 2.1 ..................................................................... 6 Setting up and starting operation2.2 .............................................................................. 6 Switching on the apparatus2.3 ................................................................................................ 6 Preadjustments

.......................................................................................................................6 2.3.1 Focusing .........................................................................................7 2.3.2 Manual intensity adjustment

3 Measurement procedures ............................................................................. 8 3.1 .............................................................................. 8 Determining the dark value3.2 ....................................................................................... 8 Static measurements3.3 ..................................................................................... 9 Kinetic measurements3.4 .................................................................................................. 12 Slow kinetics3.5 ............................................................................. 13 Saving and exporting data3.6 ........................................................................................................... 13 Printing4 Temperature-controlled sample holder TCL 50FTM................................... 14 4.1 ............................................................................................................ 14 Set-up4.2 ............................................................................................................ 15 Usage5 Addendum .................................................................................................... 16

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1 Diode array spectrometer UV-VIS 190-720nm

1.1 Setting up and starting operation The spectrometer is set and wired up as follows:

Figure 1: Diode array spectrometer UV-VIS 190-720nm

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Spectrometer Front Back

Light source (AvaLight-DHS)

Sample holder

Cable 1 (optical fiber, red) connects the lower part of the spectrometer (front) with the sample holder. Cable 2 (optical fiber) connects the light source with the sample holder. Cable 3 connects the spectrometer (back) with the flash lamp (back). Cable 4 connects the spectrometer (back) with the PC. Cable 5 and Cable 6 connect the lower spectrometer (back) with the front of the upper spectrometer.

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1.2 Switching on the apparatus The devices are switched on in the following order:

1. Start the PC 2. Start “UVVIS_HCH“ program 3. Select interface 4. Switch on the spectrometer 5. Switch on halogen and deuterium bulbs at the light source. Make sure that the

switch “TLL-Shutter“ is turned on 6. Switch on the Xenon flash lamp

Figure 2: Dialog to select the spectrometer interface

Several methods of measurement may be selected which are described in detail in chapter 3.

Figure 3: Starting screen of the “UVVIS_HCH“ application.

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2 Diode array spectrometer UV-VIS 350-1050nm

2.1 Setting up and starting operation The spectrometer is set up and wired as the UV-VIS 190-720nm. A halogen bulb is used as light source, which also changes the measurement range. No optical fibres are used. The beam is focused by a lens (on the optical bench) directly on the input of the sample holder. The output of the sample holder is connected directly to the spectrometer.

Figure 4: The order of components on the optical bench: lamp (1), lens (2), filter holder (3), flashlamp (4), sample holder (5), spectrometer (6)

2.2 Switching on the apparatus The devices are switched on in order spectrometer, light source, PC and flash lamp (the same order as described for the diode array spectrometer UV-VIS 190-720nm in »Switching on the apparatus«). Again select Com1 as interface. Caution: Do not touch the light source since it becomes very hot after a few minutes.

2.3 Preadjustments

2.3.1 Focusing Since an optical bench is used the light of the halogen bulb must be focused on the sample holder prior to the measurement. This is done as follows:

1. All components on the optical bench (light source, lens, filter, and sample holder) must be aligned on a common level.

2. The lens (between light source and filter) is positioned that way that an image of the bulb’s spiral-wound filament is visible on the sample holder.

3. The filter can be positioned anywhere between lens and sample holder.

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2.3.2 Manual intensity adjustment Even when the automatic adjustment (“Auto“) is activated in the software (see 3 Measurement procedures) it may happen that the intensity is too high. In such cases an appropriate filter suitable to the type of measurement has to be used.

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3 Measurement procedures In the following chapters all menu commands are printed in italics. Be sure that the box Auto is checked for automatic scaling of the measured data.

3.1 Determining the dark value To set the base line for the subsequently measured spectra to zero, the dark value is determined prior to the first measurement. In doing so the noise of the spectrometer’s components is subtracted. The procedure is as follows:

1. Turn of the TTL switch on the light source 2. Start Online measurement (the base line is at an intensity value of approx. 300) 3. Stop Online measurement 4. Click Dark value (the dark value is calculated). The number of averages should be at

least as high as the number of averages for the actual measurement (better even more). If the integration time is changed a new dark value has to be determined.

5. Check Dark value check box 6. Start Online measurement. By doing this one can check if the base line is at zero

now. The procedure may not be repeated as long as the check box is checked, since the determined dark value is subtracted automatically from each spectrum measured afterwards.

7. Switch on the TTL switch on the light source. Remark: It is not necessary to determine the dark value for kinetic measurements.

3.2 Static measurements Static measurements are used for samples which do not have to be excited by flashes.

Figure 5: Menu for static measurements

1. Click Static measurement 2. Activate Intensity adjustment (the intensity of the measured spectrum is adjusted

automatically).

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3. Insert the reference sample and start Reference measurement; the measured reference can be kept for subsequent measurements by activating the check box Keep reference?.

4. Insert sample in the holder and start measurement by clicking Sample.

3.3 Kinetic measurements A (fast) kinetic measurement is used for samples which run through intermediates and relax to the initial state after excitation by flash light. The sample to be measured can be excited by periodic flashes from the Xenon flash lamp in a given time interval. The kinetics at any wavelength of interest can be monitored.

1. Insert sample into holder 2. Enter averaging count (max. 1000) in the input field (determines the number of

flashes). 3. Number of spectra (max. 120) in the input field (number of recorded spectra between

two flashes) 4. Start Kinetic measurement

Figure 6: The command Kinetic measurement yields a number of spectra. The wavelength λ in nm is plotted against the relative intensity. The deviant spectrum displayed here is recorded during the exciting flash.

5. Click Calculate extinction

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Figure 7: Menu for calculating the extinction. The default spectrum_X = 1 is recorded prior to the flash.

Figure 8: Calculating the extinction again results in a number of spectra. In the figure the two marked spectra are conspicuous. These are the reference spectrum divided by itself (1) and the “flash” spectrum divided by the reference spectrum (2). Spectra can be hidden and made visible by repeated clicking of the marked ”+“ and ”-“ buttons.

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Figure 9: By clicking the “+“ button twice the spectra 1:1 and 2:1 marked in figure 8 are hidden. By clicking “-“ they are made visible again.

6. With the From and To buttons the displayed wavelength-range can be narrowed for

closer inspection. Full range will reset the displayed wavelength-range to the default values.

7. The kinetics at a certain wavelength can be monitored and plotted against time by clicking Kinetics.

Figure 10: The wavelength can be selected by the sliders (1). The number in the field below the slider (2) shows the diode of the array for which the kinetics is calculated. The corresponding wavelength in nm is also shown (3).

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A new window pops up showing the kinetics at the selected wavelength.

Figure 11: Example of the kinetics of Bacteriorhodopsin recorded at a wavelength of λ=659nm. The check box “logarithmic“ switches between a linear and a logarithmic scale of the time axis. (This feature works only with min. 31 and max. 120 spectra)

3.4 Slow kinetics In this case the sample is excited by a single flash.

1. Click Kinetic measurement (slow) 2. Set number of spectra (max. 500) 3. Get reference 4. Insert sample and Start the measurement

Figure 12: Menu for entering the parameters of a slow kinetic measurement. The “Transient of the wavelength” is the wavelength at which the course of the extinction is measured.

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3.5 Saving and exporting data A measurement can be saved to disk in the proprietary format SPK by clicking the button Save. Data saved this way can be loaded again via the button Load, e.g. for using them as reference. The data types to be saved can be:

• the measurement itself • the reference measurement • the calculated extinction • measured kinetics

To handle the measured data with different programs the export function has to be used. The same four data types can be exported.

Figure 13: The Export dialog.

The following export formats can be selected in the pull-down menu Program:

• Excel • Lambda as X value • Time as X value • Gnuplot • NDL

In case of Excel export the exported file’s path and the path to Excel have to be entered in the corresponding input fields.

3.6 Printing Click Inspect → File → Print setup

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TM4 Temperature-controlled sample holder TCL 50F

4.1 Set-up The entire set-up consists of the following components:

Temperature control unit Water cooling

Sample holder Front Left

Bezel

Wiring of the spectrometer is done as described in Setting up and starting operation. Additionally cable 6 is connected to the back of the temperature control unit.

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4.2 Usage

1. Turn on the device (on the back). 2. Turn on water cooling for the sample holder (peltier element). Make sure the control

valve is never closed completely since the water pressure would be too high. 3. Insert sample cuvette (standard: 1x1 cm) in the sample holder. 4. Insert bezel(s) into the corresponding mount(s). It is reasonable to insert one bezel in

position 1 to limit dispersion or excitation of the sample. 5. If needed put a stirrer rod in the cuvette and adjust the designated stirring speed with

the turning knob. 6. Put the opaque lid on the sample holder. 7. The temperature is adjusted by pressing the up/down key on the right side of the

device. A temperature range of -10°C to +80°C is covered. 8. Confirm with the Run/Stop key. Pressing the key again switches off the temperature

control unit. The red light flashes as long as the set temperature is not reached. When the temperature is reached (±0.02°C) the red light burns constantly.

9. After finishing the measurement, press the Run/Stop key to turn off the temperature control unit. Water cooling also is switched off.

To prevent condensation on the surface of the cuvette and optical elements the sample holder is flushed with dry gas (nitrogen or argon). The flow rate should be between 50 and 200cm³/min. Remark: The temperature controlled sample holder can only be used with the diode

array spectrometer UV-VIS 190 -720 nm!

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5 Addendum Auto Automatic scaling of measured spectra.

COM-Port Interface used for connecting the spectrometer to the PC.

Dark value The noise of the spectrometer’s components is subtracted from the measured spectrum.

IntTime The integration time for one spectrum. A range of 1 to 200ms is covered

Logarithmic Switching from linear to logarithmic time axis when displaying kinetics

Averages The number of flashes. A maximum of 1000 averages are possible

Online The dark spectrum is displayed online

Spectra The number of spectra recorded between two flashes. A maximum of 120 spectra is possible

Trigger The number of recorded spectra before the exciting flash. The default is 1

TTL Shutter. Turning off the TTL switch on light source of the diode array spectrometer UV-VIS 190-720nm prevents light from the halogen or deuterium lamp passing the fiber, i.e. only the noise of the components is recorded

From…To A certain section of the spectrum can be displayed. This section can also be saved to disk or printed

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timetable of course 09.08.07 · outline of the experiments during the course: 1. modelling i...

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