The overall goal of this procedure is to probe the dynamics of protonation and confirmational changes of two photo sensitive membrane proteins using time resolved STEP scan FTIR spectroscopy oscopy. This is accomplished by first forming a hydrated protein film whose infrared absorption will be probed either in an attenuated total reflection configuration for bacteria opsin, or in a transmission configuration for channel Rodin two. The second step is to excite the sample with a nanosecond laser flash of suitable wavelength to initiate a photo cyclic reaction in the protein detecting time resolved changes in the intensity of the infrared light interacting with the sample.
Next, the above process is repeated at discreet positions of the mobile mirror of an interferometer which controls the optical path difference between two split beams until a complete time resolved interferogram is recorded. The final step is to transform the time resolved interferogram to a time resolved IR difference absorption spectra using the Fourier transform and the Lambert beer law. Ultimately, the time evolution of bans, especially in the MI one and in the CO double bond carboxylic regions are inspected to obtain dynamics of confirmational and protonation changes in the protein.
The main advantage of the technique of a most existing experimental methods is that it resolves transient protonation changes in proteins. Moreover, it does it on the micro and millisecond time scales, the most relevant time range to probe protein functionality. This method can help to answer key questions in the molecular biophysics and protein science field, such as which residues the protonate and when they do during the functional mechanism of a protein or when major changes in proteins do take place.
Demonstrating the procedure will be done by Victor Ria research associate from my laboratory First mount, the attenuated total reflectance or a TR accessory into the sample compartment of the FTIR spectrometer. Measure a broad range energy spectrum at four inverse centimeters. Resolution of the clean internal reflection element surface by conventional rapid scan FTIR spectroscopy cover the surface of the A TR with 20 microliters of the high ionic strength buffer used later to rehydrate the sample.
Then measure the IR absorption of the buffer. Next, remove the buffer and rinse the surface with water without touching it. Remove the residual liquid by an intense airflow spread approximately three microliters of a six milligram per milliliter protein solution of bacteria Rodin in purple membrane on top of the surface.
Dry the protein suspension under a gentle stream of dry air until a film is attained. Then measure the absorption spectrum of the dry film. Following this gently add 20 to 40 microliters of the high ionic strength buffer to rehydrate the dry film.
Cover the A TR holder with a lid to prevent water evaporation measure and absorbent spectrum. Estimate the percentage of sample remaining near the surface after rehydration of the film by subtracting the absorption spectrum of the buffer. At this point, add 10 microliters of channel opsin two dissolved in dec alside in a low ionic strength buffer at the center of a 20 millimeter diameter barium fluoride window.
Spread the solution with the help of the micro pipette tip to a six to eight millimeter diameter using a gentle flow of dry air warm homogeneous film that has a diameter roughly matching the size of the IR beam at the largest aperture. Next, use a flat silicone O-ring of one millimeter thickness. Hydrate the film by adding three to five microliters of a mixture of glycerol and water distributed in three to five drops around the dry film and tightly close it with a second window.
Insert the barium fluoride sandwiched windows in a holder. Then place the holder in the sample compartment. Measure an absorption spectrum.
Confirm that the maximum absorption in the amide one region is in the range of 0.6 to 1.0 for the A TR setup. Use an optical fiber placed on top of the A TR lid to couple the laser to the sample. Adjust the energy density of the laser at the sample to two to three millijoules per square centimeter per pulse.
Using a power meter or the transmission experiments, use mirrors to bring the laser to the sample and if required, lenses to either collate or divert the laser beam to a diameter slightly above the sample film size. Synchronize the laser pulse with the data recording by using the Q switch. Sync out TTL pulse of the electronics of the neodymium dope atrium aluminum garnet laser to trigger the spectrometer analog to digital converter.
Set the excitation rate of the laser to perform time resolved. Step scan measurements in the 1800 to 850 inverse centimeter spectral range. Place a lowpass optical filter in the optical path that is opaque above 1, 950 inverse centimeters, and with good transmission below 1800 inverse centimeters.
Then change the detector from AC to DC coupled mode. At this point, bring the DC level of the INTERFEROGRAM to zero by applying a current bias to the detector. Readjust the electronic gain to make better use of dynamic range of the analog to digital converter.
Following this, start the step scan menu of the FTIR spectrometer. Set the target spectral bandwidth for the step scan measurement to one eighth of the helium neon laser wave number. Set the spectral and phase resolution to eight inverse centimeters and 64 inverse centimeters respectively.
Select an appe function. Then set the INTERFEROGRAM acquisition mode to single side forward. Set the sampling rate of the analog to digital converter to the highest available in the spectrometer.
Set the trigger for experiment to external. Then set the number of linearly spaced data points to be recorded, including 100 pret trigger points. Next, set the number of co editions or number of averages of the photo reaction per mirror position and start the experiment.
Finally, repeat the experiment 10 times for bacteria, redsin, and 35 times on three different sample films for channel redsin two to have approximately 200 co additions per mirror position characteristic bans from peptide bond amite vibrations are distinguishable in the bacteria redsin dry foam absorption spectrum. When using low ionic strength buffers, the film expands excessively reducing the amount of protein probed by the evanescent field. The exact dependence between film swelling and buffer ionic strength will depend among other factors on the nature of the lipids.
Film swelling after hydration requires time to reach stabilization for bacteria of dosin. In purple membrane, the process is mono exponential with a time constant of 12 minutes. A 3D plot from a typical time resolved step scan, FDIR experiment on bacteria.
Redsin is shown here. Spectra can be extracted at specific times. For instance, when intermediate states in the bacteria redsin photo cycle are expected to reach their highest population.
In the bacteria rod Dossin photo cycle time traces of absorbance changes at 1, 762 inverse centimeters. Reports on the protonation deprotonation dynamics of aspartate 85 and at 1, 741 inverse centimeters on hydrogen bonding changes and the deprotonation repro nation dynamics of aspartic acid.96. The time traces at 1, 670 and 1, 555 inverse centimeters.
Report on changes in MI one and two vibrations, which are both sensitive to the peptide backbone confirmation. Following this method, other techniques such as site directed neurogenesis or retinal enzyme spectroscopy can be performed in order to assign vibrational band to specific residues in the protein After its development. This technique paved the way for researchers in the field of molecular biophysics to explore the functional mechanism of plenty of different light driven proteins.
