The overall goal of the following experiment is to characterize confirmational changes associated with ligand binding to calcium transducers on the sub millisecond timescale. The confirmational transition between the APO protein to the calcium bound protein is triggered by the photo cleavage of a caged calcium compound calcium DM nitro that leads to the photo release of free calcium. The associated change in the index of reflection is measured as a displacement of the probe beam using a quadruple position sensitive detector.
Next, the photothermal beam deflection traces are measured as a function of temperature for both the sample and the reference. Compound analysis of the photo beam deflection traces provides insight into the mechanism of calcium induced confirmational switch in terms of reaction volume and enthalpy changes. The main advantage of photothermal beam deflection over the existing mast, such as top flow, is that this techniques allows for a simultaneous detection of volume and entropy changes on sub millisecond timescale.
The visual demonstration of this technique is necessary as the precise alignment of instrument is critical for the recovery of thermodynamic parameters. Demonstrating the procedure will be Walter Gonzalez, a graduate student from my laboratory, Carry out the sample preparation and all sample manipulations in a dark room to prevent any unwanted uncaging. In this video, steps are demonstrated in the light for filming purposes.
To begin solubilize DM Nitro in 50 millimolar heaps buffer 100 millimolar potassium chloride, pH 7.0 to a final concentration of 400 micromolar. Add calcium chloride from the 0.1 molar stock solution to achieve the desirable calcium to DN nitro concentration ratio. Next, solubilize the reference compound potassium cyanide or sodium chromate in the same buffer.
As for the sample, refer to the text protocol for a schematic of the basic experimental configuration for the photothermal beam deflection. When setting up the experiment, propagate the prob beam through the center of a cell, placed in a temperature controlled cell holder. Then using a pinhole, adjust the diameter of the probe beam to one millimeter, use a second mirror behind the sample to center the prob beam on the center of the position sensitive detector.
Focus the pro beam on the center of the detector so that the difference in the voltage between the top two diodes and the bottom two diodes, as well as the difference in the voltage between the two diodes on the left and right side of the detector is zero. Subsequently shape the diameter of the pump beam, a 355 nanometer output of an ND YAG laser. Using a three millimeter pinhole placed between 2 355 nanometer laser mirrors co propagate the pump beam through the center of the vete.
It is important that both laser beams are propagated through the center of the optical cell in a nearly coline manner. To obtain a measurable deflection angle and thus high amplitude of photothermal beam deflection or PBD signal, use a reference compound to align the probe and pump beam to achieve a satisfactory PBD signal characterized by a good signal to noise ratio and stable PBD amplitude on longer timescales. Then position the pump beam with respect to the probe beam by incremental adjustment of the 355 nanometer laser mirrors.
Measure the amplitude of the PBD reference signal as a difference in between two top and bottom photo diodes on the position sensitive detector. The PBD signal should exhibit a rapid increase in the amplitude on a fast time scale and remain stable on the 100 millisecond timescale. Check the linearity in the PBD signal amplitude with respect to the released heat energy by measuring the linear dependence of the PBD signal on the excitation laser power, and on the number of Einstein's absorbed.
Keep the laser power below approximately 1000 micro joules to prevent multi photon absorption. Also, keep the absorbence of the sample or reference compound at an excitation wavelength less than 0.5 to prevent decrease of the pump beam power. These steps will ensure linearity of the PBD signal.
Start with the measurement of the PBD traces. For the reference, place the solution of the reference compound in a quartz cell and position the cell in the temperature controlled holder. Detect the reference PBD signal as a function of temperature in the temperature range from 16 to 35 degrees Celsius with a temperature increment of three degrees Celsius.
Upon each temperature change. Check the position of the prob beam on the position sensitive detector and readjust the position to the center of the detector. If necessary, check the linearity of the PBD signal as described in the text protocol.
Then place the sample solution in the same optical cell as used for the reference compound keeping the same orientation of the optical cell for the reference measurement. Proceed to detect the sample PBD traces in the same temperature range as for the reference, and check the linearity of the sample PBD amplitude. Take the amplitude of the reference PBD signal as the difference between the pret trigger and post trigger PBD signal.
In a similar way, determine the amplitude of the fast and slow phase of the sample PBD signal. To eliminate the instrument response parameter, scale the amplitude of the sample PBD signal by the amplitude of the PBD signal. For the reference, the ratio of the sample signal to the reference signal gives an equation from which the heat release to the solution and non-thermal volume change associated with a photo initiated reaction can be determined from the slope and intercept of the plot of the amplitude ratio multiplied by energy versus the temperature dependent term to determine the reaction volume and enthalpy change for the fast and the slow process scale, the observed volume and enthalpy change to the appropriate quantum yield.
According to the equations found in the text protocol, the amplitudes and lifetimes for the individual steps are analyzed by fitting the data to the time dependent function that describes the volume and enthalpy changes. The amplitude of the fast step corresponds to the amplitude of the prompt phase of the sample PBD signal and the amplitude of the subsequent step corresponds to the amplitude of the slow phase of the sample PBD signal. For each individual process from the temperature dependence of the rate constant for individual processes, the activation enthalpy and entropy parameters can be readily determined using IR plots.
A representative example of PBD traces for calcium photo release from calcium DM nitro is shown here. The fast phase corresponds to the photo cleavage of calcium DM nitro and calcium liberation, whereas the slow phase reflects calcium binding to the non photoed cage. The PBD trace for the calcium association to the C terminal domain of the neuronal calcium sensor downstream regulatory element antagonist, regulator, or dream is shown here upon calcium photo dissociation.
Photo released ligand associates to the C terminal domain of dream with the time constant of 1.3 plus or minus 0.3 milliseconds at 20 degrees Celsius from the temperature dependence of the observed rate constant. The activation barrier for calcium binding to the C terminal domain of dream was determined to be 9.2 plus or minus 0.4 kilo calories per mole. While attempting this procedure is important to remember to measure sample PBD traces under conditions that are identical to those for the reference.
After watching this video, you should have a good understanding of how to prepare sample, align instrumentation, and analyze data to obtain a reproducible thermodynamic parameter.
