Tryptophan fluorescence intensity is a convenient method to measure/estimate the dissociation constant of an interaction between a peptide and a partner, as it requires very low concentrations of biological material. Many papers have described the method which relies on the fluorescence intensity measured upon addition of the partner. I'm studying the interaction between peptides and real membranes extracted from bacteria using Trp fluorescence. Upon membrane addition, the maximum emission wavelength undergoes a blue shift, indicating a binding to the membrane. The fluorescence intensity overall increases with, however, an inconsistent behavior upon membrane addition (sometimes it increases, then it decreases a little bit, and increases again...). That's why I would prefer to use the maximum emission wavelength to calculate the Kd.
Can fluorescence experts recommend me an article explaining in details how to do that?
The fluorescence of tryptophan changes because it looses energy to the solvent. The best way to measure it is not the peak intensity but the avarage distribution of the emission energy what is given by the simple calculation of the center of mass (CM) of the emission spectrum. Mathematically, the CM is the point that divides the area of the spectrum in two parts of equal area. Concerning to the emission energy, the CM is the medium point of the spectrum. It may be calculated from data in nm, and the values will be inversely proportional to the energy. Or you may convert your data to wavenumber, and then the values will be directly proportional to the energy. When you observe a blue shift of the spectra when the peptides bind to membrane, that means that the hydrophobic environment helps preserving the energy, favouring the transitions in the blue side of the spectra. The CM represents the entire shift and not only the change in intensity in one wavelength. Look for the Lakowicz book, or Gregorio weber´s papers (the pionneer), Jerson Silva´s papers, david Jameson´s papers... Those are some experts on protein fluorescence and thermodynamics.
The Stern Volmer plot could also work in what case water is the quencher and the membrane protects the peptide from being quenched. It is a little bit different from the regular quenching experiments. Anisotropy may be a problem if the membranes added sccatter too much light. Tryptophan is not really a good probe for that since the exc wavelength will be low, what increases sccattering, low signal (if you can not increase the peptide concentration) because there will be photoselection during the absorption of polarized light. The suggestion of labeling the peptide is good only if labeling does not change the membrane binding properties.
I prefer fluorescence spectroscopy in this case because it is a very sensitive method. NMR is fine but can you do it by H NMR? Or do you need to have the peptide produced with C13 or N15? In any case you will need much higher concentrations than when you use fluorescence.
Basically any signal can be used to follow binding as long as the signal is proportional to the amount bound (not necessarily the concentration). For example, the membrane phase transition temperature is not suitable as it is non-linear with the amount bound. The math is the same as if you are using the signal intensity
http://www.sciencedirect.com/science/article/pii/S0022283608014952
I am not sure that wavelength maximum is proportional to the fraction bound. It depends upon the quantum yield of the bound and the free species. Many years ago I tried to calculate but came to the conclusion that only under certain conditions it should work. The calculation is buried somewhere in my notebooks. In general you cannot use it to derive a proper binding isotherm.
Actually, before asking the question I had the same opinion as Jeffrey: why not using the wavelength variation in fluorescence while we can trust the chemical shift in NMR? And then I realized that I couldn't find any publication where the wavelength maximum was used to calculate a Kd. For that reason, I guess you're right Professor Roy. Any reference about the theoretical explanation would be welcomed!!
Using the chemical shift works for two reasons:
1. In NMR, multiple binding events can occur for each transition event (fast exchange) because the lifetime of excited states in NMR is on the order of ms. In fluorescence, where excited states have ns lifetimes, only one binding event at most is at most likely to occur for each fluorescence event (slow exchange). NMR peaks under fast exhange a single peak appears that reflects the equilibrium binding statistics while in fluorescence two peaks are actually generated for the free and bound state.
2. In NMR , the peaks are usually well resolved, at least to the extent that peak maximum for each peak can be detected. In fluorescence, this is rarely the case. The peak in fluorescence only appears to move because the second peak is not resolved. This has the effect of shifting the emission maximum.
Prof. Roy is probably right that the peak position is not strictly dependent on the fraction bound. So while it may look like a binding isotherm it may not be strictly correct. You can check for yourself by setting up two Gaussian peaks and solving for the maxima as a function of the fraction bound.
Alternatively, most peptides are unfolded and become helical upon binding to membranes. Following the CD at 222 nm upon membrane titration is an established method for determining the membrane binding.
The other possibility is to use fluorescence anisotropy. It is thermodtnamically sound. It is possible that there will be increase of FA. However, W is not a good probe for FA. A better strategy would be to use an end labeled peptide with a red wavelength probe. CD will also be good but I am a bit worried about using it with real bacterial membranes.
Kd can be calculated by using Stern volmer plot. The maximum emission values can be taken before and after the binding of the protein to the membrane and then can be put in the stern volmer modified equation. It gives the Ka from which Kd can be calculated.
The fluorescence of tryptophan changes because it looses energy to the solvent. The best way to measure it is not the peak intensity but the avarage distribution of the emission energy what is given by the simple calculation of the center of mass (CM) of the emission spectrum. Mathematically, the CM is the point that divides the area of the spectrum in two parts of equal area. Concerning to the emission energy, the CM is the medium point of the spectrum. It may be calculated from data in nm, and the values will be inversely proportional to the energy. Or you may convert your data to wavenumber, and then the values will be directly proportional to the energy. When you observe a blue shift of the spectra when the peptides bind to membrane, that means that the hydrophobic environment helps preserving the energy, favouring the transitions in the blue side of the spectra. The CM represents the entire shift and not only the change in intensity in one wavelength. Look for the Lakowicz book, or Gregorio weber´s papers (the pionneer), Jerson Silva´s papers, david Jameson´s papers... Those are some experts on protein fluorescence and thermodynamics.
The Stern Volmer plot could also work in what case water is the quencher and the membrane protects the peptide from being quenched. It is a little bit different from the regular quenching experiments. Anisotropy may be a problem if the membranes added sccatter too much light. Tryptophan is not really a good probe for that since the exc wavelength will be low, what increases sccattering, low signal (if you can not increase the peptide concentration) because there will be photoselection during the absorption of polarized light. The suggestion of labeling the peptide is good only if labeling does not change the membrane binding properties.
I prefer fluorescence spectroscopy in this case because it is a very sensitive method. NMR is fine but can you do it by H NMR? Or do you need to have the peptide produced with C13 or N15? In any case you will need much higher concentrations than when you use fluorescence.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence