Editing Protein folding (section)

===Fluorescence spectroscopy===
[[Fluorescence spectroscopy]] is a highly sensitive method for studying the folding state of proteins. Three amino acids, phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp), have intrinsic fluorescence properties, but only Tyr and Trp are used experimentally because their [[quantum yield]]s are high enough to give good fluorescence signals. Both Trp and Tyr are excited by a wavelength of 280&nbsp;nm, whereas only Trp is excited by a wavelength of 295&nbsp;nm. Because of their aromatic character, Trp and Tyr residues are often found fully or partially buried in the hydrophobic core of proteins, at the interface between two protein domains, or at the interface between subunits of oligomeric proteins. In this apolar environment, they have high quantum yields and therefore high fluorescence intensities. Upon disruption of the protein's tertiary or quaternary structure, these side chains become more exposed to the hydrophilic environment of the solvent, and their quantum yields decrease, leading to low fluorescence intensities. For Trp residues, the wavelength of their maximal fluorescence emission also depend on their environment.

Fluorescence spectroscopy can be used to characterize the [[equilibrium unfolding]] of proteins by measuring the variation in the intensity of fluorescence emission or in the wavelength of maximal emission as functions of a denaturant value.<ref name="pmid=26607240">{{cite journal | vauthors = Bedouelle H | title = Principles and equations for measuring and interpreting protein stability: From monomer to tetramer | journal = Biochimie | volume = 121 | pages = 29–37 | date = February 2016 | pmid = 26607240 | doi = 10.1016/j.biochi.2015.11.013 }}</ref><ref>{{cite journal | vauthors = Monsellier E, Bedouelle H | title = Quantitative measurement of protein stability from unfolding equilibria monitored with the fluorescence maximum wavelength | journal = Protein Engineering, Design & Selection | volume = 18 | issue = 9 | pages = 445–56 | date = September 2005 | pmid = 16087653 | doi = 10.1093/protein/gzi046 | doi-access = free }}</ref> The denaturant can be a chemical molecule (urea, guanidinium hydrochloride), temperature, pH, pressure, etc. The equilibrium between the different but discrete protein states, i.e. native state, intermediate states, unfolded state, depends on the denaturant value; therefore, the global fluorescence signal of their equilibrium mixture also depends on this value. One thus obtains a profile relating the global protein signal to the denaturant value. The profile of equilibrium unfolding may enable one to detect and identify intermediates of unfolding.<ref>{{cite journal | vauthors = Park YC, Bedouelle H | title = Dimeric tyrosyl-tRNA synthetase from Bacillus stearothermophilus unfolds through a monomeric intermediate. A quantitative analysis under equilibrium conditions | journal = The Journal of Biological Chemistry | volume = 273 | issue = 29 | pages = 18052–9 | date = July 1998 | pmid = 9660761 | doi = 10.1074/jbc.273.29.18052 | doi-access = free }}</ref><ref>{{cite journal | vauthors = Ould-Abeih MB, Petit-Topin I, Zidane N, Baron B, Bedouelle H | title = Multiple folding states and disorder of ribosomal protein SA, a membrane receptor for laminin, anticarcinogens, and pathogens | journal = Biochemistry | volume = 51 | issue = 24 | pages = 4807–21 | date = June 2012 | pmid = 22640394 | doi = 10.1021/bi300335r }}</ref> General equations have been developed by Hugues Bedouelle to obtain the thermodynamic parameters that characterize the unfolding equilibria for homomeric or heteromeric proteins, up to trimers and potentially tetramers, from such profiles.<ref name="pmid=26607240"/> Fluorescence spectroscopy can be combined with fast-mixing devices such as [[stopped flow]], to measure protein folding kinetics,<ref>{{cite journal | vauthors = Royer CA | title = Probing protein folding and conformational transitions with fluorescence | journal = Chemical Reviews | volume = 106 | issue = 5 | pages = 1769–84 | date = May 2006 | pmid = 16683754 | doi = 10.1021/cr0404390 }}</ref> generate a [[chevron plot]] and derive a [[Phi value analysis]].