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{{short description|Graph of protein folding kinetics}}
[[Image:Chevron plot.JPG|frame|right|A typical chevron plot observed in [[protein folding]] experiments.]]

A '''chevron plot''' is a way of representing [[protein folding]] [[chemical kinetics|kinetic]] data in the presence of varying concentrations of [[Denaturation (biochemistry)|denaturant]] that disrupts the protein's native [[tertiary structure]]. The plot is known as "chevron" plot because of the canonical ''v'', or [[Chevron (insignia)|chevron]] shape observed when the [[logarithm]] of the observed [[relaxation rate]] is plotted as a function of the denaturant concentration.

In a two-state system, folding and unfolding rates dominate the observed relaxation rates below and above the [[denaturation midpoint]] (Cm). This gives rise to the terminology of folding and unfolding arms for the limbs of the chevron. A priori information on the Cm of a protein can be obtained from equilibrium experiments. In fitting to a two-state model, the logarithm of the folding and unfolding rates is assumed to depend linearly on the denaturant concentration, thus resulting in the [[slope]]s m<sub>f</sub> and m<sub>u</sub>, called the folding and unfolding m-values, respectively (also called the kinetic m-values). The sum of the two rates is the observed relaxation rate. An agreement between equilibrium m-value and the absolute sum of the kinetic m-values is typically seen as a signature for two-state behavior. Most of the reported denaturation experiments have been carried out at [[298 K]] with either [[urea]] or [[guanidinium chloride]] (GuHCl) as denaturants.

==Experimental methodology==
To generate the folding limb of the chevron, the protein in a highly concentrated denaturant solution is diluted rapidly (in less than a millisecond) in an appropriate buffer to a particular denaturant concentration by means of a [[stopped flow]] apparatus. The relaxation to the new equilibrium is monitored by [[spectroscopic]] probes such as [[fluorescence]] or less frequently by [[circular dichroism]] (CD). The volume of the dilution is adjusted to obtain the relaxation rate at a specific denaturant concentration. The final protein concentration in the mixture is usually 1-20 μM, depending on the constraints imposed by the amplitude of relaxation and the signal-to-noise ratio. The unfolding limb is generated in a similar fashion by mixing denaturant-free protein with a concentrated denaturant solution in buffer. When the logarithm of these relaxation rates are plotted as a function of the final denaturant concentration, a chevron plot results.

The mixing of the solutions determines the [[dead time]] of the instrument, which is about a millisecond. Therefore, a stopped-flow apparatus can be employed only for proteins with a relaxation time of a few milliseconds. In cases where the relaxation time is shorter than the dead-time of the instrument, the experimental temperature is lowered (thus increasing the [[viscosity]] of water/buffer) to increase the relaxation time to a few milliseconds. On the other hand, for fast-folding proteins (i.e., those with a relaxation rate of 1 to 100 microseconds), [[pressure jump]] (dead time~few microseconds),<ref name="Jenkins">{{cite journal | doi = 10.1007/s00249-009-0420-6 | last1 = Jenkins | first1 = DC | last2 = Pearson | first2 = DS | last3 = Harvey | first3 = A | last4 = Sylvester | first4 = ID | last5 = Geeves | first5 = MA | last6 = Pinheiro | first6 = TJ. |name-list-style=vanc | year = 2009 | title = Rapid folding of the prion protein captured by pressure-jump | journal = Eur Biophys J | volume = 38 | issue = 5| pages = 625–35 | pmid = 19255752 | pmc = 4509520 }}</ref> [[temperature jump]] (T-jump; dead time~few nanoseconds) or continuous flow mixing (dead time~few microseconds),<ref name="Ferguson">{{cite journal | doi = 10.1073/pnas.221467198 | last1 = Ferguson | first1 = N | last2 = Johnson | first2 = CM | last3 = Macias | first3 = M | last4 = Oschkinat | first4 = H | last5 = Fersht | first5 = AR. |name-list-style=vanc | year = 2001 | title = Ultrafast folding of WW domains without structured aromatic clusters in the denatured state | journal = Proc. Natl. Acad. Sci. USA | volume = 98 | issue = 23| pages = 13002–13007 | pmid = 11687613 | pmc = 60814 |bibcode = 2001PNAS...9813002F | doi-access = free }}</ref> can be carried out at different denaturant concentrations to obtain a chevron plot.

==Chevron roll-overs==
Though the limbs of the chevron are assumed to be linear with denaturant concentration, it is not always the case. Non-linearities are usually observed in the either both the limbs or one of them and are termed chevron roll-overs. The reason for such an observation is not clear. Many interpretations including on-pathway intermediates,<ref name="Sanchez">{{cite journal | doi = 10.1016/S0022-2836(02)01230-5 | last1 = Sanchez | first1 = IE | last2 = Kiefhaber | first2 = T. |name-list-style=vanc | year = 2003 | title = Evidence for sequential barriers and obligatory intermediates in apparent two-state protein folding | journal = J. Mol. Biol. | volume = 325 | issue = 2| pages = 367–376 | pmid = 12488101 }}</ref> dead-time limitations, [[transition state]] movements ([[Hammond effect]]),<ref name="Ternstrom">{{cite journal | doi = 10.1073/pnas.96.26.14854 | last1 = Ternstrom | first1 = T | last2 = Mayor | first2 = U | last3 = Akke | first3 = M | last4 = Oliveberg | first4 = M. |name-list-style=vanc | year = 1999 | title = From snapshot to movie: φ analysis of protein folding transition states taken one step further | journal = Proc. Natl. Acad. Sci. USA | volume = 96 | issue = 26| pages = 14854–14859 | pmid = 10611302 | pmc = 24737 |bibcode = 1999PNAS...9614854T | doi-access = free }}</ref> aggregation artifacts,<ref name="Went">{{cite journal | last1 = Went | first1 = HM | last2 = Benitez-Cardoza | first2 = CG | last3 = Jackson | first3 = SE | title = Is an intermediate state populated on the folding pathway of ubiquitin? | journal = FEBS Letters | volume = 567 | issue = 2–3 | pages = 333–8 | year = 2004 | pmid = 15178347 | doi = 10.1016/j.febslet.2004.04.089 | doi-access = | bibcode = 2004FEBSL.567..333W }}</ref> [[downhill folding]],<ref name="Kaya">{{cite journal | doi = 10.1103/PhysRevLett.90.258104 | last1 = Kaya | first1 = H | last2 = Chan | first2 = HS. |name-list-style=vanc | year = 2003 | title = Origins of chevron rollovers in non-two-state protein folding kinetics | journal = Phys. Rev. Lett. | volume = 90 | issue = 258104–1| pages = 258104–4 | pmid = 12857173 | bibcode=2003PhRvL..90y8104K|arxiv = cond-mat/0302305 | s2cid = 15026414 }}</ref> and salt-induced [[Debye–Hückel equation|Debye–Hückel]] effects<ref name="Rios">{{cite journal | doi = 10.1021/bi048444l | last1 = Rios | first1 = MAD | last2 = Plaxco | first2 = KW. |name-list-style=vanc | year = 2005 | title = Apparent Debye-Huckel effects in the folding of a simple, single domain protein | journal = Biochemistry | volume = 44 | issue = 4| pages = 1243–1250 | pmid = 15667218 }}</ref> have been proposed to explain this behavior. In many cases the folding limb roll-overs are ignored as they occur at low denaturant concentrations, and the data is fit to a two-state model with a linear dependence of the rates. The folding rates reported for such proteins in the absence of denaturants are therefore an over-estimation.

==See also==
* [[Protein folding]]
* [[Denaturation (biochemistry)]]
* [[Denaturation midpoint]]
* [[Equilibrium unfolding]]
* [[Phi value analysis]]

==References==
<references />

[[Category:Protein structure]]
[[Category:Protein methods]]

Chevron plot - Revision history

imported>Citation bot: Added bibcode. | Use this bot. Report bugs. | Suggested by Dominic3203 | Category:Protein structure | #UCB_Category 95/189