A chevron plot is a way of representing

protein folding Protein folding is the physical process by which a protein chain is translated to its native three-dimensional structure, typically a "folded" conformation by which the protein becomes biologically functional. Via an expeditious and reproduci ...

kinetic Kinetic (Ancient Greek: κίνησις “kinesis”, movement or to move) may refer to: * Kinetic theory of gases, Kinetic theory, describing a gas as particles in random motion * Kinetic energy, the energy of an object that it possesses due to i ...

data in the presence of varying concentrations of denaturant that disrupts the protein's native

tertiary structure Protein tertiary structure is the three dimensional shape of a protein. The tertiary structure will have a single polypeptide chain "backbone" with one or more protein secondary structures, the protein domains. Amino acid side chains may int ...

. The plot is known as "chevron" plot because of the canonical ''v'', or

chevron Chevron (often relating to V-shaped patterns) may refer to: Science and technology * Chevron (aerospace), sawtooth patterns on some jet engines * Chevron (anatomy), a bone * '' Eulithis testata'', a moth * Chevron (geology), a fold in rock ...

shape observed when the

logarithm In mathematics, the logarithm is the inverse function to exponentiation. That means the logarithm of a number to the base is the exponent to which must be raised, to produce . For example, since , the ''logarithm base'' 10 o ...

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 Denaturation midpoint of a protein is defined as the temperature (Tm) or concentration of Denaturation (biochemistry), denaturant (Cm) at which both the Protein tertiary structure, folded and Denaturation (biochemistry), unfolded states are equall ...

(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 In mathematics, the slope or gradient of a line is a number that describes both the ''direction'' and the ''steepness'' of the line. Slope is often denoted by the letter ''m''; there is no clear answer to the question why the letter ''m'' is use ...

s m_f and m_u, 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 Urea, also known as carbamide, is an organic compound with chemical formula . This amide has two amino groups (–) joined by a carbonyl functional group (–C(=O)–). It is thus the simplest amide of carbamic acid. Urea serves an important r ...

guanidinium chloride Guanidinium chloride or guanidine hydrochloride, usually abbreviated GdmCl and sometimes GdnHCl or GuHCl, is the hydrochloride salt of guanidine. Structure Guanidinium chloride crystallizes in orthorhombic space group ''Pbca''. The crystal stru ...

(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 Stopped-flow is an experimental technique for studying chemical reactions with a half time of the order of 1 ms, introduced by Britton Chance and extended by Quentin Gibson (Other techniques, such as the temperature-jump method, are available for ...

apparatus. The relaxation to the new equilibrium is monitored by

spectroscopic Spectroscopy is the field of study that measures and interprets the electromagnetic spectra that result from the interaction between electromagnetic radiation and matter as a function of the wavelength or frequency of the radiation. Matter wav ...

probes such as

fluorescence Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, tha ...

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 For detection systems that record discrete events, such as particle and nuclear detectors, the dead time is the time after each event during which the system is not able to record another event. An everyday life example of this is what happens when ...

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 The viscosity of a fluid is a measure of its resistance to deformation at a given rate. For liquids, it corresponds to the informal concept of "thickness": for example, syrup has a higher viscosity than water. Viscosity quantifies the inte ...

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 Pressure jump is a technique used in the study of chemical kinetics. It involves making rapid changes to the pressure of an experimental system and observing the return to equilibrium or steady state. This allows the study of the shift in equilib ...

(dead time~few microseconds),

temperature jump The temperature jump method is a technique used in chemical kinetics for the measurement of very rapid reaction rates. It is one of a class of chemical relaxation methods pioneered by the German physical chemist Manfred Eigen in the 1950s. In the ...

(T-jump; dead time~few nanoseconds) or continuous flow mixing (dead time~few microseconds), 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, dead-time limitations,

transition state In chemistry, the transition state of a chemical reaction is a particular configuration along the reaction coordinate. It is defined as the state corresponding to the highest potential energy along this reaction coordinate. It is often marked wi ...

movements (

Hammond effect Hammond's postulate (or alternatively the Hammond–Leffler postulate), is a hypothesis in physical organic chemistry which describes the geometric structure of the transition state in an organic chemical reaction. First proposed by George Hammo ...

), aggregation artifacts,

downhill folding Downhill folding is a process in which a protein folds without encountering any significant macroscopic free energy barrier. It is a key prediction of the folding funnel hypothesis of the energy landscape theory of proteins. Overview Downhill ...

, and salt-induced Debye–Hückel effects{{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 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.

References

Protein structure Protein methods

Experimental methodology

Chevron roll-overs

See also

References