Chevron plot
Encyclopedia
Protein folding
Protein folding is the process by which a protein structure assumes its functional shape or conformation. It is the physical process by which a polypeptide folds into its characteristic and functional three-dimensional structure from random coil....
kinetic
Chemical kinetics
Chemical kinetics, also known as reaction kinetics, is the study of rates of chemical processes. Chemical kinetics includes investigations of how different experimental conditions can influence the speed of a chemical reaction and yield information about the reaction's mechanism and transition...
data in the presence of varying concentrations of denaturant
Denaturation (biochemistry)
Denaturation is a process in which proteins or nucleic acids lose their tertiary structure and secondary structure by application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent , or heat...
that disrupts the protein's native tertiary structure
Tertiary structure
In biochemistry and molecular biology, the tertiary structure of a protein or any other macromolecule is its three-dimensional structure, as defined by the atomic coordinates.-Relationship to primary structure:...
. The plot is known as "chevron" plot because of the canonical v, or chevron shape observed when the logarithm
Logarithm
The logarithm of a number is the exponent by which another fixed value, the base, has to be raised to produce that number. For example, the logarithm of 1000 to base 10 is 3, because 1000 is 10 to the power 3: More generally, if x = by, then y is the logarithm of x to base b, and is written...
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
Assuming two-state protein folding, denaturation midpoint is defined as that temperature or denaturant concentration at which both the folded and unfolded states are equally populated at equilibrium....
(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
Slope
In mathematics, the slope or gradient of a line describes its steepness, incline, or grade. A higher slope value indicates a steeper incline....
s mf and mu, 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
Urea or carbamide is an organic compound with the chemical formula CO2. The molecule has two —NH2 groups joined by a carbonyl functional group....
or guanidinium chloride
Guanidinium chloride
Guanidinium chloride or guanidine hydrochloride, usually abbreviated GdmCl and sometimes GndCl or GuHCl, is the hydrochloride salt of guanidine.-Use in protein denaturation:...
(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 flowStopped flow
A stopped flow instrument is a rapid mixing device used to study the chemical kinetics of a reaction in solution. After two or more solutions containing the reagents are mixed, they are studied by whatever experimental methods are deemed suitable. Different forms of spectroscopy and scattering of...
apparatus. The relaxation to the new equilibrium is monitored by spectroscopic probes such as fluorescence
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
or less frequently by circular dichroism
Circular dichroism
Circular dichroism refers to the differential absorption of left and right circularly polarized light. This phenomenon was discovered by Jean-Baptiste Biot, Augustin Fresnel, and Aimé Cotton in the first half of the 19th century. It is exhibited in the absorption bands of optically active chiral...
(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
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....
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
Viscosity
Viscosity is a measure of the resistance of a fluid which is being deformed by either shear or tensile stress. In everyday terms , viscosity is "thickness" or "internal friction". Thus, water is "thin", having a lower viscosity, while honey is "thick", having a higher 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
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...
(dead time~few microseconds), temperature-jump
Temperature Jump
Temperature jump is a technique used in the study of chemical kinetics. It usually involves the discharging of a capacitor through a small volume Temperature jump is a technique used in the study of chemical kinetics. It usually involves the discharging of a capacitor (in the kV range) through a...
(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 stateTransition state
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 energy along this reaction coordinate. At this point, assuming a perfectly irreversible reaction, colliding reactant molecules will always...
movements (Hammond effect
Hammond's Postulate
Hammond's postulate, also referred to as the Hammond–Leffler postulate, is a hypothesis, derived from transition state theory, concerning the transition state of organic chemical reactions, which states that:-Interpreting the postulate:...
), aggregation artifacts, downhill folding
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:...
, and salt-induced Debye-Huckel
Debye-Hückel equation
The Debye–Hückel equation and Debye–Hückel limiting law, were derived by Peter Debye and Erich Hückel, who developed a theory with which to calculate activity coefficients of electrolyte solutions. Activities, rather than concentrations, are needed in many chemical calculations because solutions...
effects 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 foldingProtein foldingProtein folding is the process by which a protein structure assumes its functional shape or conformation. It is the physical process by which a polypeptide folds into its characteristic and functional three-dimensional structure from random coil....
- Denaturation (biochemistry)Denaturation (biochemistry)Denaturation is a process in which proteins or nucleic acids lose their tertiary structure and secondary structure by application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent , or heat...
- Denaturation midpointDenaturation midpointAssuming two-state protein folding, denaturation midpoint is defined as that temperature or denaturant concentration at which both the folded and unfolded states are equally populated at equilibrium....
- Equilibrium unfoldingEquilibrium unfoldingIn biochemistry, equilibrium unfolding is the process of unfolding a protein or RNA molecule by gradually changing its environment, such as by changing the temperature or pressure, adding chemical denaturants, or applying force as with an atomic force microscope tip. Since equilibrium is...
- Phi value analysisPhi value analysisPhi value analysis is an experimental protein engineering method used to study the structure of the folding transition state in small protein domains that fold in a two-state manner. Since the folding transition state is by definition a transient and partially unstructured state, its structure is...