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  Section: General Biochemistry » Protein Folding
 
 
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General Experimental Strategies

 
     
 
As discussed above, experimental studies of protein folding reactions fall into the category of either equilibrium or kinetics studies, with the former yielding thermodynamic information about the energy differences between the native and denatured structural states and the latter studies providing information about the folding pathway and the height of energy barriers between important species on this pathway. In general, to perform either an equilibrium (thermodynamics) or time-dependent (kinetics) study, one must be able to experimentally monitor a signal that tracks the population of the structural states of the protein.

There are a number of ways this can be done. The most convenient experimental methods involve solution-phase spectroscopic measurements; among these methods are absorption spectroscopy, fluorescence, circular dichroism, and nuclear magnetic resonance. Other methods include differential scanning calorimetry, light scattering, electrophoresis, and chromatography. This section gives a brief description of the advantages and disadvantages of some of the above methods. These methods are not equally applicable to equilibrium and time-dependent studies of protein
unfolding, as some methods have a rapid response and some have a slow response. Methods also differ in their intrinsic sensitivity, which is related to the concentration of protein necessary to perform the measurement, their ease and economy of use, and whether they provide auxiliary information about the structure of the protein in its native and denatured states. What is meant by the last statement is that some of the spectroscopic signals can provide information about the secondary or tertiary structure of the protein species. For most types of spectroscopy, the signal arises from particular amino acid residues (e.g., aromatic side chains or peptide bond), thus differences in the signals for the conformational states can be related to differences in the local environment of these amino acid residues (e.g., tryptophan residue 140 in staphylococcal nuclease; see Fig. 1). If there are only a very few of such signal origination sites, then site-specific information can be obtained. If there are many probe sites and they are distributed throughout the protein’s structure, then the method yields global information (e.g., signal from the amide linkage in the peptide backbone; see Fig. 1). It goes without saying that the protein sample to be studied must be well defined with regard to purity, and solution conditions must be selected and controlled to be relevant to other functional studies and studies with other proteins. Neutral pH, 20°C, and an ionic strength of 0.1 to 0.2 are the most commonly employed solution conditions.


A key to most of these methods and their use in protein unfolding studies is that the signal is a mole-fraction weighted average of the signals of each protein species. That is, for the simplest case of a thermodynamics study of the transition between a native, N, and unfolded, U, state of a protein, the observed signal, S, can be expressed as:

⇒ Equation [5] S = ΣSi Xi

where Xi is the mole fraction of each species i and Si is the intrinsic signal of species i . This relationship applies to most solution optical spectroscopic methods. Clearly, for a particular spectroscopic signal to be useful for tracking a N ↔ U transition, the signal of the N and U states must be sufficiently different. The native (XN ) and unfolded (XU ) mole fractions are directly related to the equilibrium constant in Eq. (2), as:

⇒ Equation [6] XN = 1/(1 + Kun); XU = Kun /(1 + Kun).

The transition from the native state to the unfolded state, or vice versa, can be induced in several ways, essentially by varying the solution conditions in a way that changes the equilibrium between the native and unfolded state. The transition may be induced by varying temperature, adding chemical (chaotropic agent) denaturant, adding acid or base, or increasing pressure. In the case of multimeric proteins, subunit dissociation, which may be accompanied by denaturation of the subunits, can be induced by dilution of the protein. Before discussing the various spectroscopic methods, some thermodynamic relationships are presented for describing the transitions induced in the above ways.
 
     
 
 
     



     
 
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