Techniques for Characterizing the Folding Pathway

To determine the nature of any folding intermediates it is necessary to examine the folding of the protein using a series of techniques to access different information on the folding pathway. Traditionally folding studies have been

performed on a large number of protein molecules, which during the folding process adopt an ensemble of structures. More recent developments have allowed the study of the folding of single protein molecules increasing the understanding of the unfolding and refolding of complex proteins.

The study of the folding of an ensemble of molecules averages the folding of large number of molecules at once and averages the folded state of the ensemble of protein structures present under the conditions used. The folding of the ensemble of

required on the folding of the protein. The thermodynamics and the kinetics of the folding of proteins require different techniques to access. The thermodynamics of the folding of proteins is accessed using equilibrium folding experiments. In these experiments the protein being observed is perturbed from its initial conditions by a denaturing factor, such as temperature, pH or the presence of a chemical denaturant, and allowed to reach equilibrium under the new conditions. The extent of

denaturation or renaturation (if starting with denatured protein) is then measured using one or more folding probes. The process is then repeated until the protein is fully denatured or renatured. The free energy of folding can then be determined from the behaviour of the folding probes.

Once the thermodynamics of folding have been established, it is then possible to establish the kinetics of folding. The speed of folding varies dramatically between different proteins and as a result a number of different techniques are required to follow the folding of proteins. Larger proteins, however, tend to fold more slowly than smaller proteins. Due to the complexity of their structures, or a dependence on slow changes such as cis-trans proline isomerisation (Wedemeyeret al., 2002) in their folding, some proteins fold very slowly, for example collagen III (Engel and Bächinger, 2000). With these proteins it is possible to use simple instrumentation with a manual mixing of the protein and the buffer used to refold or unfold the protein. Similar folding probes are used with this type of folding experiment as are used in equilibrium folding experiments, since the time scales involved are fairly long.

For proteins which fold faster, on a timescale of seconds to milliseconds a more specialized technique is required. The mixing of the buffers required for the experiments must be performed much faster than is possible with manual mixing. This requires specialized mixers and relatively fast flow rates for the buffers used. The technique of stopped-flow is used to achieve these. Depending on the instrument used, the dead-time, that is the time during which data cannot be collected, may be as low as ~0.5 ms but is more commonly in the range of 2-3 ms. Some proteins fold faster than this dead-time. A reduction in temperature can be used to slow the folding of the protein, so that it can be captured. If the folding of the protein is still too fast for stopped-flow to capture, or a major phase of the folding is identified which is too fast to be captured, then further techniques must be used which allow for lower dead times.

Several techniques have been developed for the study of folding events which are too fast to be accessed by stopped-flow methods. The methods that have been adopted depend on the method of denaturation used. When the protein being studied has been denatured either by chemical denaturants, or by a change in the pH, then the technique of continuous-flow is used. This technique is similar to the

stopped-flow technique, but the flow is allowed to continue through the observation channel during the observation, and the flow channels and mixers are smaller than those used for stopped-flow, with the dead-times being correspondingly lower. With pressure, or temperature being used to denature the protein, the methods of P-jump or T-jump have been adopted. These techniques have been used to study the fast folding of several proteins including prions (Jenkinset al., 2009) and ribonuclease A (Fontet al., 2006). P-jump and T-jump measurements have a distinct advantage over continuous-flow methods in that they require much less protein and one can perform refolding and unfolding experiments on the same samples.

Single molecules studies are valuable in directly observing the folding of individual molecules. This technique uses a different method of unfolding the target protein. The protein being studied is unfolded by placing a force on the protein molecule to pull apart the structure. This technique is of particular value in studying the folding of large repeat proteins, such as the consensus ankyrin repeat protein N6C (Liet al., 2006). The individual repeated domains of these proteins have often been studied by the techniques outlined above. Single molecule studies are

particularly valuable in determining the extent of cooperatively between the

unfolding and refolding of the repeated domains. Both optical traps and atomic force microscopy (AFM) have been used to capture and apply force to tested proteins. The information derived on the folding of proteins is less detailed than the information that is obtained with the techniques outlined above, although single molecule techniques do have several distinct advantages over equilibrium and kinetic folding measurements. Firstly the folding of a single molecule is directly being examined as opposed to a large number of molecules adopting an ensemble of conformational states. Secondly, the study of large repeat proteins is more easily performed than is the case with methods of the study of folding which involve large numbers of molecules. This is because the unfolding and refolding is directly observed through changes in the extension of the protein relative to the force applied, as opposed to the

require the modification of the protein to introduce reporter molecules or remove extra reporters from the protein. The technique is not sensitive to the concentration of protein used, which is advantageous compared to other techniques which show a concentration sensitivity for both successful folding and for their folding probes.