Intracellular protein folding: GroEL and GroES


The intracellular protein folding problem

Only part of the information needed to fold a protein into its functional three-dimensional structure is encoded by the linear sequence of its amino acids.  The remainder of this information comes from the specific environment in which the protein is located.  Often, neither of these information inputs is, or can be, optimized for the efficient folding of a protein.  

In fact, the concentrated and complex interior of a cell is an inherently hostile environment for the efficient folding of many proteins.  These proteins—constrained by sequence, topology, size, and function—simply cannot fold by themselves and are instead prone to misfolding and aggregation.   


Folding intermediates, kinetics traps and aggregation

Protein folding is, fundamentally, a complex polymer dynamics problem.  One way to visualize the process involves relating a protein’s accessible conformational space to the free energy of each polypeptide conformation. The resulting picture of protein folding is one of trajectories across an energy hypersurface or landscape that globally slopes toward the native state and becomes dramatically narrower as a protein approaches its native conformation (a folding funnel).  Small, globular proteins (<15 kDa) tend to fold quickly and efficiently.  This folding behavior can be explained by a folding landscape where various routes to the native state are available, with no particularly large energy barriers to inhibit the efficient downward slide.


Larger and more topologically complex proteins, however, often possess slower and more complicated folding reactions.  In general, large proteins tend to populate a range of meta-stable intermediate states during folding and are more prone to misfolding by virtue of having many more possible contacts than smaller proteins, some of which are sufficiently stable that they get a large protein into serious trouble. The folding landscape of such a protein is thus replete with kinetic traps, conformational wells (either native-like or not) that can dramatically slow folding and leave the protein highly protein to aggregation.


Molecular chaperones: GroEL and GroES

For some proteins, the barriers to efficient folding are so significant that spontaneous folding is essentially impossible, at least under conditions and on time scales relevant for life. To solve this problem, cells have developed a number of specialized systems that monitor and correct protein folding mistakes.  As a group, these accessory proteins are known as molecular chaperones.

In the network of molecular chaperones that fold, monitor, and maintain cellular proteins, the large, barrel-shaped oligomers known as chaperonins play a central and essential role.  These remarkable molecular machines employ the energy of ATP hydrolysis to power a facilitated protein-folding reaction.  GroEL and GroES, the chaperonin system of the bacterium Escherichia coli, are the archetypal members of this ubiquitous family of protein folding engines.


How does GroEL facilitate protein folding?

GroEL, and its close relatives in other organisms, have been shown to assist the folding of a wide range of proteins.  The early recognition that GroEL assists the folding of many different proteins strongly implied that GroEL does not facilitate protein folding by providing a structural template for protein native states. GroEL must, therefore, target a common, folding-inhibitory property of the substrate proteins upon which it works.

Proteins that require GroEL are typically large (>20 kDa), slow folding, and aggregation prone. Thus, GroEL-dependent proteins are those for which folding kinetics have trumped the sequence-encoded thermodynamic drive to the native state. In the most general terms then, GroEL must function to alter the kinetic balance between productive folding and misfolding or aggregation. 

Studying protein dynamics with FRET and cryo-EM

Using the GroELS system as a model, our goal is to determine how chaperonin-assisted protein folding works.  It has been well established that enclosure of non-native substrate proteins inside the GroEL-GroES cavity is an important part of how GroEL facilitates folding.  However, how the assembly of this folding chamber proceeds and is controlled remains poorly understood.  In addition, it is not yet clear how binding of a non-native protein to GroEL, followed by its encapsulation inside the GroEL-GroES cavity, promotes productive folding.

To address these questions, we are using a variety of biochemical and biophysical methods, including various types of fluorescence spectroscopy, rapid-mixing methods and single molecule techniques.  One approach we employ is fluorescence resonance energy transfer, a type of fluorescence spectroscopy that permits the very sensitive measurement of molecular proximity.  By attaching small, highly fluorescent probes to GroEL and GroES, or to a non-native substrate protein, we can monitor a range of structural and dynamic properties of the GroEL-GroES system during a productive folding reaction.

In collaboration with the laboratory of Dr. Wah Chiu at the Baylor College of Medicine, we are also employing cryo-electron microscopy (cryo-EM) and 3-D particle reconstruction  to examine the structure of the GroEL-GroES complex as it encapsualtes a non-native substrate protein and initiates a protein folding reaction.      


Department of Biochemistry and Biophysics                           Texas A&M University              2016