Functional in vivo and in vitro analysis of the archaeal chaperonin complex

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Proteins have numerous roles inside living organisms. They may catalyse reactions, they may be important parts of cellular structures, they may enable cells to respond to external signals, they may turn genes on or off, and so on. Proteins are made as long chains of amino-acids, but before they can do their job inside the cell, they have to fold into a particular shape. Each shape is unique to each protein. Many problems arise when proteins fail to fold correctly. These may be health problems (for example, diseases such as BSE are associated with proteins failing to fold to their functional shape). In addition, proteins are widely produced in industry, and misfolding of these proteins is a major problem in some cases. A significant finding in recent years is that many proteins have to interact with other proteins called molecular chaperones before they reach their folded state. Molecular chaperones only interact briefly with proteins as they fold, but without this interaction many proteins fail to fold properly. There are various different classes of molecular chaperone. We are particularly interested in the class referred to as 'chaperonins'. Chaperonins are of great interest for two reasons. First, they are essential to all cells, whereas many other types of molecular chaperones can be dispensed with. Second, they have a striking structure, in that all form large complexes with many sub-units that can form cages that other proteins can fold inside. Chaperonins fall into two groups: group I and group II. Group I chaperonins are found in all bacteria and also in mitochondria and chloroplasts, and are moderately well understood. Group II chaperonins are found in the cytosol of eukaryotes (like humans), and are much less well understood. They are also found in the archaea, a group of simple organisms that look like bacteria but are more closely related to eukaryotes. Group II chaperonins are known to be important: in eukaryotic cells, they fold the key proteins actin and tubulin, which together form the internal framework of the cell (the cytoskeleton). They also help fold a protein that can suppress tumour formation, and can help to block the formation of aggregated proteins that cause diseases such as Huntington's chorea. The eukaryotic chaperonins contain eight different types of sub-unit and are hard to study; we do not know the fine details of their structure, for example. The archaeal chaperonins often function with only a single type of sub-unit, and we have an excellent knowledge of their structure. Recently in our group we have developed new ways of studying archaeal chaperonins in cells, and the current proposal aims to use these to learn a lot more about these proteins. We want to find out which parts of the chaperonin are needed for them to work, by changing different amino-acids in the proteins and then looking to see how these altered chaperonins function in cells (in vivo). Remarkably, we have shown that the archaeal chaperonins can also work in bacteria, and we want to study this unexpected finding by looking for mutated proteins that can work even better in bacteria. We will then purify some of these altered proteins and look at their properties using biochemical assays (in vitro). This will let us relate the ability of the proteins to function in vivo with particular properties that they have in vitro. We will also use some of the mutant chaperonins to try to identify other proteins with which they interact. These two approaches (genetic and biochemical) will teach us a lot about the archaeal chaperonins in particular and about chaperonins in general, and will help us to understand the eukaryotic chaperonins in more detail. This understanding has important implications for human and animal health and for biotechnological processes. The work will involve a collaborations between three research teams with highly complementary expertise in this area.