Journal articles

In this chapter we review recent studies of repeat proteins, a class of proteins consisting of tandem arrays of small structural motifs that stack approximately linearly to produce elongated structures. We discuss the observation that, despite lacking the long-range tertiary interactions that are thought to be the hallmark of globular protein stability, repeat proteins can be as stable and as co-orperatively folded as their globular counterparts.

We have investigated the use of FlAsH, a small fluorogenic molecule that binds to tetracysteine motifs, to probe folding of the 15-HEAT repeat protein PR65A. PR65A is one of a special class of modular non-globular proteins known as tandem repeat proteins, which are composed of small structural motifs that stack to form elongated, one-dimensional architectures. We were able to introduce linear and bipartite tetracysteine motifs at several sites along the α-helical HEAT array of PR65A without disrupting the structure or stability.

p13(suc1) (suc1) is a member of the CDC28 kinase specific family of cell cycle regulatory proteins that bind to the cyclin-dependent kinase CDK2 and regulate its activity. suc1 has two distinct conformational and assembly states, a compact globular monomer and a beta strand-exchanged dimer. The dimerisation is an example of domain-swapping, and is mediated by a molecular hinge mechanism that is conserved across the entire CKS family.

Solvent isotope effects have been observed on the in vitro refolding kinetics of a protein, hen lysozyme. The rates of two distinct phases of refolding resolved by intrinsic fluorescence have been found to be altered, to differing extents, in D2O compared with H2O, and experiments have been conducted aimed at assessing the contributions to these effects from various possible sources.

Ankryin repeat proteins comprise tandem arrays of a 33-residue, predominantly alpha-helical motif that stacks roughly linearly to produce elongated and superhelical structures. They function as scaffolds mediating a diverse range of protein-protein interactions, and some have been proposed to play a role in mechanical signal transduction processes in the cell. Here we use atomic force microscopy and molecular-dynamics simulations to investigate the natural 7-ankyrin repeat protein gankyrin. We find that gankyrin unfolds under force via multiple distinct pathways.

The subunits of the presumptive replicative helicase of archaea and eukaryotes, the MCM complex, are members of the AAA+ (ATPase-associated with various cellular activities) family of ATPases. Proteins within this family harness the chemical energy of ATP hydrolysis to perform a broad range of cellular processes. Here, we investigate the function of the AAA+ site in the mini-chromosome maintenance (MCM) complex of the archaeon Sulfolobus solfataricus (SsoMCM).

Repeat proteins are composed of tandem arrays of 30- to 40-residue structural motifs and are characterized by short-range interactions between residues close in sequence. Here we have investigated the equilibrium unfolding of D34, a 426-residue fragment of ankyrinR that comprises 12 ankyrin repeats. We show that D34 unfolds via an intermediate in which the C-terminal half of the protein is structured and the N-terminal half is unstructured. Surprisingly, however, we find that we change the unfolding process when we attempt to probe it.

The modular structures of repeat proteins afford them distinct properties compared with globular proteins, enabling them to function in a large and diverse range of cellular processes. Here, we show that they can also have different folding mechanisms. Myotrophin comprises four ankyrin repeats stacked linearly to form an elongated structure. Using site-directed mutagenesis, we find that folding of wild-type myotrophin is initiated at the C-terminal repeats.