Although our understanding of globular protein folding continues to advance, the irregular tertiary structures and high cooperativity of globular proteins complicates energetic dissection. Recently, proteins with regular, repetitive tertiary structures have been identified that sidestep limitations imposed by globular protein architecture. Here we review recent studies of repeat-protein folding. These studies uniquely advance our understanding of both the energetics and kinetics of protein folding. Equilibrium studies provide detailed maps of local stabilities, access to energy landscapes, insights into cooperativity, determination of nearest-neighbor interaction parameters using statistical thermodynamics, relationships between consensus sequences and repeat-protein stability. Kinetic studies provide insight into the influence of short-range topology on folding rates, the degree to which folding proceeds by parallel (versus localized) pathways, and the factors that select among multiple potential pathways. The recent application of force spectroscopy to repeat-protein unfolding is providing a unique route to test and extend many of these findings. KeywordsRepeat-protein; Ankyrin repeat; protein folding; energy landscape; atomic force microscopy In the last twenty-five years, advances in experimental studies and in computation and theory have greatly improved our understanding of protein folding. On the experimental side, advances have come from new and improved techniques, including site-directed mutagenesis, hydrogen exchange (HX) methods, improvements to rapid mixing devices, and development of single-molecule fluorescence and force spectroscopy. Experimental advances have also come in the form of generalizations and insights from expanding databases of thermodynamic and kinetic constants for protein folding [1][2][3][4][5].On the computational side, advances in our understanding of protein folding have come from huge increases in computer speed and storage capacity, in innovative methods to study energetics of folding such as ensemble-based approaches and replica exchange methods [6,7], and distributed computing methods [8]. As with experiment, computational studies of folding, and in particular, fold prediction, have greatly benefited from expanding databases of protein structure [9,10]. On the theoretical side, the application of ideas from condensed-matter *Correspondence: barrick@jhu.edu, (410) 516-0409. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptArch Biochem Biophys. Author manuscript; available in PMC 2009 January ...
Terminal deletions of units from a-helical repeat proteins have provided insight into the physical origins of their cooperativity. To test if the same principles governing cooperativity apply to b-sheet-containing repeat proteins, we have created a series of C-terminal deletion constructs from a large leucine-rich repeat (LRR) protein, YopM. We have examined the structure and stability of the resulting deletion constructs by a combination of solution spectroscopy, equilibrium denaturation studies, and limited proteolysis. Surprisingly, a high degree of nonuniformity was found in the stability distribution of YopM. Unlike previously studied repeat proteins, we identified several key LRR that on deletion disrupt nearby structure, at distances as far away as up to three repeats, in YopM. This partial unfolding model is supported by limited proteolysis studies and by point substitution in repeats predicted to be disordered as a result of deletion of adjacent repeats. We show that key internal-and terminal-caps must be present to maintain the structural integrity in adjacent regions (roughly four LRRs long) of decreased stability. The finding that full-length YopM maintains a high level of cooperativity in equilibrium unfolding underscores the importance of interfacial interactions in stabilizing locally unstable regions of structure.
Small globular proteins have many contacts between residues that are distant in primary sequence. These contacts create a complex network between sequence-distant segments of secondary structure, which may be expected to promote the cooperative folding of globular proteins. Although repeat proteins, which are made up of tandem modular units, lack sequence-distant contacts, several of considerable length have been shown to undergo cooperative two-state folding. To explore the limits of cooperativity in repeat proteins, we have studied the unfolding of YopM, a leucine-rich repeat (LRR) protein of over 400 residues. Despite its large size and modular architecture (15 repeats), YopM equilibrium unfolding is highly cooperative, and shows a very strong dependence on urea concentration. In contrast, kinetic studies of YopM folding indicate a mechanism that includes one or more transient intermediates. The urea dependence of the folding and unfolding rates suggests a relatively small transition state ensemble. As with the urea dependence, we have found an extreme dependence of the free energy of unfolding on salt concentration. This salt dependence likely results from general screening of a large number of unfavorable columbic interactions in the folded state, rather than from specific cation binding.
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