Article

Modulation of the multistate folding of designed TPR proteins through intrinsic and extrinsic factors

School of Biological and Chemical Sciences, Queen Mary, University of London, London E1 4NS, United Kingdom.
Protein Science (Impact Factor: 2.86). 03/2012; 21(3):327-38. DOI: 10.1002/pro.2018
Source: PubMed

ABSTRACT Tetratricopeptide repeats (TPRs) are a class of all alpha-helical repeat proteins that are comprised of 34-aa helix-turn-helix motifs. These stack together to form nonglobular structures that are stabilized by short-range interactions from residues close in primary sequence. Unlike globular proteins, they have few, if any, long-range nonlocal stabilizing interactions. Several studies on designed TPR proteins have shown that this modular structure is reflected in their folding, that is, modular multistate folding is observed as opposed to two-state folding. Here we show that TPR multistate folding can be suppressed to approximate two-state folding through modulation of intrinsic stability or extrinsic environmental variables. This modulation was investigated by comparing the thermodynamic unfolding under differing buffer regimes of two distinct series of consensus-designed TPR proteins, which possess different intrinsic stabilities. A total of nine proteins of differing sizes and differing consensus TPR motifs were each thermally and chemically denatured and their unfolding monitored using differential scanning calorimetry (DSC) and CD/fluorescence, respectively. Analyses of both the DSC and chemical denaturation data show that reducing the total stability of each protein and repeat units leads to observable two-state unfolding. These data highlight the intimate link between global and intrinsic repeat stability that governs whether folding proceeds by an observably two-state mechanism, or whether partial unfolding yields stable intermediate structures which retain sufficient stability to be populated at equilibrium.

Full-text

Available from: Ewan R G Main, Feb 03, 2015
0 Followers
 · 
107 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Repeat proteins have become increasingly important due to their capability to bind to almost any proteins and the potential as alternative therapy to monoclonal antibodies. In the past decade repeat proteins have been designed to mediate specific protein-protein interactions. The tetratricopeptide and ankyrin repeat proteins are two classes of helical repeat proteins that form different binding pockets to accommodate various partners. It is important to understand the factors that define folding and stability of repeat proteins in order to prioritize the most stable designed repeat proteins to further explore their potential binding affinities. Here we developed distance-dependant statistical potentials using two classes of alpha-helical repeat proteins, tetratricopeptide and ankyrin repeat proteins respectively, and evaluated their efficiency in predicting the stability of repeat proteins. We demonstrated that the repeat-specific statistical potentials based on these two classes of repeat proteins showed paramount accuracy compared with non-specific statistical potentials in: 1) discriminate correct vs. incorrect models 2) rank the stability of designed repeat proteins. In particular, the statistical scores correlate closely with the equilibrium unfolding free energies of repeat proteins and therefore would serve as a novel tool in quickly prioritizing the designed repeat proteins with high stability. StaRProtein web server was developed for predicting the stability of repeat proteins.
    PLoS ONE 03/2015; 10(3):e0119417. DOI:10.1371/journal.pone.0119417 · 3.53 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In recent decades, advances in computational methods and experimental biophysical techniques have improved our understanding of protein folding. Although some of these advances have been remarkable, the structural variability of globular proteins usually encountered makes it difficult to extract general features of their folding processes. To overcome this difficulty, experimental and computational studies of the folding of repeat (or modular) proteins are of interest. Because their native structures can be described as linear arrays of the same, repeated, supersecondary structure unit, it is possible to seek a possibly independent behavior of the different modules without taking into account the intrinsic stability associated with different secondary structure motifs. In this work we have used a Monte Carlo-based simulation to study the folding equilibrium of four repeat proteins belonging to the tetratricopeptide repeat family. Our studies provide new insights into their energy profiles, enabling investigation about the existence of intermediate states and their relative stabilities. We have also performed structural analyses to describe the structure of these intermediates, going through the vast number of conformations obtained from the simulations. In this way, we have tried to identify the regions of each protein in which the modular structure yields a different behavior and, more specifically, regions of the proteins that can stay folded when the rest of the chain has been thermally denatured.
    European Biophysics Journal 07/2014; 43(8-9). DOI:10.1007/s00249-014-0975-8 · 2.47 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: We propose a band-separated, bidirectional amplifier based on a bismuth-based erbium-doped fiber for use in long-reach hybrid dense wavelength division multiplexing-time division multiplexing passive optical networks (DWDM-TDM-PONs). We also propose a long-reach hybrid DWDM-TDM-PON architecture in which the proposed amplifier can be effectively used. The feasibility of using the proposed amplifier for long-reach hybrid DWDM-TDM-PONs is experimentally investigated by performing a series of signal transmission experiments with an exemplary PON configuration having a total reach of 75 km and 8 split users. Error-free bidirectional signal transmission at a line rate of 10 Gbit/s is successfully demonstrated. A theoretical investigation also shows that the reach/split limitation issue associated with an insufficient WDM signal gain in the experimental demonstration can be easily solved by optimizing the amplifier.
    Journal of Optical Communications and Networking 03/2012; 4(3):165-172. DOI:10.1364/JOCN.4.000165 · 1.55 Impact Factor