Site-Directed Mutations in the C-Terminal Extension of Human aB-Crystallin Affect Chaperone Function and Block Amyloid Fibril Formation Teresa M. Treweek 1,2 , Heath Ecroyd 3 , Danielle M. Williams 3 , Sarah Meehan 4 , John A. Carver 3 , Mark J. Walker 5 * 1 Department of Chemistry, University of Wollongong, Wollongong, New South Wales, Australia, 2 Graduate School of Medicine, University of Wollongong, Wollongong, New South Wales, Australia, 3 School of Chemistry and Physics, The University of Adelaide, Adelaide, South Australia, Australia, 4 The University Chemical Laboratory, University of Cambridge, Cambridge, United Kingdom, 5 School of Biological Sciences, University of Wollongong, Wollongong, New South Wales, Australia Background. Alzheimer’s, Parkinson’s and Creutzfeldt-Jakob disease are associated with inappropriate protein deposition and ordered amyloid fibril assembly. Molecular chaperones, including aB-crystallin, play a role in the prevention of protein deposition. Methodology/Principal Findings. A series of site-directed mutants of the human molecular chaperone, aB- crystallin, were constructed which focused on the flexible C-terminal extension of the protein. We investigated the structural role of this region as well as its role in the chaperone function of aB-crystallin under different types of protein aggregation, i.e. disordered amorphous aggregation and ordered amyloid fibril assembly. It was found that mutation of lysine and glutamic acid residues in the C-terminal extension of aB-crystallin resulted in proteins that had improved chaperone activity against amyloid fibril forming target proteins compared to the wild-type protein. Conclusions/Significance. Together, our results highlight the important role of the C-terminal region of aB-crystallin in regulating its secondary, tertiary and quaternary structure and conferring thermostability to the protein. The capacity to genetically modify aB-crystallin for improved ability to block amyloid fibril formation provides a platform for the future use of such engineered molecules in treatment of diseases caused by amyloid fibril formation. Citation: Treweek TM, Ecroyd H, Williams DM, Meehan S, Carver JA, et al (2007) Site-Directed Mutations in the C-Terminal Extension of Human aB- Crystallin Affect Chaperone Function and Block Amyloid Fibril Formation. PLoS ONE 2(10): e1046. doi:10.1371/journal.pone.0001046 INTRODUCTION The classic experiments of Anfinsen [1] on the folding of ribonuclease in vitro revealed that all the information required for folding of a polypeptide chain into its final native conformation is contained within the polypeptide chain itself. This is indeed evident with small proteins used for in vitro folding studies (e.g. barnase, 110 residues) which are able to refold to their active conformation in the absence of other proteins. In the case of large, multi-domain proteins encoded by long sequences, however, only a limited proportion achieve their native state unassisted [2]. Most are prevented from reaching this state by incorrect intermolecular interactions that occur when the protein is in a partially folded, intermediate state, whereby hydrophobic regions on their surface interact resulting in protein aggregation and precipitation. Thus, protein folding and unfolding are exquisitely regulated in the cell and involve molecular chaperone proteins that assist other proteins in adopting their correct, native state. The small heat shock proteins (sHsps) act in a chaperone manner by recognizing and stabilizing the intermediate states of target proteins, thereby preventing improper or incorrect folding that would otherwise result in protein misfolding, aggregation, precipitation and possibly disease [3,4,5,6]. aB-Crystallin is a sHsp that is capable of interacting with a multitude of target proteins to prevent their aggregation and precipitation [7]. However, unlike the classical bacterial chaperonin GroEL, sHsps (including aB-crystallin) do not directly participate in refolding of the denatured proteins, except in the presence of another chaperone protein, e.g. Hsp70 [8]. sHsps act specifically with target proteins that are on their off- folding pathway [9]. aB-Crystallin is primarily found in the eye lens, where it associates with the closely related aA-crystallin, which has 57% sequence homology with aB-crystallin and shares the conserved ‘a-crystallin domain’ (reviewed in [10]), to form large hetero-oligomeric species. However, it is also constitutively expressed in many non-lenticular tissues, including the brain, lung and cardiac and skeletal muscle where it forms complexes with other sHsps [11]. As with other members of the sHsp family, the expression of aB-crystallin is dramatically up-regulated in response to stress and pathological conditions such as Alzheimer’s, Parkinson’s and Creutzfeldt-Jakob diseases [4,12,13,14]. The sHsps form a structurally divergent protein family with members present in archaea, bacteria and eukarya [10]. Monomeric molecular masses of the sHsps range between 12 and 40 kDa, however, most form large oligomeric assemblies of 150–800 kDa. All members are characterized by the presence of a homologous sequence of 80–100 residues, referred to as the ‘a- crystallin’ domain [15]. This domain is preceded by an N-terminal domain, which is highly variable in size and sequence, and is followed by a C-terminal extension. Whilst the C-terminal Academic Editor: Edathara Abraham, University of Arkansas, United States of America Received March 7, 2007; Accepted September 21, 2007; Published October 17, 2007 Copyright: ß 2007 Treweek et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants (to JAC) from the National Health and Medical Research Council (NHMRC) of Australia and the Australian Research Council. TT was supported by an Australian Postgraduate Award, HE is supported by an NHMRC Peter Doherty Postdoctoral Training Fellowship and SM was supported by a Royal Society International Fellowship. Competing Interests: The authors have declared that no competing interests exist. * To whom correspondence should be addressed. E-mail: mwalker@uow.edu.au PLoS ONE | www.plosone.org 1 October 2007 | Issue 10 | e1046