The induction of α-helical structure in partially unfolded HypF-N does not affect its aggregation propensity

Protein Engineering, Design & Selection vol. 24 no. 7 pp. 553– 563, 2011 Published online April 25, 2011 doi:10.1093/protein/gzr018 The induction of a-helical structure in partially unfolded HypF-N does not affect its aggregation propensity B.Ahmad 1,4, I.Vigliotta 1, F.Tatini 1, S.Campioni 1,5, B.Mannini 1, J.Winkelmann 1,6, B.Tiribilli 2 and F.Chiti 1,3,7 Keywords: aggregation mechanism/amyloid fibrils/ on-pathway/protein misfolding/a-helical intermediate 1 Received March 18, 2011; revised March 29, 2011; accepted March 29, 2011 Edited by Daniel Otzen. The conversion of proteins into structured fibrillar aggregates is a central problem in protein chemistry, biotechnology, biology and medicine. It is generally accepted that aggregation takes place from partially structured states of proteins. However, the role of the residual structure present in such conformational states is not yet understood. In particular, it is not yet clear as to whether the ahelical structure represents a productive or counteracting structural element for protein aggregation. We have addressed this issue by studying the aggregation of pHunfolded HypF-N. It has previously been shown that the two native a-helices of HypF-N retain a partial a-helical structure in the pH-unfolded state and that these regions are also involved in the formation of the cross-b structure of the aggregates. We have introduced mutations in such stretches of the sequence, with the aim of increasing the a-helical structure in the key regions of the pHunfolded state, while minimizing the changes of other factors known to influence protein aggregation, such as hydrophobicity, b-Sheet propensity, etc. The resulting HypF-N mutants have higher contents of a-helical structure at the site(s) of mutation in their pH-unfolded states, but such an increase does not correlate with a change of aggregation rate. The results suggest that stabilisation of a-helical structure in amyloidogenic regions of the sequence of highly dynamic states does not have remarkable effects on the rate of protein aggregation from such conformational states. Comparison with other protein systems indicate that the effect of increasing a-helical propensity can vary if the stabilised helices are in non-amyloidogenic stretches of initially unstructured peptides (accelerating effect), in amyloidogenic stretches of initially unstructured peptides (no effect) or in amyloidogenic stretches of initially stable helices (decelerating effect). Introduction Proteins and peptides have a generic propensity to form wellorganised fibrillar aggregates characterised by an extended cross-b structure, generally referred to as amyloid-like fibrils (Dobson, 1999; Dobson and Stefani, 2003; Uversky and Fink, 2004; Chiti and Dobson, 2006). From a physicochemical perspective, this process represents an essential feature of the behaviour of polypeptide chains that needs to be fully understood for a thorough characterisation of the nature of proteins (Jahn and Radford, 2008). From a more biological perspective, formation of amyloid fibrils, or intracellular inclusions with structurally related characteristics, is associated with a large number of pathological conditions in humans (Chiti and Dobson, 2006). It is also a major problem in biotechnology as the large-scale expression of proteins potentially interesting to the market often results in their selfassembly in inclusion bodies with amyloid-like structural features (Ventura and Villaverde, 2006). It is well accepted that the process of amyloid fibril formation by normally globular proteins requires a partial or global unfolding of the native structure across the major freeenergy barrier for unfolding or, alternatively, structural and transient fluctuations from the native state ensemble (Chiti and Dobson, 2009). Several studies have shown that aggregation of highly flexible, partially folded states is promoted by regions of the sequence with a high hydrophobicity and high propensity to form b-sheet structure, resulting in mathematical algorithms able to predict the aggregation-promoting regions in a protein from the knowledge of its sequence (Fernandez-Escamilla et al., 2004; Yoon and Welsh, 2004; Pawar et al., 2005; Tartaglia et al., 2005; Trovato et al., 2006; Galzitskaya et al., 2006; Conchillo-Solé et al., 2007; Maurer-Stroh et al., 2010). It is not clear, however, if the residual structure present in the aggregation-competent state plays an important role in the process. While it is widely recognised that an efficient mechanism to neutralise the amyloidogenic potential of aggregation-promoting regions is to promote their folding into a stable and persistent structure, such as that of the native state of a globular protein (Dobson, 1999; Uversky and Fink, 2004; Monsellier and Chiti, 2007; Tzotzos and Doig, 2010), it is still unclear as to whether the flexible structure present in partially folded states of proteins plays a role in the process of aggregation from such states. Similarly, it is not clear as to whether the formation of structure in intrinsically disordered proteins plays a role in the process of amyloid formation by such systems. # The Author 2011. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: 553 Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze, Viale Morgagni 50, 50134, Firenze, Italy, 2Consiglio Nazionale delle Ricerche (CNR), Istituto dei Sistemi Complessi, Via Madonna del Piano 10, 50019 Sesto Fiorentino, Firenze, Italy, 3Consorzio Interuniversitario, Istituto Nazionale Biostrutture e Biosistemi (I.N.B.B.), Viale delle Medaglie d’Oro 305, 00136 Roma, Italy, 4Present address: Department of Physics and Astronomy, Michigan State University, East Lansing, Michigan 48824, USA, 5Present address: Department of Chemistry and Applied Biosciences, Laboratory of Physical Chemistry, Eidgenössische Technische Hochschule Zürich, Wolfgang Pauli Str. 10, 8093 Zurich, Switzerland and 6 Present address: Magnetic Resonance Center (CERM), University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Firenze, Italy 7 To whom correspondence should be addressed. Email: B.Ahmad et al. 554 not completely unfolded, as monitored with far-UV circular dichroism (CD) and nuclear magnetic resonance (NMR) (Campioni et al., 2008; Calloni et al., 2008). Importantly, both regions of the sequence have been shown to play an important role in the process of amyloid-like protofibril formation, as deduced from the ability of mutations located in these two regions to significantly affect such a process (Calloni et al., 2008). In addition, structural information indicates that such regions form the structural core of the resulting aggregates, meaning that most, if not all, of their residues form the b-sheet structure stabilising the aggregates (Campioni et al., 2010). These findings make HypF-N an ideal system to explore the role of a-helical structure in th (...truncated)