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

B.Ahmad 2 5 I.Vigliotta 2 F.Tatini 2 S.Campioni 2 4 B.Mannini 2 J.Winkelmann 2 3 B.Tiribilli 1 F.Chiti 0 2 0 Consorzio Interuniversitario, Istituto Nazionale Biostrutture e Biosistemi (I.N.B.B.) , Viale delle Medaglie d'Oro 305, 00136 Roma, Italy 1 Consiglio Nazionale delle Ricerche (CNR), Istituto dei Sistemi Complessi , Via Madonna del Piano 10, 50019 Sesto Fiorentino, Firenze, Italy 2 Dipartimento di Scienze Biochimiche, Universita` degli Studi di Firenze , Viale Morgagni 50, 50134, Firenze, Italy 3 Present address: Magnetic Resonance Center (CERM), University of Florence , Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Firenze, Italy 4 Present address: Department of Chemistry and Applied Biosciences, Laboratory of Physical Chemistry , Eidgenossische Technische Hochschule Zurich, Wolfgang Pauli Str. 10, 8093 Zurich, Switzerland 5 Present address: Department of Physics and Astronomy, Michigan State University , East Lansing, Michigan 48824, USA - 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-Sole 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. In particular, the results describing the role of a-helical structure are contradictory. The formation of amyloid fibrils by the 40- and 42-residue forms of the amyloid b (Ab) peptide has been shown to be preceded by the formation of oligomeric species with a-helical structure, leading to the conclusion that an a-helical containing oligomer is an on-pathway species necessary to form fibrils (Kirkitadze et al., 2001). Such a hypothesis was reinforced by the finding that small-to-moderate concentrations of the a-helical inducer 2,2,2-trifluoroethanol (TFE) is able to shorten the lag phase of amyloid fibril formation by the Ab peptide (Fezoui and Teplow, 2002). Another widely studied intrinsically disordered peptide, namely the islet amyloid polypeptide (IAPP), was shown to form transiently a-helical structure and aggregate more rapidly in the presence of model phospholipid membranes (Knight et al., 2006; Ling et al., 2009). Fibril formation by IAPP was also found to be accelerated in the presence of small concentrations of hexafluoroisopropanol (Padrick and Miranker, 2002; Abedini and Raleigh, 2005). In contrast to these findings, other reports have shown that the increase in the a-helical propensity of specific regions of the sequence by protein engineering results in a significant deceleration of the process of amyloid fibril formation (Villegas et al., 2000; Paivio et al., 2004). None of s (...truncated)