Tyrosine side chains as an electrochemical probe of stacked β-sheet protein conformations Anna Loksztejn a,b , Wojciech Dzwolak a,b , Paweł Krysiński a, ⁎ a Faculty of Chemistry, Warsaw University, Pasteura 1, 02-093, Warsaw, Poland b Institute of High Pressure Physics, Polish Academy of Sciences, Sokolowska 29/37, 01-142 Warsaw, Poland Received 26 February 2007; received in revised form 18 June 2007; accepted 19 July 2007 Available online 27 July 2007 Abstract The in vivo formation of β-pleated protein aggregates underlies a number of fatal neurodegenerative disorders, such as Alzheimer disease. Since molecular mechanisms of protein misfolding and aggregation remain poorly understood, this has been calling for many diverse biophysical tools capable of addressing different dynamic and conformational aspects of the phenomenon. The two model polypeptides used in this study are poly(L-tyrosine) and insulin. According to FT-IR spectra, poly(L-tyrosine) produced two distinct types of films with dominant either disordered or antiparallel β-sheet conformations depending on carrier solvent used for film's deposition. Electrochemical analysis of both the types of polypeptide films by the means of cyclic voltammetry and differential pulse voltammetry proved that different electrochemical behaviour of the tyrosine residues is determined by the conformation of polypeptide chains. We have rationalized this difference in terms of varying electrochemical accessibility of Tyr residues in each structure. We have also carried out spectral and electrochemical characterization of insulin β-sheet-rich amyloid fibrils. It appears that the detectable electrochemical response of the protein stems from the presence of four tyrosine residues per insulin monomer. Since hydrophobic residues, among them tyrosines play an important role in the formation of protein amyloid fibrils, but, on a molecular level, may be also critical in explaining neurotoxic properties of aggregates, their electrochemical properties may become a very valuable complementary tool in biophysical studies on protein misfolding. © 2007 Elsevier B.V. All rights reserved. Keywords: Amyloids; Polyaminoacids; Tyrosine; Electrochemistry; Differential pulse voltammetry 1. Introduction Aggregation and formation of β-pleated fibrils are what often follow when native protein structure is destabilized. The importance of this phenomenon stems from the fact that such β-sheet-rich, non-native protein assemblies (the so-called amyloids) were implicated in the etiology of several degener- ative disorders, such as Alzheimer disease [1,2]. Many proteins, even with a marginal sequential propensity to the β-sheet fold (e.g., myoglobin) [3] may acquire the ability to form fibrils under destabilizing conditions. One of the most interesting, yet least understood aspects of protein aggregation, is its polymorphism, which often manifests in a number of distinct (in terms of morphology, conformation, and biological activity) types of fibrils being formed out of a single amino acid sequence [4] This problem is particularly important in light of the so-called “prion strains”, when subtle, self-propagating conformational variabil- ity is accompanied by dramatic clinical consequences. As it can be rationalized that non-polar amino acids, among them tyrosines, are critical in determining stacking modes associated with particular types of protein fibrils [5], adequate biophysical tools that would address this problem are sought. Electrochem- istry is an as yet unexplored approach in this field. Because electrochemical activity of tyrosine is expected to depend strongly on fine topological and nano-environmental features of its surroundings, this has motivated our interest as to whether electrochemistry can provide conformation-sensitive tools for biophysics. Given that burial of non-polar amino acid residues and the ensuing increase in solvent entropy [6] is thought to be Available online at www.sciencedirect.com Bioelectrochemistry 72 (2008) 34 – 40 www.elsevier.com/locate/bioelechem ⁎ Corresponding author. Tel.: +488220211x389; fax: +48228225996. E-mail address: pakrys@chem.uw.edu.pl (P. Krysiński). 1567-5394/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bioelechem.2007.07.004