Non-random coil behavior as a consequence of extensive PPII structure in the denatured state

1 Non-random coil behavior as a consequence of extensive PPII structure in the denatured state Aitziber L. Cortajarena¶, Gregg Lois‡, Eilon Sherman§, Corey S. O’Hern‡, Lynne Regan¶,†,*, and Gilad Haran§,* ¶Department of Molecular Biophysics & Biochemistry, †Department of Chemistry, and ‡Department of Mechanical Engineering and Department of Physics, Yale University, New Haven, CT 06520, Phone: 203 432 9843, Fax: 203 432 5175 §Department of Chemical Physics, Weizmann Institute of Science, P.O.B. 26, Rehovot 76100, Israel, Phone: 972 8 9342625, Fax: 972-8-934-2749 E-mail: lynne.regan@yale.edu; Gilad.Haran@weizmann.ac.il Running title: Compact conformation in a protein’s denatured state. Keywords: Protein folding, fluorescence correlation spectroscopy, PPII helix, denatured state, self-avoiding random walk, hydrodynamic radius. manuscript Click here to view linked References 2 Abstract Unfolded proteins may contain native or non-native residual structure, which has important implications for the thermodynamics and kinetics of folding as well as for misfolding and aggregation diseases. However, it has been universally accepted that residual structure should not affect the global size scaling of the denatured chain, which obeys the statistics of random coil polymers. Here we use a single-molecule optical technique, fluorescence correlation spectroscopy, to probe the denatured state of set of repeat proteins containing an increasing number of identical domains, from two to twenty. The availability of this set allows us to obtain the scaling law for the unfolded state of these proteins, which turns out to be unusually compact, strongly deviating from random-coil statistics. The origin of this unexpected behavior is traced to the presence of extensive non-native polyproline II helical structure, which we localize to specific segments of the polypeptide chain. We show that the experimentally observed effects of PPII on the size scaling of the denatured state can be well-described by simple polymer models. Our findings suggest an hitherto unforeseen potential of non-native structure to induce significant compaction of denatured proteins, affecting significantly folding pathways and kinetics. 3 Introduction During the folding process, proteins reach a defined, unique, native structure from a poorly defined ensemble of unfolded conformations. Although we have an atomistic description of the structures of the native states of a multitude of proteins, much less is known about the unfolded ensemble. The unfolded state is often treated as some version of a random coil, a realistic model of which is the self-avoiding random walk (SARW), in which different chain segments cannot occupy the same volume element. For a SARW, the relationship between the radius of gyration (Rg) (or the hydrodynamic radius (Rh)), and the length of the chain is given by the expression Rg  N, where  = 0.59 and N is the number of units in the chain.1; 2 For many different denatured proteins the experimentally-measured relationship between Rg or Rh and N is consistent with the behavior of a SARW.3; 4; 5; 6 This observation has been interpreted as support for an unstructured unfolded state. Conversely, there have been several reports that describe the existence of ‘residual structure’ in the unfolded state 7; 8; 9; 10 which, in general, does not change the global size scaling of the chain.11; 12 A detailed understanding of the nature of the unfolded state is essential for a complete understanding of protein folding, because any residual structure in the ‘unfolded state’ will clearly play a role in modulating both the thermodynamics and kinetics of folding, and might affect the formation of alternative structures, such as amyloid. Repeat proteins are composed of tandem arrays of a small structural motif. 13 they are widespread in nature and in many cases function as mediators of protein-protein interactions. Their simplified modular structures make them ideal systems for understanding basic principles that drive protein folding.14; 15; 16 The tetratricopeptide repeat is a 34 amino-acid motif in which two anti-parallel -helices stack together.17 4 We have previously reported the design and characterization of a consensus tetratricopeptide (CTPR) sequence, and have generated a series of proteins with different numbers of tandem repeats of this sequence.17; 18; 19 Tandem arrays of CTPR units form a regular, superhelical, extended structure (Fig. 1A).18; 19 It has been suggested that the folding mechanism of these proteins may be different than that of globular proteins.18 Here we combine spectroscopy and modeling to shed light on the structure of the denatured state of the CTPR proteins. Surprisingly, we find that extensive non-native structure leads to a compact denatured state, with size scaling strongly deviating from that expected for a random coil. Results and Discussion The denatured state of CT

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