Distribution of molecular size within an unfolded state ensemble using small-angle X-ray scattering and pulse field gradient NMR techniques¹

Abstract

The size distribution of molecules within an unfolded state of the N-terminal SH3 domain of drk (drkN SH3) has been studied by small-angle X-ray scattering (SAXS) and pulsed-field-gradient NMR (PFG-NMR) methods. An empirical model to describe this distribution in the unfolded state ensemble has been proposed based on (i) the ensemble-averaged radius of gyration and hydrodynamic radius derived from the SAXS and PFG-NMR data, respectively, and (ii) a histogram of the size distribution of structures obtained from preliminary analyses of structural parameters recorded on the unfolded state. Results show that this unfolded state, U_exch, which exists in equilibrium with the folded state, F_exch, under non-denaturing conditions, is relatively compact, with the average size of conformers within the unfolded state ensemble only 30–40 % larger than the folded state structure. In addition, the model predicts a significant overlap in the size range of structures comprising the U_exch state with those in a denatured state obtained by addition of 2 M guanidinium chloride.

Introduction

Contrary to a simplistic structure-function paradigm, an increasing number of proteins that are unstructured under physiological conditions yet biologically active are being described1. For example, the p21 Cdk inhibitors and the C-terminal activation domain of c-Fos are functionally active but intrinsically disordered in the absence of binding to target proteins2, 3. It has been suggested that conformational disorder may play an important role in the diversity of interactions of these and other such proteins. In addition to the experimentally demonstrated examples, a significant fraction of protein sequences in various genomes are predicted to code for disordered or partially disordered “structures”4.

To better understand the potential biological role of disorder, structural information on unfolded and partially unfolded states of proteins under physiological conditions is crucial. Unlike a protein in its folded state, the unfolded state is an ensemble of rapidly interconverting conformers. A complete characterization requires not only descriptions of average local and global properties but details on the various conformers that contribute to the disordered state ensemble and their relative populations5. Even though unfolded states have much less persistent structure than folded states, recently developed NMR and other spectroscopic techniques have allowed characterization of the residual structure present. Information on backbone conformational preferences can be derived from NMR parameters including scalar J-coupling constants6, 7, 8, sequential HN-HN and Hα-HN NOEs9 as well as chemical shifts10, 11. Long range HN-HN NOEs can also be observed in the unfolded state ensemble using extensive deuteration to reduce relaxation12 and paramagnetic relaxation enhancement13, 14 and residual dipolar couplings15 can be used to probe structural features of partially unfolded or unfolded molecules. Details on specific interactions in populated conformers of the unfolded state ensemble can thus be obtained.

A full description of the unfolded state, however, requires information about the ensemble distribution of molecular sizes, shapes and the extent of compactness. Small-angle X-ray scattering (SAXS) is one of the few techniques that provides a direct measure of these properties16. Recent advances in X-ray sources and instrumentation make SAXS a powerful tool for studying the conformations and interactions of biological macromolecules with a number of studies focussing on disordered states. In particular, SAXS results have shown that thermally and chemically denatured states of ribonuclease A are more compact than a random coil of the same length17. A time-resolved approach to SAXS has also been applied to study the changes in compactness of lysozyme during different stages of the folding process18, 19.

Pulsed-field-gradient NMR (PFG-NMR) is another useful technique to study molecular size20, 21. While SAXS yields the radius of gyration (R_g), PFG-NMR provides a measure of the translational diffusion coefficient, which can be used to determine the hydrodynamic radius (R_h). This method has been applied to the study of folded and unfolded states of a number of proteins21, 22, 23.

Here, we have used SAXS and PFG-NMR to measure the radius of gyration and hydrodynamic radius of the unfolded state ensemble of the isolated N-terminal SH3 domain of the Drosophilia signal transduction protein drk (drkN SH3) under non-denaturing conditions. The Drosophilia protein drk functions to couple activated receptor tyrosine kinases to Ras signaling via binding of the guanine nucleotide exchange factor Sos to this SH3 domain24. SH3 domains, in general, mediate protein recognition in signal transduction and cellular localization by binding to proline-rich targets25. Most isolated SH3 domains are stably folded; however, the drkN SH3 domain is unstable in the absence of its binding target Sos and exists in equilibrium between a folded (F_exch) and an unfolded form (U_exch) in aqueous buffer and near neutral pH. The protein can be stabilized to the folded state (F_s) by addition of 0.4 M sodium sulfate or denatured (U_gdn) by adding 2.0 M guanidinium chloride (GdnCl). Extensive structural studies of these various states have been performed using NMR and other spectroscopic techniques5, 12, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35. To enable a more complete understanding of the U_exch state, which coexists with the folded state under non-denaturing conditions, we have applied SAXS and PFG-NMR techniques to determine the molecular size distribution in the U_exch ensemble. In particular, we have taken advantage of the difference in modes of ensemble averaging of the radius of gyration and hydrodynamic radius, along with additional information provided by preliminary structures of the unfolded ensemble5, to describe the size distribution of conformers in this state.