Differential Dynamics of Extracellular and Cytoplasmic Domains in Denatured States of Rhodopsin

Abstract

Rhodopsin is a model system for understanding membrane protein folding. Recently, conditions that allow maximally denaturing rhodopsin without causing aggregation have been determined, opening the door to the first structural characterization of denatured states of rhodopsin by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy. One-dimensional 1H NMR spectra confirm a progressive increase in flexibility of resonances in rhodopsin with increasing denaturant concentrations. Two-dimensional 1H–15N HSQC spectra of [15N]-α-lysine-labeled rhodopsin in which signals arise primarily from residues in the cytoplasmic (CP) domain and of [15N]-α,ε-tryptophan-labeled rhodopsin in which signals arise only from transmembrane (TM) and extracellular (EC) residues indicate qualitatively that EC and CP domains may be differentially affected by denaturation. To obtain residue-specific information, particular residues in EC and CP domains were investigated by site-directed spin labeling. EPR spectra of the spin-labeled samples indicate that the EC residues retain more rigidity in the denatured states than the CP residues. These results support the notion of residual structure in denatured states of rhodopsin.

Slow Interconversion in a Heterogeneous Unfolded-State Ensemble of Outer-Membrane Phospholipase A

“…Of note, this non-random-coil behavior does not change the unfolded-state expansion in terms of a continuous shift in transfer efficiency with increasing denaturant concentration, in line with previous reports (49)(50)(51). Residual structure in denatured proteins is well established for both soluble (4,5,11,52) and membrane proteins (22,(53)(54)(55)(56). In many cases, nonrandom structural elements in unfolded proteins have been ascribed to hydrophobic clustering (5,20,22,23,52).…”

Dynamics of proteins in solution

“…Several additional studies with complementary non-neutron-based techniques were carried out in order to characterize the dynamics of proteins in native, molten and denatured states (Buck et al ., 1996; Bai et al ., 2000; Dilg et al ., 2002; Kuzmenkina et al ., 2005; Nienhaus, 2006; Ramboarina and Redfield, 2008; Santos et al ., 2010; Dutta et al ., 2014; Yadav et al ., 2014; Ghosh et al ., 2015; Mondal et al ., 2015; Aznauryan et al ., 2016). In general, FRET (Kuzmenkina et al ., 2005; Nienhaus, 2006; Yadav et al ., 2014; Mondal et al ., 2015) as well as NMR (Buck et al ., 1996; Bai et al ., 2000; Ramboarina and Redfield, 2008; Dutta et al ., 2014) and Mössbauer (Dilg et al ., 2002) as well as NMR (Buck et al ., 1996; Bai et al ., 2000; Ramboarina and Redfield, 2008; Dutta et al ., 2014) and Mössbauer (Dilg et al ., 2002) studies indicate a higher flexibility and dynamic heterogeneity of denatured proteins and molten globules compared with natively folded proteins on timescales ranging from nanoseconds (Buck et al ., 1996; Ramboarina and Redfield, 2008; Dutta et al ., 2014; Yadav et al ., 2014; Mondal et al ., 2015) to micro- to milli-seconds (Buck et al ., 1996; Bai et al ., 2000; Dutta et al ., 2014), and even several seconds, as evidenced by a significant ‘dynamic’ heterogeneity of the structure of the unfolded proteins (Kuzmenkina et al ., 2005; Nienhaus, 2006). By analyzing the average fluorescence lifetime of labeled HSA at different GnHCl concentrations and temperatures, Yadav et al .…”

“…In summary, most experiments performed with different techniques accessing protein internal dynamics on an extremely large range of timescales from picoseconds to several seconds indicate that molten globules and denatured proteins are characterized by an increased flexibility, a loss of local confinement and a larger fraction of mobile atoms (Buck et al ., 1996; Receveur et al ., 1997; Kataoka et al ., 1999 a ; Bai et al ., 2000; Bu et al ., 2000, 2001; Dilg et al ., 2002; Russo et al ., 2002; Tarek et al ., 2003; Fitter, 2003 a , 2003 b ; Jansson and Swenson, 2008; Gibrat et al ., 2008; Ramboarina and Redfield, 2008; Santos et al ., 2010; Hennig et al ., 2012; Dutta et al ., 2014; Yadav et al ., 2014; Mondal et al ., 2015; Grimaldo et al ., 2015 a ; Aznauryan et al ., 2016; Stadler et al ., 2016 a ; Ameseder et al ., 2018 b ). Moreover, the dynamics of the mobile atoms is generally characterized by an increased dynamic heterogeneity in the molten and denatured structures compared with the native conformations (Bai et al ., 2000; Tarek et al ., 2003; Kuzmenkina et al ., 2005; Nienhaus, 2006; Ramboarina and Redfield, 2008; Santos et al ., 2010; Dutta et al ., 2014; Mondal et al ., 2015; Aznauryan et al ., 2016). This, however, might not be true in all denaturing environments (Ghosh et al ., 2015).…”

Characterisation of denatured states of sensory rhodopsin II by solution-state NMR

“…A range of NMR methods are available for studying protein folding events which occur at different timescales and for probing the properties of low--population folding intermediates, hence yielding detailed insights into folding landscapes, misfolding, aggregation and function [22][23][24][25][26][27][28]. Comprehensive studies on the denatured states of full--length polytopic α--helical membrane proteins are scarce [29], and have mostly been limited to sparsely--labelled samples [30][31][32][33] or fragments of polytopic α--helical membrane proteins [34][35][36]. Insights on unfolding pathways and unfolded states could only be derived from a small subset of amino acids or chemical groups in the protein.…”