Structural Changes in Bacteriorhodopsin during In Vitro Refolding from a Partially Denatured State

Abstract

We report on the formation of the secondary and tertiary structure of bacteriorhodopsin during its in vitro refolding from an SDS-denatured state. We used the mobility of single spin labels in seven samples, attached at various locations to six of the seven helical segments to engineered cysteine residues, to follow coil-to-helix formation. Distance measurements obtained by spin dipolar quenching in six samples labeled at either the cytoplasmic or extracellular ends of pairs of helices revealed the time dependence of the recovery of the transmembrane helical bundle. The secondary structure in the majority of the helical segments refolds with a time constant of <100-140 ms. Recovery of the tertiary structure is achieved by sequential association of the helices and occurs in at least three distinct steps with time constants of 1), well below 1 s; 2), 3-4 s; and 3), 60-130 s (the latter depending on the helical pair). The slowest of these processes occurs in concert with recovery of the retinal chromophore.

Kinetic Folding Mechanism of an Integral Membrane Protein Examined by Pulsed Oxidative Labeling and Mass Spectrometry

“…35 Nonetheless, the occurrence of parallel events as suggested in some studies 72,86 cannot be ruled out. The retinal binding mechanism observed here may be different from the processes occurring during BR reconstitution from apomembrane.…”

“…Unfortunately, the data obtained in this way provide only global information, and details of the structural changes remain hidden. Engineered fluorescence tags 32 or spin labels [33][34][35] can offer additional insights. Nonetheless, there remains a need to establish robust techniques capable of monitoring the kinetics of membrane protein structural changes in a spatially resolved fashion.…”

The safety dance: biophysics of membrane protein folding and misfolding in a cellular context

“…The rates of soluble protein folding reactions vary greatly but are generally rapid (Plaxco et al, 2000); folding of soluble proteins generally requires anywhere from microseconds to minutes in vitro . Similar to soluble proteins (Brockwell & Radford, 2007), kinetic intermediates of helical membrane proteins can form within milliseconds of the initiation of folding from detergent-denatured states (Allen et al, 2004b; Booth et al, 1995; Krishnamani & Lanyi, 2011; Lu & Booth, 2000; Otzen, 2003). However, complete refolding and/or oligomerization from can require anywhere from minutes to days in vitro (Allen et al, 2004b; Cao et al, 2011; Jefferson et al, 2013; Krishnamani & Lanyi, 2011; Riley et al, 1997; Schlebach et al, 2012; Schlebach et al, 2013).…”

“…Similar to soluble proteins (Brockwell & Radford, 2007), kinetic intermediates of helical membrane proteins can form within milliseconds of the initiation of folding from detergent-denatured states (Allen et al, 2004b; Booth et al, 1995; Krishnamani & Lanyi, 2011; Lu & Booth, 2000; Otzen, 2003). However, complete refolding and/or oligomerization from can require anywhere from minutes to days in vitro (Allen et al, 2004b; Cao et al, 2011; Jefferson et al, 2013; Krishnamani & Lanyi, 2011; Riley et al, 1997; Schlebach et al, 2012; Schlebach et al, 2013). The folding of the soluble denatured forms of β-barrel membrane proteins into membranes also appears to be quite slow (Burgess et al, 2008; Gessmann et al, 2014; Huysmans et al, 2010; Huysmans et al, 2012), though in this case the rate-limiting step seems to involve the transfer of the unfolded protein from the aqueous phase to the membrane phase (Gessmann et al, 2014; Huysmans et al, 2010; Huysmans et al, 2012).…”

Lipid bilayer composition modulates the unfolding free energy of a knotted α-helical membrane protein

“…As for urea-denatured states, the residual structure in the SDS-denatured state of membrane proteins is not well understood. bR is the best characterized with respect to folding studies, with far-UV CD indicating a significant reduction in helicity (21,23) and DEER suggesting a smaller reduction in helix structure and more or less complete loss of helix, helix tertiary interactions (59). Less direct information is available for SDS denatured states of other proteins that have been the subject of unfolding studies; for example, GlpG in SDS exhibits no loss of helical structure and very little change in fluorescence emission band, which in any case is challenging to assign to definitive structural change (10,60,61).…”