Deuterium Labeling Together with Contrast Variation Small-Angle Neutron Scattering Suggests How Skp Captures and Releases Unfolded Outer Membrane Proteins

Abstract

In gram-negative bacteria, the chaperone protein Skp forms specific and stable complexes with membrane proteins while they are transported across the periplasm to the outer membrane. The jellyfish-like architecture of Skp is similar to the eukaryotic and archeal prefoldins and the mitochondrial Tim chaperones, that is α-helical ‘tentacles’ extend from a β-strand ‘body’ to create an internal cavity. Contrast variation small-angle neutron scattering (SANS) experiments on Skp alone in solution and bound in two different complexes to unfolded outer membrane proteins (uOMPs), OmpA and OmpW, demonstrate that the helical tentacles of Skp bind their substrate in a clamp-like mechanism in a conformation similar to that previously observed in the apo crystal structure of Skp. Deuteration of the uOMP component combined with contrast variation analysis allowed the shapes of Skp and uOMP as well as the location of uOMP with respect to Skp to be determined in both complexes. This represents unique information that could not be obtained without deuterium labeling of the uOMPs. The data yield the first direct structural evidence that the α-helical Skp tentacles move closer together on binding its substrate and that the structure of Skp is different when binding different uOMPs. This work presents, by example, a tutorial on performing SANS experiments using both deuterium labeling and contrast variation, including SANS theory, sample preparation, data collection, sample quality validation, data analysis and structure modeling.

SurA is a “Groove-y” Chaperone That Expands Unfolded Outer Membrane Proteins

“…uOMPs are expected to exist in a relatively collapsed, molten globule state in the absence of chaperones (50). This collapsed conformation is maintained when uOmpA171 is bound by the other major chaperone in the uOMP biogenesis pathway, Skp (60,61). To determine whether the overall size and shape of a uOMP changes when bound to SurA, we measured the hydrodynamic properties using small angle neutron scattering (SANS).…”

Investigating Structure and Dynamics of Proteins in Amorphous Phases Using Neutron Scattering

“…Examples of this methodology in biological systems are presented in the literature [25], [47], [48], [49], [50], [51]. The basic idea of this method is that the scattered intensity depends on Δρ (see Eq.…”

“…These techniques are useful for gaining information on the structural arrangement and dynamics of proteins as well as establishing correlations with stability in amorphous phases. Readers interested in other neutron techniques, or other applications of neutron scattering in biology, are referred to general reviews discussing the fundamentals of neutron scattering [15], [16], [17], [18], [19], neutron scattering for structural biology studies [13], [14], [20], [21], and contrast matching techniques for biological samples [22], [23], [24], [25].…”

From Chaperones to the Membrane with a BAM!

“…Small-angle Neutron Scattering (SANS , See Glossary) experiments have demonstrated that the α-helical arms of Skp are flexible to accommodate the unstructured uOMP [19]. Structural modeling of uOMP bound to Skp revealed that the uOMP is not entirely encapsulated but protrudes from the Skp cavity (Figure 3).…”