Folding studies of purified LamB protein, the maltoporin from the Escherichia coli outer membrane: Trimer dissociation can be separated from unfolding

Abstract

The folding mechanisms for β-barrel membrane proteins present unique challenges because acquisition of both secondary and tertiary structure is coupled with insertion into the bilayer. For the porins in Escherichia coli outer membrane, the assembly pathway also includes association into homotrimers. We study the folding pathway for purified LamB protein in detergent and observe extreme hysteresis in unfolding and refolding, as indicated by the shift in intrinsic fluorescence. The strong hysteresis is not seen in unfolding and refolding a mutant LamB protein lacking the disulfide bond, as it unfolds at much lower denaturant concentrations than wild type LamB protein. The disulfide bond is proposed to stabilize the structure of LamB protein by clasping together the two sides of Loop 1 as it lines the inner cavity of the barrel. In addition we find that low pH promotes dissociation of the LamB trimer to folded monomers, which run at about one third the size of the native trimer during SDS PAGE and are much more resistant to trypsin than the unfolded protein. We postulate the loss at low pH of two salt bridges between Loop 2 of the neighboring subunit and the inner wall of the monomer barrel destabilizes the quaternary structure.

Engineered Oligomerization State of OmpF Protein through Computational Design Decouples Oligomer Dissociation from Unfolding

“…This indicates that the dissociation of the dimer was not coupled with the unfolding of the protein. This phenomenon is consistent with recent unfolding studies on LamB, another β -barrel membrane protein, where the trimeric protein disassociated into folded monomeric species at low pH values, suggesting that trimer dis-association and unfolding of the protein may not occur simultaneously [44]. …”

“…Trypsin resistance has been used in previous studies as a useful indicator of protein folding [44, 23]. We observed that the folded oligomeric and monomeric bands (Figure 4a) were resistant to trypsin digestion, while the unfolded band disappeared after trypsin treatment (Figure 4b).…”

“…The mixture was incubated overnight at 37° C using Isotemp Hybridization Incubator (Fisher Scientific). After overnight refolding, subsequent degradation of the unfolded protein was induced by the addition of trypsin (trypsin/protein 1:100 w/w) [44]. Final protein was purified and concentrated using Amicon Ultra-0.5 mL Centrifugal Filters 30K (Millipore).…”

“…The mutant R100L/G135W exists in folded dimeric (D), monomeric, as well as an unfolded state. b) After overnight refolding, subsequent degradation of the unfolded protein was induced by the addition of trypsin (trypsin/protein 1:100 w/w) [44]. Final protein was purified and concentrated using Centrifugal Filters.…”

Quantifying the co-solvent effects on trypsin from the digestive system of carp Catla catla by biophysical techniques and molecular dynamics simulations

“…Folding and unfolding of protein have been observed with the help of banding pattern obtained from the SDS-PAGE. 52-54 Baldwin et al 52 focused on the folded and unfolded proteins on SDS-PAGE and used to quantitate their unfolding.…”