Kell K. Andersen

The Role of Decorated SDS Micelles in Sub-CMC Protein Denaturation and Association

We have combined spectroscopy, chromatography, calorimetry, and small-angle X-ray scattering (SAXS) to provide a comprehensive structural and stoichiometric description of the sodium dodecyl sulfate (SDS)-induced denaturation of the 86-residue alpha-helical bovine acyl-coenzyme-A-binding protein (ACBP). Denaturation is a multistep process. Initial weak binding of 1-3 SDS molecules per protein molecule below 1.3 mM does not perturb the tertiary structure. Subsequent binding of approximately 13 SDS molecules per ACBP molecule leads to the formation of SDS aggregates on the protein and changes in both tertiary and secondary structures. SAXS data show that, at this stage, a decorated micelle links two ACBP molecules together, leaving about half of the polypeptide chain as a disordered region protruding into the solvent. Further titration with SDS leads to the additional uptake of 26 SDS molecules, which, according to SAXS, forms a larger decorated micelle bound to a single ACBP molecule. At the critical micelle concentration, we conclude from reduced mobility and increased fluorescence anisotropy that each ACBP molecule becomes associated with more than one micelle. At this point, 56-60 SDS molecules are bound per ACBP molecule. Our data provide key structural insights into decorated micelle complexes with proteins, revealing a remarkable diversity in the different conformations they can stabilize. The data highlight that a minimum decorated micelle size, which may be a key driving force for intermolecular protein association, exists. This may also provide a structural basis for the known ability of submicellar surfactant concentrations to induce protein aggregation and fibrillation.

Folding of outer membrane proteins.

Outer membrane proteins (OMPs) represent a large group of β-barrel proteins found both in the membranes of both bacteria and eukaryotes. Their general ease of expression and refolding and straightforward methods to monitor their degree of folding conspire to make OMPs excellent model systems to investigate how the membrane environment and other biological factors modulating the membrane insertion and folding of OMPs influence the folding pathway. This review attempts to provide an overview of how these proteins are studied in vitro and what kind of information can reliably be extracted. Numerous choices have to be made in setting the conditions for successful folding/unfolding, and here a major challenge remains to identify conditions that lead to completely reversible folding without any hysteresis. Recent progress indicates that this is possible through rigorous optimization, such as the use of relatively extreme pH and phospholipids with short chain lengths. OMPs are generally kinetically very stable, which means that they both fold and unfold very slowly. Many OMPs cannot even unfold when embedded in lipid vesicles, but recent work has demonstrated that surfactants can provide a useful alternative which can lead to a complete description of the kinetics of folding and unfolding of an OMP. The recent report of the first protein engineering study of an OMP has demonstrated that it may soon be possible to have almost atomic-level resolution of an OMP folding mechanism. Combining this insight with the biological complexity of the membrane environment constitutes an exciting new frontier in membrane protein science.

How Chain Length and Charge Affect Surfactant Denaturation of Acyl Coenzyme A Binding Protein (ACBP)

Using intrinsic tryptophan fluorescence, equilibria and kinetics of unfolding of acyl coenzyme A binding protein (ACBP) have been investigated in sodium alkyl sulfate surfactants of different chain length (8-16 carbon atoms) and with different proportions of the nonionic surfactant dodecyl maltoside (DDM). The aim has been to determine how surfactant chain length and micellar charge affect the denaturation mechanism. ACBP denatures in two steps irrespective of surfactant chain length, but with increasing chain length, the potency of the denaturant rises more rapidly than the critical micelle concentration (cmc) declines. Increasing proportions of DDM, which significantly reduce the amount of monomeric sodium dodecyl sulfate (SDS), make the first denaturation step occur at lower concentrations but weaken and eventually remove the second denaturation step. The logarithm of the unfolding rate constants increases linearly with denaturant concentration below the cmc but declines at higher concentrations. Both shortening chain length and decreasing micellar charge reduce the overall kinetics of unfolding and makes the dependence of unfolding rate constants on surfactant concentration more complex. This behavior contrasts with the simplicity of unfolding in chemical denaturants and highlights the changing properties of surfactant micelles. We suggest that the transition from spherical to more elongated micelles leads to inhibition of unfolding kinetics, while weaker binding sites may cause a subsequent rise in unfolding rate constants at higher surfactant concentrations. We propose that shifting micellar binding sites on globular proteins such as ACBP, as opposed to the predefined binding sites on membrane protein surfaces, may lead to nonlinear correlations between activation unfolding energies and SDS mole fraction.

Folding of outer membrane protein A in the anionic biosurfactant rhamnolipid

Folding and stability of bacterial outer membrane proteins (OMPs) are typically studied in vitro using model systems such as phospholipid vesicles or surfactant. OMP folding requires surfactant concentrations above the critical micelle concentration (cmc) and usually only occurs in neutral or zwitterionic surfactants, but not in anionic or cationic surfactants. Various Gram‐negative bacteria produce the anionic biosurfactant rhamnolipid. Here we show that the OMP OmpA can be folded in rhamnolipid at concentrations above the cmc, though the thermal stability is reduced compared to the non‐ionic surfactant dodecyl maltoside. We discuss implications for possible interactions between OMPs and biosurfactants in vivo.

OmpA can form folded and unfolded oligomers.

The monomeric outer membrane protein OmpA from Escherichia coli has long served as a model protein for studying the folding and membrane insertion of β-barrel membrane proteins. Here we report that when OmpA is refolded in limiting amounts of surfactant (close to the cmc), it has a high propensity to form folded and unfolded oligomers. The oligomers exist both in a folded and (partially) unfolded form which both dissociate under denaturing conditions. Oligomerization does not require the involvement of the periplasmic domain and is not strongly affected by ionic strength. The folded dimers can be isolated and show native-like secondary structure; they are resistant to proteolytic attack and do not dissociate in high surfactant concentrations, indicating high kinetic stability once formed. Remarkably, OmpA also forms significant amounts of higher order structures when refolding in the presence of lipid vesicles. We suggest that oligomerization occurs by domain swapping favored by the high local concentration of OmpA molecules congregating on the same micelle or vesicle. In this model, the unfolded oligomer is stabilized by a small number of intermolecular β-strand contacts and subsequently folds to a more stable state where these intermolecular contacts are consolidated in a native-like fashion by contacts between complementary β-strands from different molecules. Our model is supported by the ability of complementary fragments to associate with each other in vitro. Oligomerization is probably avoided in the cell by the presence of cellular chaperones which maintain the protein in a monomeric state.

The anionic biosurfactant rhamnolipid does not denature industrial enzymes

Biosurfactants (BS) are surface-active molecules produced by microorganisms. Their combination of useful properties and sustainable production make them promising industrial alternatives to petrochemical and oleochemical surfactants. Here we compare the impact of the anionic BS rhamnolipid (RL) and the conventional/synthetic anionic surfactant sodium dodecyl sulfate (SDS) on the structure and stability of three different commercially used enzymes, namely the cellulase Carezyme® (CZ), the phospholipase Lecitase Ultra® (LT) and the α-amylase Stainzyme® (SZ). Our data reveal a fundamental difference in their mode of interaction. SDS shows great diversity of interaction toward the different enzymes. It efficiently unfolds both LT and CZ, but LT is unfolded by SDS through formation of SDS clusters on the enzyme well below the cmc, while CZ is only unfolded by bulk micelles and on average binds significantly less SDS than LT. SDS binds with even lower stoichiometry to SZ and leads to an increase in thermal stability. In contrast, RL does not affect the tertiary or secondary structure of any enzyme at room temperature, has little impact on thermal stability and only binds detectably (but at low stoichiometries) to SZ. Furthermore, all enzymes maintain activity at both monomeric and micellar concentrations of RL. We conclude that RL, despite its anionic charge, is a surfactant that does not compromise the structural integrity of industrially relevant enzymes. This makes RL a promising alternative to current synthetic anionic surfactants in a wide range of commercial applications.

Low-Resolution Structures of OmpA⋅DDM Protein–Detergent Complexes

We have used SAXS to determine the low‐resolution structure of the outer‐membrane protein OmpA from E. coli solubilized by the surfactant dodecyl maltoside (DDM). We have studied three variants of the transmembrane domain of OmpA—namely monomers, self‐associated dimers, and covalently linked dimers—as well as the monomeric species of the full‐length protein with the periplasmic domain. We can successfully model the structures of the monomeric and covalently linked dimer as one and two natively folded proteins in a DDM micelle, respectively, whereas the noncovalently linked dimer presents a more complicated structure, possibly due to higher‐order species. We have determined the structure of the full‐length protein to be that of a globular periplasmic domain attached through a flexible linker to the transmembrane domain. This approach provides valuable information about how membrane proteins are embedded in amphiphilic environments.

Stable intermediates determine proteins' primary unfolding sites in the presence of surfactants

Despite detailed knowledge of the overall structural changes and stoichiometries of surfactant binding, little is known about which protein regions constitute the preferred sites of attack for initial unfolding. Here we have exposed three proteins to limited proteolysis at anionic (SDS) and cationic (DTAC) surfactant concentrations corresponding to specific conformational transitions, using the surfactant‐robust broad‐specificity proteases Savinase and Alcalase. Cleavage sites are identified by SDS‐PAGE and N‐terminal sequencing. We observe well‐defined cleavage fragments, which suggest that flexibility is limited to certain regions of the protein. Cleavage sites for α‐lactalbumin and myoglobin correspond to regions identified in other studies as partially unfolded at low pH or in the presence of organic solvents. For Tnfn3, which does not form partially folded structures under other conditions, cleavage sites can be rationalized from the structure of the proteins folding transition state and the position of loops in the native state. Nevertheless, they are more sensitive to choice of surfactant and protease, probably reflecting a heterogeneous and fluctuating ensemble of partially unfolded structures. Thus, for proteins accumulating stable intermediates on the folding pathway, surfactants encourage the formation of these states, while the situation is more complex for proteins that do not form these intermediates.

pH Regulation of the Kinetic Stability of the Lipase from Thermomyces lanuginosus

Thermomyces lanuginosus lipase (TlL) is a kinetically stable protein, resistant toward both denaturation and refolding in the presence of the ionic surfactant sodium dodecyl sulfate (SDS) and the nonionic surfactant decyl maltoside (DecM). We investigate the pH dependence of this kinetic stability. At pH 8, TlL remains folded and enzymatically active at multimillimolar surfactant concentrations but fails to refold from the acid urea-denatured state at submillimolar concentrations of SDS and DecM, indicating a broad concentration range of kinetic trapping or hysteresis. At pH 8, very few SDS molecules bind to TlL. The hysteresis SDS concentration range shrinks when moving to pH 4-6; in this pH range, SDS binds as micellelike clusters. Although hysteresis can be eliminated by reducing disulfide bonds, destabilizing the native state, and lowering the unfolding activation barrier, SDS sensitivity is not directly linked to intrinsic kinetic stability [its resistance to the general chemical denaturant guanidinium chloride (GdmCl)], because TlL unfolds more slowly in GdmCl at pH 6.0 than at pH 8.0. However, the estimated net charge drops from approximately -12 to approximately -5 between pH 8 and 6. SDS denatures TlL at pH 6.0 by nucleating via a critical number of bound SDS molecules on the surface of native TlL to form clusters. These results imply that SDS sensitivity is connected to the availability of appropriately charged regions on the protein. We suggest that conformational rigidity is a necessary but not sufficient feature of SDS resistance, because this has to be combined with sufficient negative electrostatic potential to avoid extensive SDS binding.

Glycolipid Biosurfactants Activate, Dimerize, and Stabilize Thermomyces lanuginosus Lipase in a pH-Dependent Fashion

We present a study of the interactions between the lipase from Thermomyces lanuginosus (TlL) and the two microbially produced biosurfactants (BSs), rhamnolipid (RL) and sophorolipid (SL). Both RL and SL are glycolipids; however, RL is anionic, while SL is a mixture of anionic and non-ionic species. We investigate the interactions of RL and SL with TlL at pH 6 and 8 and observe different effects at the two pH values. At pH 8, neither RL nor SL had any major effect on TlL stability or activity. At pH 6, in contrast, both surfactants increase TlLs thermal stability and fluorescence and activity measurements indicate interfacial activation of TlL, resulting in 3- and 6-fold improved activity in SL and RL, respectively. Nevertheless, isothermal titration calorimetry reveals binding of only a few BS molecules per lipase. Size-exclusion chromatography and small-angle X-ray scattering suggest formation of TlL dimers with binding of small amounts of either RL or SL at the dimeric interface, forming an elongated complex. We conclude that RL and SL are compatible with TlL and constitute promising green alternatives to traditional surfactants.