Investigating the Relationship Between Physical Properties of Detergents and Membrane Protein Structure Determination

Biochemistry, 2007

Surfactant protein B (SP-B) is essential for normal lung surfactant function, which is in itself essential to life. However, the molecular basis for SP-B's activity is not understood and a high-resolution structure for SP-B has not been determined. Mini-B is a 34-residue peptide with internal disulfide linkages that is composed of the N-and C-terminal helical regions of SP-B. It has been shown to retain similar activity to full-length SP-B in certain in vitro and in vivo studies. We have used solution NMR to determine the structure of Mini-B in the presence of micelles composed of the anionic detergent sodium dodecyl sulfate (SDS). Under these conditions, Mini-B forms two R-helices connected by an unstructured loop. Mini-B possesses a strikingly amphipathic surface with a large positively charged patch on one face of the peptide and a large hydrophobic patch on the opposite face. A tryptophan side chain extends outward from the peptide in a position to interact with lipids at the polar/apolar interface. Interhelix interactions are stabilized by both disulfide bonds and by interleaving of hydrophobic side chains from the two helices. Surfactant protein B (SP-B 1) is an essential component of lung surfactant, a material that is indispensable for normal breathing. Lung surfactant is a mixture of lipids and proteins that lines the air-water interface in alveoli. One of its main functions is to drastically reduce the surface tension at the air-water interface, thus preventing alveolar collapse during expiration and reducing the work of breathing (1, 2). Lung surfactant components are also important in the innate immune response to microbes in the lungs (3). Deficiency or inactivation of lung surfactant leads to potentially fatal respiratory disorders such as neonatal respiratory distress syndrome (NRDS) in premature newborns (4) and acute respiratory distress syndrome (ARDS) in adults with acute injury and illness (5). Development of lung surfactant replacement treatments in the early 1990s greatly improved the outlook for NRDS (6), but successful treatment of ARDS with endogenous surfactant has proved more challenging, probably because the conditions that lead to ARDS in the first place lead to rapid deactivation of the replacement surfactant (7). Clinical trials have shown artificial surfactants to be much more effective if they include the proteins SP-B or SP-C, as compared to protein-free preparations (8). The requirement for SP-B's presence in effective surfactant replacement therapy is in keeping with the lethality of hereditary SP-B deficiency in humans (9) and knockout mice (10). Improvement of surfactant replacement preparations, for example, to avoid the use of animalderived surfactant and to improve its activity in the context of ARDS, is hampered by a lack of understanding of the structural bases for the activity of the lung surfactant proteins. Lung surfactant is synthesized and secreted into the alveolar fluid by epithelial type II pneumocytes (11). The composition of lung surfactant varies among different vertebrates and also throughout the physiological develop-† We acknowledge financial support from the Canadian Institutes of Health Research and the National Institutes of Health (Grant R01 HL55534) and salary support for V.B. from the Parker B. Francis Foundation. ‡ The structures of Mini-B have been deposited in the Protein Data Bank as entries 2JOU (in hexafluoro-2-propanol) and 2DWF (in sodium dodecyl sulfate micelles). The chemical shifts of Mini-B have been deposited in the BMRB data bank as entry 6741.

Journal of the …, 2009

One major obstacle to membrane protein structure determination is the selection of a detergent micelle that mimics the native lipid bilayer. Currently, detergents are selected by exhaustive screening because the effects of protein-detergent interactions on protein structure are poorly understood. In this study, the structure and dynamics of an integral membrane protein in different detergents is investigated by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy, and small angle X-ray scattering (SAXS). The results suggest that matching of the micelle dimensions to the protein's hydrophobic surface avoids exchange processes that reduce the completeness of the NMR observations. Based on these dimensions, several mixed micelles were designed that improved the completeness of NMR observations. These findings provide a basis for the rational design of mixed micelles that may advance membrane protein structure determination by NMR.

The Journal of Physical Chemistry B, 2008

Membrane proteins present major challenges for structural biology. In particular, the production of suitable crystals for high-resolution structural determination continues to be a significant roadblock for developing an atomic-level understanding of these vital cellular systems. The use of detergents for extracting membrane proteins from the native membrane for either crystallization or reconstitution into model lipid membranes for further study is assumed to leave the protein with the proper fold with a belt of detergent encompassing the membrane-spanning segments of the structure. Small-angle x-ray scattering was used to probe the detergent-associated solution conformations of three membrane proteins, namely bacteriorhodopsin (BR), the Ste2p G-protein coupled receptor from S. cerevisiae, and the E. coli porin OmpF. The results demonstrate that, contrary to the traditional model of a detergent-associated membrane protein, the helical proteins BR and Ste2p are not in the expected, compact conformation and associated with detergent micelles, while the beta-barrel OmpF is indeed embedded in a disk-like micelle in a properly-folded state. The comparison provided by the BR and Ste2p, both members of the 7TM family of helical membrane proteins, further suggests that the inter-helical interactions between the transmembrane helices of the two proteins differ, such that BR, like other rhodopsins, can properly refold to crystallize, while Ste2p continues to prove resistant to crystallization from an initially detergent-associated state.

ACS Omega, 2021

Membrane proteins are frequently reconstituted in different detergents as a prerequisite to create a phospholipid environment reminiscent of their native environment. Different detergent characteristics such as their chain length and bond types could affect the structure and function of proteins. Yet, they are seldom taken into account when choosing a detergent for structural studies. Here, we explore the effect of different detergents and lipids with varying degrees of double-or single-bond composition on 1 H− 15 N transverse relaxation optimized spectroscopy spectra of the outer membrane protein W (OmpW). We observed changes in nuclear magnetic resonance chemical shifts for OmpW reconstituted in micelles, bicelles, and nanodiscs, depending on their detergent/ lipid composition. These results suggest that a careful evaluation of detergents is necessary, so as not to jeopardize the structure and function of the protein.

Biochemistry, 2017

There remains a need for new non-ionic detergents that are suitable for use in biochemical and biophysical studies of membrane proteins. Here we explore the properties of n-dodecyl-β-melibioside (β-DDMB) micelles as a medium for membrane proteins. Melibiose is d-galactose-α(1→6)-d-glucose. Light scattering showed the β-DDMB micelle to be roughly 30 kDa smaller than micelles formed by the commonly used n-dodecyl-β-maltoside (β-DDM). β-DDMB stabilized diacylglycerol kinase (DAGK) against thermal inactivation. Moreover, activity assays conducted using aliquots of DAGK purified into β-DDMB yielded activities that were 40% higher than those of DAGK purified into β-DDM. β-DDMB yielded similar or better TROSY-HSQC NMR spectra for two single-pass membrane proteins and the tetraspan membrane protein peripheral myelin protein 22. β-DDMB appears be a useful addition to the toolbox of non-ionic detergents available for membrane protein research.

2006

2.1 Scope of this Thesis This thesis represents an attempt to enlighten the role of the detergent in reconstitution and more specifically in two-dimensional (2D) crystallogenesis of membrane proteins. The construction of a tool for precise and routine measurements of detergent concentrations provided a valuable tool for better understanding and controlling the detergent issue. Additionally, a novel approach for detergent removal in 2D crystallization, i.e. the use of cyclodextrins was explored and a nanoliter dispensing high throughput tool was developed allowing for profound and sophisticated screening of optimal conditions for protein reconstitution and crystallization. 2.2 Combining Electron Microscopy and Atomic Force Microscopy Although electron crystallography has proven to be a powerful approach to structure determination of membrane proteins (for a recent example see (Gonen et al., 2005)) successes are somehow restricted to certain classes of membrane proteins (e.g., outer m...

The journal of physical chemistry. B, 2017

Although fundamentally significant in structural, chemical, and membrane biology, the interfacial protein-detergent complex (PDC) interactions have been modestly examined because of the complicated behavior of both detergents and membrane proteins in aqueous phase. Membrane proteins are prone to unproductive aggregation resulting from poor detergent solvation, but the participating forces in this phenomenon remain ambiguous. Here, we show that using rational membrane protein design, targeted chemical modification, and steady-state fluorescence polarization spectroscopy, the detergent desolvation of membrane proteins can be quantitatively evaluated. We demonstrate that depleting the detergent in the sample well produced a two-state transition of membrane proteins between a fully detergent-solvated state and a detergent-desolvated state, the nature of which depended on the interfacial PDC interactions. Using a panel of six membrane proteins of varying hydrophobic topography, structura...

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2000

Detergents are indispensable in the isolation of integral membrane proteins from biological membranes to study their intrinsic structural and functional properties. Solubilization involves a number of intermediary states that can be studied by a variety of physicochemical and kinetic methods ; it usually starts by destabilization of the lipid component of the membranes, a process that is accompanied by a transition of detergent binding by the membrane from a noncooperative to a cooperative interaction already below the critical micellar concentration (CMC). This leads to the formation of membrane fragments of proteins and lipids with detergent-shielded edges. In the final stage of solubilization membrane proteins are present as protomers, with the membrane inserted sectors covered by detergent. We consider in detail the nature of this interaction and conclude that in general binding as a monolayer ring, rather than as a micelle, is the most probable mechanism. This mode of interaction is supported by neutron diffraction investigations on the disposition of detergent in 3-D crystals of membrane proteins. Finally, we briefly discuss the use of techniques such as analytical ultracentrifugation, size exclusion chromatography, and mass spectrometry relevant for the structural investigation of detergent solubilized membrane proteins. ß

Biophysical Journal, 2005

The structure and flexibility of the outer membrane protein X (OmpX) in a water-detergent solution and in pure water are investigated by molecular dynamics simulations on the 100-ns timescale and compared with NMR data. The simulations allow for an unbiased determination of the structure of detergent micelles and the protein-detergent mixed micelle. The short-chain lipid dihexanoylphosphatidylcholine, as a detergent, aggregates into pure micelles of ;18 molecules, or alternatively, it binds to the protein surface. The detergent binds in the form of a monolayer ring around the hydrophobic b-barrel of OmpX rather than in a micellar-like oblate; ;40 dihexanoylphosphatidylcholine lipids are sufficient for an effective suppression of water from the surface of the b-barrel region. The phospholipids bind also on the extracellular, protruding bsheet. Here, polar interactions between charged amino acids and phosphatidylcholine headgroups act as condensation seed for detergent micelle formation. The polar protein surface remains accessible to water molecules. In total, ;90-100 detergent molecules associate within the protein-detergent mixed micelle, in agreement with experimental estimates. The simulation results indicate that OmpX is not a water pore and support the proposed role of the protruding b-sheet as a ''fishing rod''.

Molecular membrane biology, 2016

Detergents are amphiphilic compounds that have crucial roles in the extraction, purification and stabilization of integral membrane proteins and in experimental studies of their structure and function. One technique that is highly dependent on detergents for solubilization of membrane proteins is solution-state NMR spectroscopy, where detergent micelles often serve as the best membrane mimetic for achieving particle sizes that tumble fast enough to produce high-resolution and high-sensitivity spectra, although not necessarily the best mimetic for a biomembrane. For achieving the best quality NMR spectra, detergents with partial or complete deuteration can be used, which eliminate interfering proton signals coming from the detergent itself and also eliminate potential proton relaxation pathways and strong dipole-dipole interactions that contribute line broadening effects. Deuterated detergents have also been used to solubilize membrane proteins for other experimental techniques inclu...