Ryan C. Oliver

Dependence of micelle size and shape on detergent alkyl chain length and head group

Micelle-forming detergents provide an amphipathic environment that can mimic lipid bilayers and are important tools for solubilizing membrane proteins for functional and structural investigations in vitro. However, the formation of a soluble protein-detergent complex (PDC) currently relies on empirical screening of detergents, and a stable and functional PDC is often not obtained. To provide a foundation for systematic comparisons between the properties of the detergent micelle and the resulting PDC, a comprehensive set of detergents commonly used for membrane protein studies are systematically investigated. Using small-angle X-ray scattering (SAXS), micelle shapes and sizes are determined for phosphocholines with 10, 12, and 14 alkyl carbons, glucosides with 8, 9, and 10 alkyl carbons, maltosides with 8, 10, and 12 alkyl carbons, and lysophosphatidyl glycerols with 14 and 16 alkyl carbons. The SAXS profiles are well described by two-component ellipsoid models, with an electron rich outer shell corresponding to the detergent head groups and a less electron dense hydrophobic core composed of the alkyl chains. The minor axis of the elliptical micelle core from these models is constrained by the length of the alkyl chain, and increases by 1.2–1.5 Å per carbon addition to the alkyl chain. The major elliptical axis also increases with chain length; however, the ellipticity remains approximately constant for each detergent series. In addition, the aggregation number of these detergents increases by ∼16 monomers per micelle for each alkyl carbon added. The data provide a comprehensive view of the determinants of micelle shape and size and provide a baseline for correlating micelle properties with protein-detergent interactions.

Dehaloperoxidase-hemoglobin from Amphitrite ornata is primarily a monomer in solution.

The crystal structures of the dehaloperoxidase-hemoglobin from A. ornata (DHP A) each report a crystallographic dimer in the unit cell. Yet, the largest dimer interface observed is 450 Å(2), an area significantly smaller than the typical value of 1200-2000 Å(2) and in contrast to the extensive interface region of other known dimeric hemoglobins. To examine the oligomerization state of DHP A in solution, we used gel permeation by fast protein liquid chromatography and small-angle X-ray scattering (SAXS). Gel permeation experiments demonstrate that DHP A elutes as a monomer (15.5 kDa) and can be separated from green fluorescent protein, which has a molar mass of 27 kDa, near the 31 kDa expected for the DHP A dimer. By SAXS, we found that DHP A is primarily monomeric in solution, but with a detectable level of dimer (~10%), under all conditions studied up to a protein concentration of 3.0 mM. These concentrations are likely 10-100-fold lower than the K(d) for dimer formation. Additionally, there was no significant effect either on the overall conformation of DHP A or its monomer-dimer equilibrium upon addition of the DHP A inhibitor, 4-iodophenol.

Tuning Micelle Dimensions and Properties with Binary Surfactant Mixtures

Detergent micelles are used in many areas of research and technology, in particular, as mimics of the cellular membranes in the purification and biochemical and structural characterization of membrane proteins. Applications of detergent micelles are often hindered by the limited set of properties of commercially available detergents. Mixtures of micelle-forming detergents provide a means to systematically obtain additional micellar properties and expand the repertoire of micelle features available; however, our understanding of the properties of detergent mixtures is still limited. In this study, the shape and size of binary mixtures of seven different detergents commonly used in molecular host-guest systems and membrane protein research were investigated. The data suggests that the detergents form ideally mixed micelles with sizes and shapes different from those of pure individual micelles. For most measurements of size, the mixtures varied linearly with detergent mole fraction and therefore can be calculated from the values of the pure detergents. We propose that properties such as the geometry, size, and surface charge can be systematically and predictably tuned for specific applications.

Designing Mixed Detergent Micelles for Uniform Neutron Contrast

Micelle-forming detergents provide an amphipathic environment that mimics lipid bilayers and are important tools used to solubilize and stabilize membrane proteins in solution for in vitro structural investigations. Small-angle neutron scattering (SANS) at the neutron contrast match point of detergent molecules allows observing the signal from membrane proteins unobstructed by contributions from the detergent. However, we show that even for a perfectly average-contrast matched detergent there arises significant core-shell scattering from the contrast difference between aliphatic detergent tails and hydrophilic head groups. This residual signal interferes with interpreting structural data of membrane proteins. This complication is often made worse by the presence of excess empty (protein-free) micelles. We present an approach for the rational design of mixed micelles containing a deuterated detergent analog, which eliminates neutron contrast between core and shell and allows the micelle scattering to be fully contrast-matched to unambiguously resolve membrane protein structure using solution SANS.

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the investigation of biological materials, biofuel processing, and bio-inspired materials covering nanometer to micrometer length scales. The methods presented here for investigating physical properties (i.e., size and shape) of membrane proteins (here, MmIAP, an intramembrane aspartyl protease from Methanoculleus marisnigri) in solutions of micelle-forming detergents are well-suited for this small-angle neutron scattering instrument, among others. Other biophysical characterization techniques are hindered by their inability to address the detergent contributions in a protein-detergent complex structure. Additionally, access to the Bio-Deuteration Lab provides unique capabilities for preparing large-scale cultivations and expressing deuterium-labeled proteins for enhanced scattering signal from the protein. While this technique does not provide structural details at high-resolution, the structural knowledge gap for membrane proteins contains many addressable areas of research without requiring near-atomic resolution. For example, these areas include determination of oligomeric states, complex formation, conformational changes during perturbation, and folding/unfolding events. These investigations can be readily accomplished through applications of this method.

Detector upgrade drives new scientific capabilities at the Bio-SANS instrument

The Bio-SANS instrument is ideally suited for studies of biomacromolecules including proteins, DNA/RNA, lipid membranes and other hierarchical complexes. A wide-angle detector bank was installed on Bio-SANS in 2016. The combination of the main detector and new wing detector has extended the q range to ~1 Å, which is unprecedented for reactor-based SANS and expands capabilities beyond the small-angle regime. Length scales spanning ~0.7 300 nm (0.002 0.9 Å) or ~3 600 nm (0.001 – 0.2 Å) can now be obtained in a single measurement. This detector expansion nearly doubles the active detector area and increases the Bio-SANS dynamic scattering vector range 15-fold to a new dynamic range greater than 200. In addition, data collection times are decreased by up to a factor of ~2. New science opportunities include, in situ kinetic processes using time-resolved SANS, and studies of hierarchical and complex biological systems with simultaneous access to multiple length scales. The added detector bank builds on ORNL in-house technology that has been demonstrated to be successful on Bio-SANS since 2012 when its main detector was replaced. The main Bio-SANS detector has count rate capabilities (>10 Hz) that enable utilization of the full potential of the high neutron flux from the cold source. Several sample environment capabilities are available for studies of biological systems using neutrons. These include a pressure cell to monitor chemical reactions in situ such as biomass pretreatment studies, a multiposition sample holder with rotational (tumbling) capability especially useful for studying suspensions, a humidity-controlled chamber critical for membrane studies and a flow cell for systems that partition to multiple phases (e.g. microemulsions) with additional capability of flowing one or two phases during measurement. Furthermore, grazing-incidence SANS in conjunction with a humidity chamber is available for studies of biomembranes and substrate-supported biosensors. Remote data reduction for the Bio-SANS user community is now available from a centralized analysis cluster at the SNS. The BDL features a bioreactor system that supports working volumes from 700 ml to 7.5L in addition to a parallel bioreactor system (4 x 250mL). Both systems allow high-density cell growth with precise control and monitoring of dissolved oxygen, pH, agitation, and feeding rates. Other new laboratory capabilities include a Rigaku single-crystal diffractometer, a Rigaku BioSAXS small-angle x-ray scattering instrument, liquid handling robots for preparing crystallization screens, and a suite of incubators for temperature-controlled protein crystallization.

Contrast matching of detergent micelles for membrane protein studies with SANS

Detergents represent the most frequently employed tool to solubilize membrane proteins for structural and functional investigations in solution. However, an excess of empty (protein-free) detergent micelles are often present in addition to the desired protein-detergent complexes. For SANS experiments, which record the ensemble average of particles in solution, the signal from these empty micelles has a negative impact on the scattering of interest. Additionally, the core-shell architecture of micelles produces a structure factor which remains present at the total detergent match point. We present one approach for the rational design of mixed micelles containing a deuterated detergent analog, which produce a negligible contrast between core and shell, and can be fully contrast matched to better resolve membrane protein structure with SANS.