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1.
J Am Chem Soc ; 2024 Apr 11.
Article in English | MEDLINE | ID: mdl-38604609

ABSTRACT

Lipopolysaccharide (LPS) is vital for maintaining the outer membrane barrier in Gram-negative bacteria. LPS is also frequently obtained in complex with the inner membrane proteins after detergent purification. The question of whether or not LPS binding to inner membrane proteins not involved in outer membrane biogenesis reflects native lipid environments remains unclear. Here, we leverage the control of the hydrophilic-lipophilic balance and packing parameter concepts to chemically tune detergents that can be used to qualitatively differentiate the degree to which proteins copurify with phospholipids (PLs) and/or LPS. Given the scalable properties of these detergents, we demonstrate a detergent fine-tuning that enables the facile investigation of intact proteins and their complexes with lipids by native mass spectrometry (nMS). We conclude that LPS, a lipid that is believed to be important for outer membranes, can also affect the activity of membrane proteins that are currently not assigned to be involved in outer membrane biogenesis. Our results deliver a scalable detergent chemistry for a streamlined biophysical characterization of protein-lipid interactions, provide a rationale for the high affinity of LPS-protein binding, and identify noncanonical associations between LPS and inner membrane proteins with relevance for membrane biology and antibiotic research.

2.
RSC Adv ; 12(16): 9671-9680, 2022 Mar 25.
Article in English | MEDLINE | ID: mdl-35424940

ABSTRACT

Electrospray ionization mass spectrometry is increasingly applied to study the structures and interactions of membrane protein complexes. However, the charging mechanism is complicated by the presence of detergent micelles during ionization. Here, we show that the final charge of membrane proteins can be predicted by their molecular weight when released from the non-charge reducing saccharide detergents. Our data indicate that PEG detergents lower the charge depending on the number of detergent molecules in the surrounding micelle, whereas fos-choline detergents may additionally participate in ion-ion reactions after desolvation. The supercharging reagent sulfolane, on the other hand, has no discernible effect on the charge of detergent-free membrane proteins. Taking our observations into the context of protein-detergent interactions in the gas phase, we propose a charge equilibration model for the generation of native-like membrane protein ions. During ionization of the protein-detergent complex, the ESI charges are distributed between detergent and protein according to proton affinity of the detergent, number of detergent molecules, and surface area of the protein. Charge equilibration influenced by detergents determines the final charge state of membrane proteins. This process likely contributes to maintaining a native-like fold after detergent release and can be harnessed to stabilize particularly labile membrane protein complexes in the gas phase.

3.
PNAS Nexus ; 1(4): pgac153, 2022 Sep.
Article in English | MEDLINE | ID: mdl-36714824

ABSTRACT

Despite tremendous advances in sample preparation and classification algorithms for electron cryomicroscopy (cryo-EM) and single-particle analysis (SPA), sample heterogeneity remains a major challenge and can prevent access to high-resolution structures. In addition, optimization of preparation conditions for a given sample can be time-consuming. In the current work, it is demonstrated that native electrospray ion-beam deposition (native ES-IBD) is an alternative, reliable approach for the preparation of extremely high-purity samples, based on mass selection in vacuum. Folded protein ions are generated by native electrospray ionization, separated from other proteins, contaminants, aggregates, and fragments, gently deposited on cryo-EM grids, frozen in liquid nitrogen, and subsequently imaged by cryo-EM. We demonstrate homogeneous coverage of ice-free cryo-EM grids with mass-selected protein complexes. SPA reveals that the complexes remain folded and assembled, but variations in secondary and tertiary structures are currently limiting information in 2D classes and 3D EM density maps. We identify and discuss challenges that need to be addressed to obtain a resolution comparable to that of the established cryo-EM workflow. Our results show the potential of native ES-IBD to increase the scope and throughput of cryo-EM for protein structure determination and provide an essential link between gas-phase and solution-phase protein structures.

4.
Curr Opin Struct Biol ; 69: 78-85, 2021 08.
Article in English | MEDLINE | ID: mdl-33930613

ABSTRACT

Structure determination of membrane proteins has highlighted the many roles played by lipids in influencing overall protein architecture. It is now widely accepted that lipids surrounding membrane proteins play crucial roles by modulating their conformational, structural, and functional properties. Capturing often transient lipid interactions and defining their chemical identity, however, remains challenging. Recent advances in mass spectrometry have resolved questions concerning lipid interactions by providing the molecular composition of intact complexes in association with lipids. Together with other biophysical tools, a picture is emerging of the dynamic nature of lipid-mediated interactions and their effects on conformation, interactions, and signaling.


Subject(s)
Membrane Lipids , Membrane Proteins , Cell Membrane , Mass Spectrometry
5.
J Am Chem Soc ; 143(11): 4085-4089, 2021 03 24.
Article in English | MEDLINE | ID: mdl-33711230

ABSTRACT

The use of mass spectrometry to investigate proteins is now well established and provides invaluable information for both soluble and membrane protein assemblies. Maintaining transient noncovalent interactions under physiological conditions, however, remains challenging. Here, using nanoscale electrospray ionization emitters, we establish conditions that enable mass spectrometry of two G protein-coupled receptors (GPCR) from buffers containing high concentrations of sodium ions. For the Class A GPCR, the adenosine 2A receptor, we observe ligand-induced changes to sodium binding of the receptor at the level of individual sodium ions. We find that antagonists promote sodium binding while agonists attenuate sodium binding. These findings are in line with high-resolution X-ray crystallography wherein only inactive conformations retain sodium ions in allosteric binding pockets. For the glucagon receptor (a Class B GPCR) we observed enhanced ligand binding in electrospray buffers containing high concentrations of sodium, as opposed to ammonium acetate buffers. A combination of native and -omics mass spectrometry revealed the presence of a lipophilic negative allosteric modulator. These experiments highlight the advantages of implementing native mass spectrometry, from electrospray buffers containing high concentrations of physiologically relevant salts, to inform on allosteric ions or ligands with the potential to define their roles on GPCR function.


Subject(s)
Receptors, G-Protein-Coupled/chemistry , Sodium/chemistry , Humans , Ions/chemistry , Ligands , Mass Spectrometry , Models, Molecular
6.
Nat Methods ; 17(5): 505-508, 2020 05.
Article in English | MEDLINE | ID: mdl-32371966

ABSTRACT

Ligands bound to protein assemblies provide critical information for function, yet are often difficult to capture and define. Here we develop a top-down method, 'nativeomics', unifying 'omics' (lipidomics, proteomics, metabolomics) analysis with native mass spectrometry to identify ligands bound to membrane protein assemblies. By maintaining the link between proteins and ligands, we define the lipidome/metabolome in contact with membrane porins and a mitochondrial translocator to discover potential regulators of protein function.


Subject(s)
Lipids/analysis , Mass Spectrometry/methods , Membrane Proteins/metabolism , Metabolome , Proteome/analysis , Humans , Ligands
7.
Methods Mol Biol ; 2073: 287-299, 2020.
Article in English | MEDLINE | ID: mdl-31612447

ABSTRACT

In native mass spectrometry, non-covalent interactions are preserved in solution and through transfer to the gas phase. This technique can be used to characterize the composition, stoichiometry, and architecture of protein nano-assemblies, such as those observed in vivo or constructed through protein engineering in nanotechnology and synthetic biology. Here we describe an implementation of native mass spectrometry for studying protein-based nanostructures, including membrane proteins. Unambiguous structural details of assemblies can be rapidly determined due to the high resolution and mass accuracy afforded by mass spectrometry measurements including protein nano-assembly stoichiometry, heterogeneity, and ligand binding characteristics.


Subject(s)
Mass Spectrometry/methods , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Membrane Proteins/ultrastructure , Nanotechnology/methods , Protein Conformation , Protein Multimerization , Synthetic Biology/methods
8.
ACS Appl Mater Interfaces ; 11(37): 34463-34470, 2019 Sep 18.
Article in English | MEDLINE | ID: mdl-31496217

ABSTRACT

Microfluidic instrumentation offers unique advantages in biotechnology applications including reduced sample and reagent consumption, rapid mixing and reaction times, and a high degree of process automation. As dimensions decrease, the ratio of surface area to volume within a fluidic architecture increases, which gives rise to some of the unique advantages inherent to microfluidics. Thus, manipulation of surface characteristics presents a promising approach to tailor the performance of microfluidic systems. Microfluidic valves are essential components in a number of small volume applications and for automated microfluidic platforms, but rigorous evaluation of the sealing quality of these valves is often overlooked. In this work, the glass valve seat of hybrid glass/PDMS microfluidic valves was surface modified with hydrophobic silanes, octyldimethylchlorosilane (ODCS) or (tridecafluoro-1,1,2,2-tetrahydrooctyl)dimethylchlorosilane (PFDCS), to investigate the effect of surface energy on electrical resistance of valves. Valves with ODCS- or PFDCS-modified valve seats both exhibited >70-fold increases in electrical resistance (>500 GΩ) when compared to the same valve design with unmodified glass valve seats (7 ± 3 GΩ), indicative of higher sealing capacity. The opening times for valves with ODCS- or PFDCS-modified valve seats was ca. 5× shorter compared to unmodified valve seats, whereas the closing time was up to 8× longer for modified valve seats, although the total closing time was ≤1.5 s, compatible with numerous microfluidic valving applications. Surface modified valve assemblies offered sufficient electrical resistance to isolate sub-pA current signals resulting from electrophysiology measurement of α-hemolysin conductance in a suspended lipid bilayer. This approach is well-suited for the design of novel microfluidic architectures that integrate fluidic manipulations with electrophysiological or electrochemical measurements.

9.
Annu Rev Biochem ; 88: 85-111, 2019 06 20.
Article in English | MEDLINE | ID: mdl-30901263

ABSTRACT

Membrane proteins that exist in lipid bilayers are not isolated molecular entities. The lipid molecules that surround them play crucial roles in maintaining their full structural and functional integrity. Research directed at investigating these critical lipid-protein interactions is developing rapidly. Advancements in both instrumentation and software, as well as in key biophysical and biochemical techniques, are accelerating the field. In this review, we provide a brief outline of structural techniques used to probe protein-lipid interactions and focus on the molecular aspects of these interactions obtained from native mass spectrometry (native MS). We highlight examples in which lipids have been shown to modulate membrane protein structure and show how native MS has emerged as a complementary technique to X-ray crystallography and cryo-electron microscopy. We conclude with a short perspective on future developments that aim to better understand protein-lipid interactions in the native environment.


Subject(s)
Glycerophospholipids/metabolism , Glycolipids/metabolism , Mass Spectrometry/methods , Membrane Proteins/metabolism , Sphingolipids/metabolism , Sterols/metabolism , Bacteria/chemistry , Bacteria/metabolism , Binding Sites , Cell Membrane/chemistry , Cell Membrane/metabolism , Cryoelectron Microscopy/instrumentation , Cryoelectron Microscopy/methods , Fungi/chemistry , Fungi/metabolism , Glycerophospholipids/chemistry , Glycolipids/chemistry , Magnetic Resonance Spectroscopy/instrumentation , Magnetic Resonance Spectroscopy/methods , Mass Spectrometry/instrumentation , Membrane Proteins/chemistry , Membrane Proteins/ultrastructure , Models, Molecular , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Sphingolipids/chemistry , Sterols/chemistry
10.
Protein Expr Purif ; 146: 61-68, 2018 06.
Article in English | MEDLINE | ID: mdl-29409958

ABSTRACT

The inwardly rectifying K+ (Kir) channel, Kir6.2, plays critical roles in physiological processes in the brain, heart, and pancreas. Although Kir6.2 has been extensively studied in numerous expression systems, a comprehensive description of an expression and purification protocol has not been reported. We expressed and characterized a recombinant Kir6.2, with an N-terminal decahistidine tag, enhanced green fluorescent protein (eGFP) and deletion of C-terminal 26 amino acids, in succession, denoted eGFP-Kir6.2Δ26. eGFP-Kir6.2Δ26 was expressed in HEK293 cells and a purification protocol developed. Electrophysiological characterization showed that eGFP-Kir6.2Δ26 retains native single channel conductance (64 ±â€¯3.3 pS), mean open times (τ1 = 0.72 ms, τ2 = 15.3 ms) and ATP affinity (IC50 = 115 ±â€¯25 µM) when expressed in HEK293 cells. Detergent screening using size exclusion chromatography (SEC) identified Fos-choline-14 (FC-14) as the most suitable surfactant for protein solubilization, as evidenced by maintenance of the native tetrameric structure in SDS-PAGE and western blot analysis. A two-step scheme using Co2+-metal affinity chromatography and SEC was implemented for purification. Purified protein activity was assessed by reconstituting eGFP-Kir6.2Δ26 in black lipid membranes (BLMs) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), l-α-phosphatidylinositol-4,5-bisphosphate (PIP2) in a 89.5:10:0.5 mol ratio. Reconstituted eGFP-Kir6.2Δ26 displayed similar single channel conductance (61.8 ±â€¯0.54 pS) compared to eGFP-Kir6.2Δ26 expressed in HEK293 membranes; however, channel mean open times increased (τ1 = 7.9 ms, τ2 = 61.9 ms) and ATP inhibition was significantly reduced for eGFP-Kir6.2Δ26 reconstituted into BLMs (IC50 = 3.14 ±â€¯0.4 mM). Overall, this protocol should be foundational for the production of purified Kir6.2 for future structural and biochemical studies.


Subject(s)
Potassium Channels, Inwardly Rectifying/genetics , Potassium Channels, Inwardly Rectifying/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Chromatography, Affinity , Chromatography, Gel , Gene Expression , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/isolation & purification , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Lipid Bilayers/metabolism , Patch-Clamp Techniques , Potassium Channels, Inwardly Rectifying/analysis , Potassium Channels, Inwardly Rectifying/isolation & purification , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Sequence Deletion , Solubility , Transfection/methods
11.
Anal Chem ; 89(2): 1315-1322, 2017 01 17.
Article in English | MEDLINE | ID: mdl-27981836

ABSTRACT

The binding of a target analyte to an ion channel (IC), which is readily detected electrochemically in a label-free manner with single-molecule selectivity and specificity, has generated widespread interest in using natural and engineered ICs as transducers in biosensing platforms. To date, the majority of developments in IC-functionalized sensing have focused on IC selectivity or sensitivity or development of suitable membrane environments and aperture geometries. Comparatively little work has addressed analytical performance criteria, particularly criteria required for temporal measurements of dynamic processes. We report a measurement protocol suitable for rapid, time-resolved monitoring (≤30 ms) of IC-modulated membrane conductance. Key features of this protocol include the reduction of membrane area and the use of small voltage steps (10 mV) and short duration voltage pulses (10 ms), which have the net effect of reducing the capacitive charging and decreasing the time required to achieve steady state currents. Application of a conductance protocol employing three sequential, 10 ms voltage steps (-10 mV, -20 mV, -30 mV) in an alternating, pyramid-like arrangement enabled sampling of membrane conductance every 30 ms. Using this protocol, dynamic IC measurements on black lipid membranes (BLMs) functionalized with gramicidin A were conducted using a fast perfusion system. BLM conductance decreased by 76 ± 7.5% within 30 ms of switching from solutions containing 0 to 1 M Ca2+, which demonstrates the feasibility of using this approach to monitor rapid, dynamic chemical processes. Rapid conductance measurements will be broadly applicable to IC-based sensors that undergo analyte-specific gating.


Subject(s)
Biosensing Techniques/instrumentation , Electric Conductivity , Gramicidin/chemistry , Immobilized Proteins/chemistry , Membrane Lipids/chemistry , Equipment Design , Membranes, Artificial , Transducers
12.
ACS Appl Mater Interfaces ; 5(22): 11918-26, 2013 Nov 27.
Article in English | MEDLINE | ID: mdl-24187929

ABSTRACT

The development of next-generation transmembrane protein-based biosensors relies heavily on the use of black lipid membranes (BLMs); however, electrical, mechanical, and temporal instability of BLMs poses a limiting challenge to biosensor development. In this work, micrometer-sized glass apertures were modified with silanes of different chain length and fluorine composition, including 3-cyanopropyldimethychlorosilane (CPDCS), ethyldimethylchlorosilane (EDCS), n-octyldimethylchlorosilane (ODCS), (tridecafluoro-1, 1, 2, 2-tetrahydrooctyl)dimethylchlorosilane (PFDCS), or (heptadecafluoro-1,1,2,2-tetrahydrodecyl)dimethylchlorosilane (PFDDCS), to explore the effect of substrate surface energy on BLM stability. Low energy silane-modified surfaces promoted enhanced lipid-substrate interactions that facilitate the formation of low-leakage, stable BLMs. The surface energies of silane-modified substrates were 30 ± 3, 16 ± 1, 14 ± 2, 11 ± 1, and 7.1 ± 2 mJ m(-2) for CDCS, EDCS, ODCS, PFDCS, and PFDDCS, respectively. Decreased surface energy directly correlated to improved electrical, mechanical, and temporal BLM stability. Amphiphobic perfluorinated surface modifiers yielded superior performance compared to traditional hydrocarbon modifiers in terms of stability and BLM formation, with only marginal effects on BLM membrane permeability. Leakage currents obtained for PFDCS and PFDDCS BLMs were elevated only 10-30%, though PFDDCS modification yielded >5-fold increase in electrical stability as indicated by breakdown voltage (> 2000 mV vs 418 ± 73 mV), and >25-fold increase in mechanical stability as indicated by air-water transfers (> 50 vs 2 ± 0.2) when compared to previously reported CPDCS modification. Importantly, the dramatically improved membrane stabilities were achieved with no deleterious effects on reconstituted ion channel function, as evidenced by α-hemolysin activity. Thus, this approach provides a simple, low cost, and broadly applicable alternative for BLM stabilization and should contribute significantly toward the development of next-generation ion-channel-functionalized biosensors.


Subject(s)
Hemolysin Proteins/chemistry , Lipids/chemistry , Membranes, Artificial , Surface Tension
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