Beyond detergent micelles: The advantages and applications of non-micellar and lipid-based membrane mimetics for solution-state NMR

Klöpfer, Kai; Hagn, Franz

doi:10.1016/j.pnmrs.2019.08.001

Cited by 32 publications

(33 citation statements)

References 146 publications

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“…Due to the fact that TREM2-TMH harbors a charged lysine residue at position 186 (K186) that is located in the hydrophobic transmembrane region, we anticipate that this energetically unfavorable location is a key factor that determines its conformation and dynamics. In order to rule out the influence of the nonplanar detergent micelle surface, we inserted the TREM2-TMH into di-myristoyl-glycero-phosphocholine/di-myristoyl-glycero-phosphoglycerol (DMPC/DMPG, 3:1) phospholipid nanodiscs assembled with MSP1D1DH5 (Hagn et al, 2013(Hagn et al, , 2018Klöpfer & Hagn, 2019) ( Fig EV1C and D) and obtained high-quality 2D NMR spectra that enabled backbone resonance assignments ( Fig EV2A), as well as the acquisition of 3D-NOESY spectra ( Fig EV2C). Similar to the DPC micelle environment, TREM2-TMH shows the same chemical shiftderived secondary structure content ( Fig EV2B) and an identical NOE pattern in lipid nanodiscs, where no contacts can be observed within the amino acid stretch between residues 190 and 192 ( Fig EV2C).…”

Section: Resultsmentioning

confidence: 99%

γ‐Secretase cleavage of the Alzheimer risk factor TREM 2 is determined by its intrinsic structural dynamics

et al. 2020

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Sequence variants of the microglial expressed TREM2 (triggering receptor expressed on myeloid cells 2) are a major risk factor for late onset Alzheimer's disease. TREM2 requires a stable interaction with DAP12 in the membrane to initiate signaling, which is terminated by TREM2 ectodomain shedding and subsequent intramembrane cleavage by c-secretase. To understand the structural basis for the specificity of the intramembrane cleavage event, we determined the solution structure of the TREM2 transmembrane helix (TMH). Caused by the presence of a charged amino acid in the membrane region, the TREM2-TMH adopts a kinked structure with increased flexibility. Charge removal leads to TMH stabilization and reduced dynamics, similar to its structure in complex with DAP12. Strikingly, these dynamical features match with the site of the initial c-secretase cleavage event. These data suggest an unprecedented cleavage mechanism by c-secretase where flexible TMH regions act as key determinants of substrate cleavage specificity.

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Section: Resultsmentioning

confidence: 99%

γ‐Secretase cleavage of the Alzheimer risk factor TREM 2 is determined by its intrinsic structural dynamics

et al. 2020

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“…In addition, paramagnetic ions can be added to the lipid mixtures, so the resulting bicelles can align in an external magnetic field, aiding magnetic resonance studies on IMPs [155,156]. Notably, the presence of detergent-like short-chain lipids and a bilayer size is insufficient to provide membrane-like lateral pressure and may perturb the structure and dynamics of bicelle-residing IMPs [54,69,157]. Another disadvantage of conventional bicelles is that their size and geometry depend on the total lipid concentration in the solution; therefore, any dilution changes the system properties.…”

Section: Bicelles In Studies Of Integral Membrane Proteins 221 General Properties Of Bicellesmentioning

confidence: 99%

Lipid Membrane Mimetics in Functional and Structural Studies of Integral Membrane Proteins

et al. 2021

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Integral membrane proteins (IMPs) fulfill important physiological functions by providing cell–environment, cell–cell and virus–host communication; nutrients intake; export of toxic compounds out of cells; and more. However, some IMPs have obliterated functions due to polypeptide mutations, modifications in membrane properties and/or other environmental factors—resulting in damaged binding to ligands and the adoption of non-physiological conformations that prevent the protein from returning to its physiological state. Thus, elucidating IMPs’ mechanisms of function and malfunction at the molecular level is important for enhancing our understanding of cell and organism physiology. This understanding also helps pharmaceutical developments for restoring or inhibiting protein activity. To this end, in vitro studies provide invaluable information about IMPs’ structure and the relation between structural dynamics and function. Typically, these studies are conducted on transferred from native membranes to membrane-mimicking nano-platforms (membrane mimetics) purified IMPs. Here, we review the most widely used membrane mimetics in structural and functional studies of IMPs. These membrane mimetics are detergents, liposomes, bicelles, nanodiscs/Lipodisqs, amphipols, and lipidic cubic phases. We also discuss the protocols for IMPs reconstitution in membrane mimetics as well as the applicability of these membrane mimetic-IMP complexes in studies via a variety of biochemical, biophysical, and structural biology techniques.

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“…The bicelle concentration was calculated based on the number of DMPC molecules in one bicelle. The radius R of the bilayer region of the bicelle (for q = 0.2) was calculated to be 2.04 nm using the formula R = 1/2rq[π + (π 2 + 8/q) 1/2 ] assuming a bilayer thickness of 4 nm with a radius r = 2 nm (Klopfer and Hagn, 2019). Thus the calculated surface area of the bicelle is 1307 Å 2 , which corresponds to 46 DMPC molecules (given a surface area of 57 Å 2 per DMPC molecule).…”

Section: Bicelles and Nanodiscs Preparationmentioning

confidence: 99%

Membrane Interactions of the Peroxisomal Proteins PEX5 and PEX14

Gaussmann

Gopalswamy

Eberhardt

et al. 2021

Front. Cell Dev. Biol.

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Human PEX5 and PEX14 are essential components of the peroxisomal translocon, which mediates import of cargo enzymes into peroxisomes. PEX5 is a soluble receptor for cargo enzymes comprised of an N-terminal intrinsically disordered domain (NTD) and a C-terminal tetratricopeptide (TPR) domain, which recognizes peroxisomal targeting signal 1 (PTS1) peptide motif in cargo proteins. The PEX5 NTD harbors multiple WF peptide motifs (WxxxF/Y or related motifs) that are recognized by a small globular domain in the NTD of the membrane-associated protein PEX14. How the PEX5 or PEX14 NTDs bind to the peroxisomal membrane and how the interaction between the two proteins is modulated at the membrane is unknown. Here, we characterize the membrane interactions of the PEX5 NTD and PEX14 NTD in vitro by membrane mimicking bicelles and nanodiscs using NMR spectroscopy and isothermal titration calorimetry. The PEX14 NTD weakly interacts with membrane mimicking bicelles with a surface that partially overlaps with the WxxxF/Y binding site. The PEX5 NTD harbors multiple interaction sites with the membrane that involve a number of amphipathic α-helical regions, which include some of the WxxxF/Y-motifs. The partially formed α-helical conformation of these regions is stabilized in the presence of bicelles. Notably, ITC data show that the interaction between the PEX5 and PEX14 NTDs is largely unaffected by the presence of the membrane. The PEX5/PEX14 interaction exhibits similar free binding enthalpies, where reduced binding enthalpy in the presence of bicelles is compensated by a reduced entropy loss. This demonstrates that docking of PEX5 to PEX14 at the membrane does not reduce the overall binding affinity between the two proteins, providing insights into the initial phase of PEX5-PEX14 docking in the assembly of the peroxisome translocon.

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Beyond detergent micelles: The advantages and applications of non-micellar and lipid-based membrane mimetics for solution-state NMR

Cited by 32 publications

References 146 publications

γ‐Secretase cleavage of the Alzheimer risk factor TREM 2 is determined by its intrinsic structural dynamics

γ‐Secretase cleavage of the Alzheimer risk factor TREM 2 is determined by its intrinsic structural dynamics

Lipid Membrane Mimetics in Functional and Structural Studies of Integral Membrane Proteins

Membrane Interactions of the Peroxisomal Proteins PEX5 and PEX14

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