Structural Biochemistry/Membrane Proteins/Methods for Studying Membrane Proteins/Solution NMR Spectroscopy

Understanding how membrane proteins function has posed difficulty because they cannot be purified or removed from the membrane without losing vital information. To study them, they must be placed in an environment that mimics conditions they are typically found in and then analyzed through solution NMR spectroscopy. Solution NMR spectroscopy requires certain compounds and assemblies to stabilize membrane proteins as they are analyzed.

Using Detergents

Detergent in aqueous solution forms micelle with membrane protein. Red components signify detergent monomer. Gray area is the hydrophobic region of membrane protein. Orange area is hydrophilic region of membrane protein.

Typically, micelle-forming detergents are used to stabilize the proteins when they are in aqueous solution. Detergents are molecules that contain both hydrophobic and hydrophilic regions. Each detergent has a critical micellar concentration (cmc) where if detergent molecules are in concentrations below their cmc, they will exist as monomers; if they are above, they will exist as detergent micelles that are in equilibrium with detergent monomers.

However, detergents are not ideal for studying membrane proteins. Because they are highly dynamic, they can cause the protein to unfold and aggregate. Membrane proteins that contain sections which extend onto the aqueous space are particularly difficult to study with detergents because these regions unfold and become destabilized. The spherical shape of micelles also poses a problem as the membrane the proteins are found in are planar. Detergent molecules can also interfere with experimental conditions used to study membrane proteins and it takes quite a bit of time to find a compatible detergent for the protein to be studied.

Using detergents overall can be time consuming and problematic.

Lipopeptide Detergents (LPD's)

Detergents are essential tools used by scientists for the biochemical and structural study of membrane proteins. The structural study of membrane proteins requires detergents that can effectively mimic lipid bi-layers. Scientists are always struggling between what choice of detergent they want to use. Do scientists want detergents that promote protein stability or detergents that form small micelles?

Schematic representations of membrane protein/amphiphile associations. Green areas represent hydrophobic surfaces, and red areas are hydrophilic. (a) An integral membrane protein embedded in a phospholipid bilayer. (b) Possible mode of detergent binding in a traditional protein–detergent complex (PDC). (c) Representation of a membrane protein solubilized in an LPD. (d) End-on view of a protein/ LPD complex through a plane indicated by the dashed line in panel (c). The number of LPD molecules required to form a ring around the protein depends on the circumference of the protein transmembrane region.^[1]

Dodecyl-β-D-maltopyranoside (DDM) is generally considered as one of the best detergents for maintaining protein stability, and the use of this detergent has been influential in the crystallization of landmark proteins.^[1] Although DDM is great for maintaining protein stability, its large micelle size is one of the reasons why most membranes proteins crystallized from DDM diffract only low to moderate resolutions.

Lipopeptide Detergents (LPD’s) are a new class of amphiphile (a water loving and fat loving molecule) that consists of a peptide scaffold that supports two alkyl chains, one anchored to each end of an α-helix. ^[2] The LPD design is an attempt to create an amphiphile that can form small micelles, but create an acyl packing environment that is more comparable to the interior of a bi-layer that of a micelle. Eventually scientists were able to create a Lipopeptide detergent with this exact design. The LPDs were able to self-assemble into small micelles, and are gentle, nondenaturing detergents that preserve the structure of the membrane protein in solution for an extended period of time. ^[1] The target conformation for an LPD monomer is that of a wedge, with the inside edge consisting of the two hydrophobic acyl chains opposite from a wider surface consisting of the polar face of the helix (Shown in the 2nd picture) LPDs are highly effective at stabilizing proteins, because of the formation of favorable membrane-like cylindrical acyl packing interactions at the hydrophobic surface of the target protein. (shown in part c and d picture on the left). ^[1]

LPDs in NMR Normal detergents used in NMR are generally harsh and aggressive. Conformational exchange in PDCs(protein detergent complex) is a common problem. LPD are useful in NMR because the membrane-like environment achieved from PDC, while the mass stays at a minimum.

Practicality of LPDs Although LPDs are relatively expensive compared to other detergents, LPD is effective at very low molar mass ratios of 12-20 LPD monomers per protein, very little LPD is required to fully solubilize a target membrane protein. All the added LPD is used in the final sample for crystallization trials or NMR spectroscopy. This makes LPD highly efficient in what it does.^[1]

Amphipols

An alternative to detergents are amphipols, or amphiphatic polymers. Developed mainly by Jean-Luc Popot and colleagues, amphipols have “polymeric backbones that are covalently modified with stochastic distribution of hydrophobic and hydrophilic groups”. Amphipols are used as a detergent-free membrane substitute that conserve the membrane protein function.

Lipid Bilayer Systems

Another technique is using lipid bilayer systems. Lipid bilayer systems oftentimes preserve the integrity and structure of the membrane protein much better than amphipols and detergents. Three classes of membrane mimics fall under this category: liposomes, bicelles and nanolipoprotein particles (NLPs).

Bicelles

Protein membrane in bicelle assembly in aqueous solutoin. Red components are detergents. Blue components are lipids. Gray area is the hydrophobic area of the membrane protein. Orange area is the hydrophillic area of the membrane protein.

Bicelles are also used to study membrane proteins. Bicelles are binary, water-soluble assemblies of lipids and detergents. Ideally, the lipids will form the central part of the bicelle and the detergents will form the edge of the assembly. The assembly itself is a roughly circular patch of lipid bilayer in aqueous solution.

A way to study membrane proteins in their native environment uses nanolipoprotein particles (NLPs), or nano discs or reconstituted high density lipoprotein particles (rHDLs). NLPs are made of non-covalent assemblies of phospholipids arranged as a discoidal bilayer.

Nanolipoprotein Particles

A way to study membrane proteins in their native environment uses nanolipoprotein particles (NLPs), or nano discs or reconstituted high density lipoprotein particles (rHDLs). NLPs are made of "noncovalent assemblies of phospholipids arranged as a discoidal bilayer, surrounded by amphipathic apolipoproteins".

References

2. [1] http://www.ncbi.nlm.nih.gov/pubmed/19682888
3. [2] http://www.nature.com/nbt/journal/v21/n2/full/nbt776.html