NMR Spectroscopy of Large Proteins:
The performance of reverse micelles can be maximized by encapsulation in liquid ethane such that the full complement of triple resonance NMR spectroscopy can be applied to large proteins. This can be done without using TROSY or sacrificing data by deuteration. Shown below are 15N-HSQC spectra of eGFP a 54 kDa homodimer, and of DsRed2 a 104 kDa homotetramer. Both spectra were collected on a 600 MHz spectrometer equipped with a cryo-probe.
Integral Membrane Proteins:
In principle, solubilization of an integral membrane protein in a reverse micelle system could avoid these two major barriers. On the one hand, the anticipated particle is expected to tumble much more quickly than the corresponding micelle in water. On the other hand, back exchange will no longer be an issue because deuteration will not be required. In addition, the TROSY effect will not be needed and the full power of the triple resonance spectroscopy will be applicable. We envisage the reverse micelle particle containing an integral membrane protein to be quite different than that of an encapsulated soluble protein. The distinct advantage of the reverse micelle system, in contrast to simple dissolution in organic solvents like chloroform, is that the system provides both hydrophobic (alkane solvent, surfactant tail; tightly bound lipid) and polar (surfactant head groups, water) molecules to support the structure of the protein. Shown below is a schematic representation of the postulated organization of a reverse micelle particle solubilizing an integral membrane protein.
Forced Folding of Marginally Stable Proteins:
It is becoming increasingly clear that a significant fraction of the proteins encoded by the human and other genomes are likely to be significantly unfolded in vitro. This has hampered attempts to characterize their structure by classical crystallographic or solution NMR methods. There are several possible reasons for this relative instability of the folded state. On the one hand, some proteins are “intrinsically unfolded” in the absence of target ligands, i.e. their biologically relevant structure need not be the lowest free energy state of the protein in the absence of ligand. On the other hand, some proteins must simply be considered unstable in the absence of stabilizing excluded volume effects arising from the dense packing conditions of the cellular milieu. In that case, the folded native state may indeed be the lowest energy structure in concentrated in vivo-like solution but it is not sufficiently removed in free energy from non-native structures to be the dominant species in dilute solution.
Encapsulation in a reverse micelle provides a restricted environment of defined geomety to force the folding of a marginally stable protein that is largely unfolded in free solution. The physical principle for forced folding is schematically illustrated below. Here we are using the preference (free energy) for a specific size of the reverse micelle for a given water and surfactants to confine the ensemble of states explored by a protein to a restricted volume.
Encapsulation in a reverse micelle can also isolate proteins that tend to aggregate thus increasing the longevity of a sample. This makes possible the application of triple resonance NMR spectroscopy for structural studies.
