Neurofibromatosis type 1 (NF1), a genetic disorder linked to inactivating mutations or a homozygous deletion of the Nf1 gene, is characterized by tumorigenesis, cognitive dysfunction, seizures, migraine, and pain. Omic studies on human NF1 tissues identified an increase in the expression of collapsin response mediator protein 2 (CRMP2), a cytosolic protein reported to regulate the trafficking and activity of presynaptic N-type voltage-gated calcium (Cav2.2) channels. Because neurofibromin, the protein product of the Nf1 gene, binds to and inhibits CRMP2, the neurofibromin-CRMP2 signaling cascade will likely affect Ca channel activity and regulate nociceptive neurotransmission and in vivo responses to noxious stimulation. Here, we investigated the function of neurofibromin-CRMP2 interaction on Cav2.2. Mapping of >275 peptides between neurofibromin and CRMP2 identified a 15-amino acid CRMP2-derived peptide that, when fused to the tat transduction domain of HIV-1, inhibited Ca influx in dorsal root ganglion neurons. This peptide mimics the negative regulation of CRMP2 activity by neurofibromin. Neurons treated with tat-CRMP2/neurofibromin regulating peptide 1 (t-CNRP1) exhibited a decreased Cav2.2 membrane localization, and uncoupling of neurofibromin-CRMP2 and CRMP2-Cav2.2 interactions. Proteomic analysis of a nanodisc-solubilized membrane protein library identified syntaxin 1A as a novel CRMP2-binding protein whose interaction with CRMP2 was strengthened in neurofibromin-depleted cells and reduced by t-CNRP1. Stimulus-evoked release of calcitonin gene-related peptide from lumbar spinal cord slices was inhibited by t-CNRP1. Intrathecal administration of t-CNRP1 was antinociceptive in experimental models of inflammatory, postsurgical, and neuropathic pain. Our results demonstrate the utility of t-CNRP1 to inhibit CRMP2 protein-protein interactions for the potential treatment of pain.
Membrane proteins play critical physiological roles and make up the majority of drug targets. Due to their generally low expression levels and amphipathic nature, membrane proteins represent challenging molecular entities for biophysical study. Mass spectrometry offers several sensitive approaches to study the biophysics of membrane proteins. By preserving noncovalent interactions in the gas phase and using collisional activation to remove solubilization agents inside the mass spectrometer, native mass spectrometry (MS) is capable of studying isolated assemblies that would be insoluble in aqueous solution, such as membrane protein oligomers and protein-lipid complexes. Conventional methods use detergent to solubilize the protein prior to electrospray ionization. Gas-phase activation inside the mass spectrometer removes the detergent to yield the isolated proteins with bound ligands. This approach has proven highly successful for ionizing membrane proteins. With the appropriate choice of detergents, membrane proteins with bound lipid species can be observed, which allows characterization of protein-lipid interactions. However, detergents have several limitations. They do not necessarily replicate the native lipid bilayer environment, and only a small number of protein-lipid interactions can be resolved. In this Account, we summarize the development of different membrane mimetics as cassettes for MS analysis of membrane proteins. Examples include amphipols, bicelles, and picodiscs with a special emphasis on lipoprotein nanodiscs. Polydispersity and heterogeneity of the membrane mimetic cassette is a critical issue for study by MS. Ever more complex data sets consisting of overlapping protein charge states and multiple lipid-bound entities have required development of new computational, theoretical, and experimental approaches to interpret both mass and ion mobility spectra. We will present the rationale and limitations of these approaches. Starting with the early work on empty nanodiscs, we chart developments that culminate in recent high-resolution studies of membrane protein-lipid complexes ejected from nanodiscs. For the latter, increasing collision energies allowed progressive removal of nanodisc components, beginning with the scaffold proteins and continuing through successive shells of lipids, allowing direct characterization of the stoichiometry of the annular lipid belt that surrounds the membrane protein. We consider future directions for the study of membrane proteins in membrane mimetics, including the development of mixed lipid systems and native bilayer environments. Unambiguous assignment of these heterogeneous systems will rely heavily upon further enhancements in both data analysis protocols and instrumental resolution. We anticipate that these developments will provide new insights into the factors that control dynamic protein-lipid interactions in a variety of tailored and natural lipid environments.
Conventional methods to probe the binding kinetics of macromolecules at biosensor surfaces employ a stepwise titration of analyte concentrations and measure the association and dissociation to the immobilized ligand at each concentration level. It has previously been shown that kinetic rates can be measured in a single step by monitoring binding as the analyte concentration increases over time in a linear gradient. We report here the application of nonlinear analyte concentration gradients for determining kinetic rates and equilibrium binding affinities in a single experiment. A versatile nonlinear gradient maker is presented, which is easily applied to microfluidic systems. Simulations validate that accurate kinetic rates can be extracted for a wide range of association and dissociation rates, gradient slopes, and curvatures, and with models for mass transport. The nonlinear analyte gradient method is demonstrated with a silicon photonic microring resonator platform to measure prostate specific antigen-antibody binding kinetics.
Nanodiscs have become a leading technology to solubilize membrane proteins for biophysical, enzymatic, and structural investigations. Nanodiscs are nanoscale, discoidal lipid bilayers surrounded by an amphipathic membrane scaffold protein (MSP) belt. A variety of analytical tools has been applied to membrane proteins in nanodiscs, including several recent mass spectrometry studies. Mass spectrometry of full-length proteins is an important technique for analyzing protein modifications, for structural studies, and for identification of proteins present in binding assays. However, traditional matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry methods for analyzing full-length membrane proteins solubilized in nanodiscs are limited by strong signal from the MSP belt and weak signal from the membrane protein inside the nanodisc. Herein, we show that an optimized ultra-thin layer MALDI sample preparation technique dramatically enhances the membrane protein signal and nearly completely eliminates the MSP signal. First-shot MALDI and MALDI imaging are used to characterize the spots formed by the ultra-thin layer method. Furthermore, the membrane protein enhancement and MSP suppression are shown to be independent of the type of membrane protein and are applicable to mixtures of membrane proteins in nanodiscs.
Nanodiscs are a promising system for studying gas-phase and solution complexes of membrane proteins and lipids. We previously demonstrated that native electrospray ionization allows mass spectral analysis of intact Nanodisc complexes at single lipid resolution. This report details an improved theoretical framework for interpreting and deconvoluting native mass spectra of Nanodisc lipoprotein complexes. In addition to the intrinsic lipid count and charge distributions, Nanodisc mass spectra are significantly shaped by constructive overlap of adjacent charge states at integer multiples of the lipid mass. We describe the mathematical basis for this effect and develop a probability-based algorithm to deconvolute the underlying mass and charge distributions. The probability-based deconvolution algorithm is applied to a series of dimyristoylphosphatidylcholine Nanodisc native mass spectra and used to provide a quantitative picture of the lipid loss in gas-phase fragmentation.