Nanodisc Interactions with KCNE4: Heart Kv Channel Modulator

An image of the current KCEN4 protein model. One-third from the top of the image a blue field indicates where a cellular membrane would be. Several amino acids, represented by circles, extend above the field, through the field, and extend below the field. One amino acid in each region is colored orange. Two amino acids approximately two-thirds from the top of the image are colored grey.

KCNE4 (E4)

KCNE4 is a membrane protein most commonly found within the human heart and lymph notes. Not much is known about its structure, which is the primary objective of our lab's spectroscopy studies. My specific project investigates its interaction when the polymer SMALP 300, is used to incorporate E4 into nanodiscs.

The two native cystines (mutated to alanines) are indicated in grey. My selected mutation points (mutated to a cystine) are indicated in orange (one mutation site per mutant). The intermembrane region is indicated by the central blue field.

Abstract

KCNE4 (E4) is a protein and member of an accessory subunit family, which interacts with various voltage-gated potassium channels within the human body. When the processes of these channels malfunction, numerous diseases can develop, such as: long QT syndrome, acute lymphoblastic leukemia, and allergic rhinitis. E4 itself is known to inhibit potassium channels; however, the structure and dynamics of this protein before and after the interaction is largely unknown. To explore conformational changes in E4 mutants, different membrane mimetics will be applied to E4, and any structural or dynamic changes will be observed with continuous-wave electron paramagnetic resonance (CW-EPR) spectroscopy. Characterization of E4 within lipid vesicles and lipid nanoparticles will be done using dynamic light scattering (DLS). In addition, the exploration of E4 within SMALP 300 will be used to investigate structural, dynamic, and incorporation differences with various mutants along the proposed E4 structure. This Styrene-Maleic Acid (SMA) derivative is one of many which were designed to overcome some of the difficulties with traditional SMA, such as lack of versatility to pH and divalent metals. It has been shown through previous studies that SMA derivatives can produce similar structural and dynamic features to lipid vesicles. As a result of this procedure, more information about the effects of membrane characteristics on the structure of E4 is hoped to be gained, potentially shedding more light on the inhibition of potassium channels by E4 within a biological system.

Methods

Our laboratory uses MTSL spin labeling of our proteins in order to make them EPR reactive. They are attached to a specific site on our protein via sulfide bonds to our mutated cysteine residue, as shown to the right.

The molecular structure of an MTSL spin label is drawn next to a helical shape which has an SH group attached. These groups are then depicted as one MTSL molecule attached to the S atom off of the helical shape.

A graphical depiction of a membrane protein within a micelle, a vesicle, and a nanodisc membrane mimetic

In order to establish native-like conformation of E4, we utilize lipid vesicles as a membrane mimetic. Our lipid vesicles are made from 3:1 POPC:POPG.

Shown to the left are examples of different membrane mimetics.

Results

Three sites of interest were chosen: A16C (extracellular), G47C (intermembrane), and S151C (intracellular). These were selected in order to better gain a holistic sense for how E4 would interact when incorporated into the SMALP 300 nanodisc-forming polymer. DLS was used to confirm successful sample incorporation into nanodiscs prior to collecting CW-EPR spectra for each mutant.

Each spectra was collected with approximately 30 uM of sample. Black lines indicate the vesicle-only control sample while the green lines represent each of the triplicate nanodisc incorporated sample runs that were conducted. For the EPR spectra, the green line shown is the average of the data that was collected for each mutant, in triplicate.

A16C

G47C

S151C

Conclusions

From analysis of CW-EPR spectra, it is observed that incorporation into SMALP 300 nanodisc-forming polymer produced near-identical data to our vesicle-only samples, thus indicating that this is a viable option for a membrane mimetic. The line broadening present within the first peak of G47C indicates some potential dimerization of the protein, however ithe driving force for the dimerization of E4 is still being investigated so we are unsure as to why multiple species of my chosen E4 mutants may be present within the examined samples. It may also be likely that due to the location of the spin label, being within the inter-membrane region, the range of motion experienced by the spin label is further reduced by the polymer wrapping around the protein.

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