(449bk) Membrane Protein Insertion and Compatibility with Biomimetic Membranes

Ren, T., Penn State University
Shen, Y., University of California
Erbakan, M., Bozok University
Grzelakowski, M., Applied Biomimetic Inc.
Butler, P. J., Penn State University
Kumar, M., The Pennsylvania State University
Cell membrane matrices are mainly composed of lipids and membrane proteins. Membrane proteins, present in the membrane, performing a vast majority of functions including signal reception1, material transport and biochemical reactions2,3 that occur at membrane-liquid interfaces. High compatibility between cell membranes and membrane proteins are necessary for living organisms to conduct complicated tasks and thrive in the nature4. Inspired by living cell systems, biomimetic membrane-matrices are developed for mimicking cell membrane matrix functions. Biomimetic membrane matrices are composed of membrane proteins (or membrane protein-mimics) and lipids (or lipid-like block copolymers). Similar to nature cell membrane matrices, only compatible biomimetic membranes specific to different membrane proteins will provide desired membrane performance5. Highly compatible biomimetic membranes have high membrane protein (or membrane protein-mimic) reconstitution density without compromising membrane protein (or membrane protein-mimic) single molecule properties. However, there are few accurate methods for compatibility evaluation4.

Here, we propose and test methods to evaluate membrane protein and membrane protein-mimic compatibility with biomimetic membranes. We conducted biophysical activity characterization using stopped-flow spectroscopy, membrane protein density determination using Fluorescence Correlation Spectroscopy (FCS), and evaluation of the effect of matrix hydrophobicity on protein insertion using a new distyrylbenzene chromophore optical characterization technique. Combined the methods utilized provided single protein activity, and for a first time a quantitative measure of the chemical and physical compatibility between proteins and membranes. Water transport protein (aquaporins, AQPs), rhodopsins, and artificial water channels (specifically peptide-appended pillar[n]arene (PAP) channels) were reconstituted into a range of biomimetic membrane matrices to evaluate the proposed platform. Compatibility measurement results showed that both AQPs and PAP8 channels preserve their single molecule water transport rates in different biomimetic membranes while their reconstitution density changes leading to different membrane permeability. We also show that membrane protein reconstitution density correlates with hydrophobicity mismatch between membrane protein and biomimetic membranes: smaller hydrophobicity mismatch between the membrane matrix and the proteins lead to higher membrane protein (or membrane protein-mimic) reconstitution densities.


1. Broselid, S. et al. G protein-coupled Receptor 30 (GPR30) Forms a Plasma Membrane Complex with Membrane-associated Guanylate Kinases (MAGUKs) and Protein Kinase A-anchoring Protein 5 (AKAP5) That Constitutively Inhibits cAMP Production. Journal of Biological Chemistry 289, 22117-22127,(2014).

2.Parker, M. D., Myers, E. J. & Schelling, J. R. Na+-H+ exchanger-1 (NHE1) regulation in kidney proximal tubule. Cell. Mol. Life Sci. 72, 2061-2074,(2015).

3.Krall, J. F. & Korenman, S. G. Soluble Precursor of Membrane-Associated Protein-Kinase in Uterine Smooth-Muscle Cells. Arch. Biochem. Biophys. 199, 479-487,(1980).

4.Shen, Y.-x., Saboe, P. O., Sines, I. T., Erbakan, M. & Kumar, M. Biomimetic membranes: A review. Journal of Membrane Science 454, 359-381,(2014).

5.Nichols, S. P., Koh, A., Storm, W. L., Shin, J. H. & Schoenfisch, M. H. Biocompatible Materials for Continuous Glucose Monitoring Devices. Chem. Rev. 113, 2528-2549,(2013).

6.Erbakan, M., Shen, Y.-x., Butler, P. J., Kumar, M. & Curtis, W. R. Molecular cloning, overexpression and characterization of a novel water channel protein from Rhodobacter sphaeroides. PLoS ONE 9, e86830,(2014).

7.Garner, L. P., J ; Dyar, SM ; Chworos, A ; Sumner, JJ ; Bazan, GC Modification of the Optoelectronic Properties of Membranes via Insertion of Amphiphilic Phenylenevinylene Oligoelectrolytes. J. Am. Chem. Soc. 132, 10042-10052,(2010).

8.Shen, Y. X. et al. Highly permeable artificial water channels that can self-assemble into two-dimensional arrays. Proceedings of the National Academy of Sciences of the United States of America 112, 9810-9815,(2015).