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Lipid Modulation of Membrane Proteins
Membrane proteins are vital for almost all cellular functions. The main aim of the research is to understand how these proteins are modulated by lipids in the membrane. This is accomplished on one hand by using a bottom-up approach involving simple, artificial model systems of designed transmembrane peptides in synthetic lipid bilayers. In a new and complementary line of research, the group explores the use of amphipathic polymers of styrene and maleic acid, which have the ability to solubilize membrane proteins directly from their native membrane in the form of nanodiscs. This allows the proteins to be captured, purified and studied together with their native lipids. A more applied research line concerns the interaction of membranes with amyloid forming proteins, which have been associated with a wide range of diseases. The focus here lies on the mode of action of inhibitors of amyloid formation and how their effect is modulated by membranes.
Designed peptides as models for membrane proteins
Here we use artificial model systems of designed transmembrane peptides in synthetic lipid bilayers as mimics for real biological membranes. The most important advantages of using these systems are that they are relatively easy to study, that they allow systematic variation of many different parameters (e.g. peptide hydrophobic length, peptide hydrophobicity, bilayer thickness) and they can easily be modified with any desired label to allow biophysical studies. Thus, these systems are ideal to uncover general principles of protein-lipid in¬teractions in membranes. In particular we are focussing on effects of hydrophobic mismatch, i.e. the situation that the membrane spanning domains are shorter or longer than the hydrophobic thickness of the bilayer. Peptides as well as lipids can react to this in several ways. Understanding these responses allows establishment of basic rules that govern membrane structure and organization.The systems are studied by advanced biophysical techniques, including solid state NMR, fluorescence, calorimetry, mass spectrometry, circular dichroism and AFM.
WALP peptide that is used as mimic for a transmembrane alpha-helix
In a complementary line of research we have used synthetic transmembrane peptides to investigate the mode of action of the thermosensor DesK. This is a membrane-embedded sensor, which is involved in regulation of membrane fluidity in Bacillus subtillis. The discovery of a minimal sensor that consist of only one membrane spanning segment and retains its activity in a pure lipid bilayer [Cybulski LE, Martín M, Mansilla MC, Fernández A, de Mendoza D. (2010) Curr Biol. 20, 1539-1544.1] made it possible to simplify the system to a synthetic transmembrane helix in a membrane and thus allowed detailed investigation of the mode of action by biophysical and molecular biological techniques.
Model of the mode of action of DesK
1. Cybulski LE, Ballering J, Moussatova A, Inda ME, Vazquez DB, Wassenaar TA, de Mendoza D, Tieleman DP, Killian JA Activation of the bacterial thermosensor DesK involves a serine zipper dimerization motif that is modulated by bilayer thickness. Proc Natl Acad Sci U S A. 2015 ; 112(20):6353-8
2. Doux JPF, Hall BA, Killian JA How lipid head groups sense the membrane environment: an application of 14N NMR , Biophys. J. 2012 ; 103(6):1245-1253.
3. Nyholm TKM, Van Duyl BY, Rijkers DTS, Liskamp RMJ, Killian JA Probing the lipid-protein interface using model transmembrane peptides with a covalently linked acyl chain. Biophys J. 2011 ; 101(8):1959-1967.
4. Schäfer LV, de Jong DH, Holt A, Rzepiela AJ, de Vries AH, Poolman B, Killian JA, Marrink SJ Lipid packing drives the segregation of transmembrane helices into disordered lipid domains in model membranes. Proc Natl Acad Sci USA 2011 ; 108(4):1343-1348.
5. Holt A, Rougier L, Réat V, Jolibois F, Saurel O, Czaplicki J, Killian JA, Milon A Order parameters of a transmembrane helix in a fluid bilayer: case study of a WALP peptide. Biophys J. 2010 ; 98(9):1864-1872.
6. Holt A, Koehorst RBM, Rutters-Meijneke T, Gelb MH, Rijkers DTS, Hemminga MA and Killian JA Tilt and rotation angles of a transmembrane model peptide as studied by fluorescence spectroscopy Biophys J. 2009 ; 97(8):2258-2266
7. Özdirekcan S, Etchebest C, Killian JA, Fuchs PFJ On the orientation of a designed transmembrane peptide: toward the right tilt angle? J Am Chem Soc. 2007 ; 129(49):15174-15181.
8. De Planque, MRR, Demmers JAA, Bonev BB, Koeppe II RE, Greathouse DV, Separovic F, Watts A, Killian JA Interfacial anchor properties of tryptophan residues in transmembrane peptides can dominate over hydrophobic mismatch effects in peptide-lipid interactions Biochemistry 42 2003 ; 42(18):5341-5348.
9. Van der Wel, PCA, Strandberg, E. Killian, JA, Koeppe II, RE Geometry and intrinsic tilt of a tryptophan anchored transmembrane alpha-helix determined using deuterium NMR spectroscopy. Biophys. J. 2002 ; 83(3):1479-1488.
10. Killian JA and Von Heijne G How proteins adapt to a membrane-water interface. TIBS 25 2000 ; 25(9):429-434.
Nanodiscs to study protein/lipid interactions
Much of our research efforts are focussed on the exploration of styrene-maleic acid (SMA) polymers as novel tool in membrane research (for review see ref.1). SMA polymers are able to solubilize membranes in the form of small nanodiscs, allowing purification and characterization of membrane proteins directly in their native environment without the use of detergent. On one hand we use the SMA technology to solubilize, purify, and characterize membrane proteins in their native lipid environment. Examples include the tetrameric potassium channel KcsA from E.coli and the Reaction Center from Rhodobacter sphearoides. On the other hand, we are investigating the mode of action of SMA using model membrane systems. This allows systematic and straightforward variation of many potentially important parameters. These experiments lead to new insights into the molecular mechanism of nanodisc formation and they help to optimize the use of SMA for solubilization of proteins from different native membranes. In addition, we are exploring new applications of SMA in membrane research, such as isolation of liquid-ordered domains and analysis of helix-helix interactions in membranes.
Schematic representation of ways of reconstituting membrane proteins in a bilayer environment after purification in micelles (black arrows) versus direct solubilization of membrane proteins from their native environment in the form of native nanodiscs (red arrow). Adapted from ref. 1 below.
1. Dörr JM, Scheidelaar S, Koorengevel MC, Dominguez JJ, Schäfer M, van Walree CA, Killian JA The styrene-maleic acid copolymer: a versatile tool in membrane research Eur Biophys J. 2016 ; 45(1):3-21
2. Scheidelaar S, Koorengevel MC, Dominguez-Pardo J, Meeldijk JD, Breukink E, Killian JA Molecular model for the solubilization of membranes into nanodisks by styrene maleic acid copolymers Biophys. J. 2015 ; 108(2):279-290.
3. Dörr JM, Koorengevel MC, Schäfer M, Prokofyev AV, Scheidelaar S, Van der Cruijsen EAW, Dafforn TR, Baldus M, Killian JA Detergent-free isolation, characterization and functional reconstitution of a tetrameric K+ channel: the power of native nanodiscs Proc Natl Acad Sci USA. 2014 ; 111(52):18607-18612
4. Swainsbury DJ, Scheidelaar S, van Grondelle R, Killian JA, Jones MR Bacterial Reaction Centers Purified with Styrene Maleic Acid Copolymer Retain Native Membrane Functional Properties and Display Enhanced Stability Angew Chem Int Ed Engl. 2014 ; 53(44): 11803-11807.
Amyloid membrane interactions
In this line of research we focus on amyloid forming proteins. Membranes may play an important role in the lethal action of these proteins, either by catalyzing amyloid formation, and/or by loosing their integrity upon interaction with the proteins and becoming permeable. To gain insight into these possible mechanisms, several strategies are being used, varying from advanced biophysical techniques and model systems to molecular biological and synthetic approaches. In particular we focus on the islet amyloid polypeptide (IAPP) involved in type II diabetes. This is particularly relevant with the increasing occurrence of obesitas all over the world, as obesitas is one of the risk factors in development of this disease.
Models for the hIAPP-membrane interaction in relation to membrane damage and hIAPP cytotoxicity (from ref.5 below).
1. Khemtémourian L, Doménech E, Doux JP, Koorengevel MC, Killian JA Low pH acts as inhibitor of membrane damage induced by human islet amyloid polypeptide. J Am Chem Soc. 2011 ; 133(39):15598-155604.
2. Khemtémourian L, Engel MF, Liskamp RM, Höppener JW, Killian JA The N-terminal fragment of human islet amyloid polypeptide is non-fibrillogenic in the presence of membranes and does not cause leakage of bilayers of physiologically relevant lipid composition. Biochim Biophys Acta 2010 ; 1798(9):1805-1811.
3. Khemtémourian L, Engel MF, Kruijtzer JA, Höppener JW, Liskamp RM, Killian JA The role of the disulfide bond in the interaction of islet amyloid polypeptide with membranes. Eur Biophys J. 2010 ; 39(9):1359-1364.
4. Khemtémourian L, Lahoz Casarramona G, Suylen DPL, Hackeng TM, Meeldijk HDJ, de Kruijff B, Höppener JWM, Killian JA Impaired processing of human pro-islet amyloid polypeptide is not a causative factor for fibril formation or membrane damage in vitro Biochemistry 2009 ; 48(46):10918-10925.
5. Engel MFM, Khemtémourian L, Kleijer CC, Meeldijk HJ, Jacobs J, Verkleij AJ, De Kruijff B, Killian JA, Höppener JWM Membrane damage by human islet amyloid polypeptide through fibril growth at the membrane. Proc. Natl. Acad. Sci. 2008 ; 105(16):6033-6038.
6. Khemtémourian L, Killian JA., Höppener JWM. and Engel MFM Recent insights in Islet Amyloid Polypeptide-induced membrane disruption and its role in ?-cell death in type 2 diabetes mellittus Exp Diabetes Res. 2008 ; 421287.
7. Engel MFM, Yigitop H, Elgersma RC, Rijkers DTS, Liskamp RMJ, De Kruijff B, Höppener JWM, Killian JA Islet amyloid polypeptide inserts into phospholipid monolayers as monomer. J. Mol. Biol. 2006 ; 356(3):783-789.
8. Sparr E, Engel MFM, Sakharov DV, Sprong M, Jacobs J, De Kruijff B, Höppener JWM, Killian JA Islet amyloid polypeptide-induced membrane leakage involves uptake of lipids by forming amyloid fibers. FEBS Lett. 2004 ; 577(1-2):117-120.