DOI: 10.1002/cphc.201200295 Water-in-Oil Micro-Emulsion Enhances the Secondary Structure of a Protein by Confinement Stepan Shipovskov,* [a, e] Cristiano L. P. Oliveira, [b, f] Søren Vrønning Hoffmann, [c] Leif Schauser, [a] Duncan S. Sutherland, [a] Flemming Besenbacher, [d] and Jan Skov Pedersen* [b] 1. Introduction A prerequisite to obtain unique insight into the function and activity of a wide range of proteins is the ability to gain de- tailed structural information. [1] In general, structure determina- tion requires purification of the desired proteins and their study in aqueous media. However, this traditional approach is very questionable for proteins, for which the crowded cell en- vironment is highly important for structure stabilisation. In these cases, simulation of the cell environment is important to gain information on the stability and activity of the proteins. Although it was proposed almost three decades ago to use re- verse micelles or microemulsions in organic solvents for mim- icking cell-like environment, [2] this method has only very re- cently been used for solving the structure of membrane and intracellular proteins. [3–5] Herein, we present an approach in which an organic solvent environment in combination with surfactants is used to restrict the mobility by confining the protein inside an inverse microemulsion droplet. The confine- ment enhances the secondary structural features of the pro- tein, and the study thus provides unique insight into the dy- namic structure of an unfolded, flexible protein under confine- ment, which is still solvated and thus under near-physiological conditions. This novel approach may aid in the study of the structure of a wide range of natively unfolded proteins. As model system we use osteopontin (OPN), a highly phos- phorylated glycoprotein that is expressed in a wide range of cells and tissues [6] for which limited structural data exist due to the high degree of flexibility and large number of post-transla- tional modifications. OPN is broadly implicated in tissue func- tions such as inflammation, mineralisation, angiogenesis and wound healing, as well as playing a pivotal role in tumour metastasis and progression. [6, 7] By encapsulation of OPN into water-in-oil microemulsion droplets, the crowded intracellular environment can be mim- icked and structural features of the protein can be induced and determined. By means of circular dichroism (CD), dynamic A scheme is presented in which an organic solvent environ- ment in combination with surfactants is used to confine a na- tively unfolded protein inside an inverse microemulsion drop- let. This type of confinement allows a study that provides unique insight into the dynamic structure of an unfolded, flexi- ble protein which is still solvated and thus under near-physio- logical conditions. In a model system, the protein osteopontin (OPN) is used. It is a highly phosphorylated glycoprotein that is expressed in a wide range of cells and tissues for which limited structural analysis exists due to the high degree of flexibility and large number of post-translational modifications. OPN is implicated in tissue functions, such as inflammation and miner- alisation. It also has a key function in tumour metastasis and progression. Circular dichroism measurements show that con- finement enhances the secondary structural features of the protein. Small-angle X-ray scattering and dynamic light scatter- ing show that OPN changes from being a flexible protein in aqueous solution to adopting a less flexible and more compact structure inside the microemulsion droplets. This novel ap- proach for confining proteins while they are still hydrated may aid in studying the structure of a wide range of natively un- folded proteins. [a] Dr. S. Shipovskov, Dr. L. Schauser, Prof. D. S. Sutherland Interdisciplinary Nanoscience Center (iNANO) Aarhus University Ny Munkegade 8000 Aarhus (Denmark) E-mail : stepan.shipovskov@inano.au.dk [b] Prof. C. L. P. Oliveira, Prof. J. S. Pedersen Interdisciplinary Nanoscience Center (iNANO) and Department of Chemistry Aarhus University Langelandsgade 130, 8000 Aarhus (Denmark) E-mail : jsp@chem.au.dk [c] S. V. Hoffmann Institute for Storage Ring Facilities (ISA) Aarhus University Ny Munkegade, DK-8000 Aarhus (Denmark) [d] Prof. F. Besenbacher Interdisciplinary Nanoscience Center (iNANO) and Department of Physics and Astronomy Aarhus University Ny Munkegade 8000 Aarhus (Denmark) [e] Dr. S. Shipovskov Danisco A/S, Genencor Division Edwin Rahrs Vej 38 8220 Brabrand (Denmark) E-mail : stepan.shipovskov@danisco.com [f] Prof. C. L. P. Oliveira Complex Fluids Group Department of Experimental Physics Institute of Physics University of S¼o Paulo 05314-970 S¼o Paulo (Brazil) ChemPhysChem 0000, 00, 1 – 7  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim &1& These are not the final page numbers! ÞÞ