Circular dichroism measurements show that con- let. This type of confinement allows a study that provides finement enhances the secondary structural features of the unique insight into the dynamic structure of an unfolded, flexi- protein. Small-angle X-ray scattering and dynamic light scatter- ble protein which is still solvated and thus under near-physio- ing show that OPN changes from being a flexible protein in logical conditions. In a model system, the protein osteopontin aqueous solution to adopting a less flexible and more compact OPN is used.
It is a highly phosphorylated glycoprotein that is structure inside the microemulsion droplets. This novel ap- expressed in a wide range of cells and tissues for which limited proach for confining proteins while they are still hydrated may structural analysis exists due to the high degree of flexibility aid in studying the structure of a wide range of natively un- and large number of post-translational modifications.
OPN is folded proteins. Introduction A prerequisite to obtain unique insight into the function and By encapsulation of OPN into water-in-oil microemulsion activity of a wide range of proteins is the ability to gain de- droplets, the crowded intracellular environment can be mim- tailed structural information. By means of circular dichroism CD , dynamic study in aqueous media. However, this traditional approach is very questionable for proteins, for which the crowded cell en- [a] Dr. Shipovskov, Dr. Schauser, Prof. Sutherland vironment is highly important for structure stabilisation.
E-mail: stepan. Oliveira , Prof. Pedersen verse micelles or microemulsions in organic solvents for mim- Interdisciplinary Nanoscience Center iNANO icking cell-like environment, this method has only very re- and Department of Chemistry Aarhus University cently been used for solving the structure of membrane and Langelandsgade , Aarhus Denmark intracellular proteins. Hoffmann surfactants is used to restrict the mobility by confining the Institute for Storage Ring Facilities ISA protein inside an inverse microemulsion droplet. Besenbacher tein, and the study thus provides unique insight into the dy- Interdisciplinary Nanoscience Center iNANO namic structure of an unfolded, flexible protein under confine- and Department of Physics and Astronomy ment, which is still solvated and thus under near-physiological Aarhus University conditions.
This novel approach may aid in the study of the Ny Munkegade Aarhus Denmark structure of a wide range of natively unfolded proteins. Oliveira tional modifications. Pedersen, S. Shipovskov et al. The mass transfer of protein into the organ- hexane A maximal concentration of OPN of 3.
We expect that the all, while in the organic solvent a-R are clearly present. Appa- protein in hexane has a layer of hydrating water as well as rently, the main suppliers of residues to formation of a-R are a layer of AOT. CD spectra of 1. Inset: CD spectra in the range — nm.
The spectral region of — nm shows a deeper minimum for OPN in organic solution than in water, while the peak at nm is smaller than in water and the peak at nm is more pronounced, which corresponds to a decrease of Figure 2. The filled symbols. The concentrations are 10 circles , 5 squares , and fractions of the various secondary structures were calculated 2.
The flexibility of OPN is further a-D , b-regular b-R , b-distorted b-D , turns and others confirmed by a Kratky plot of the intensity multiplied by q2 random coil. The remaining residues were counted as belong- versus q, which shows a monotonic increase inset of Figure 3. In the OPN molecules. The peak decreases in size and moves to dilute aqueous solution OPN does not reveal any a-R units at smaller q for decreasing concentration.
Enhancing Protein Secondary Structure by Confinement particles with internal flexibility. In this approach, one can con- sider conformations with respective Gaussian random statistics or native-like folded conformations.
The best fit of the experimental OPN SAXS data was ob- tained for the native-like folded conformations with average radius of gyration closest to the value obtained from the fit using the semiflexible excluded volume polymer model. En- semble distributions of the radius of gyration and of the maxi- mum sizes are obtained from this modelling Figure 4 togeth- er with a set of 3D structural models Figure 5. Figure 3. SAXS data for OPN in water lower data and in 10 mm NaCl upper data, multiplied by ten extrapolated to zero concentration and cor- responding fits using the scattering function for self-avoiding flexible chains.
The insert shows a Kratky plot of q2I q versus q. Figure 4. Ensemble distribution of the radius of gyration c and of the with the distance between the molecules becoming larger and maximum size a as obtained from the ensemble-based approach for an- the electrostatic interactions becoming less important as the alysing the SAXS data for OPN in water.
Both data sets were extrapolated to zero concentration and the resulting data fitted by the scat- tering function for self-avoiding flexible chains Figure 3. The Rg value is in reasonable agree- ment with those obtained by Holt et al. The dark grey spheres are for one of the models, and the semi-transparent spheres for a few other models from the ensemble. Table 2. The hydrodynamic radii from DLS are given as spheres and a superposition of the ensemble components weight-average values with the number-average values given in paren- theses.
The observed flexibility of hexane solution. KGaA, Weinheim www. The models derived from the SAXS data can also be checked by comparing the hydrodynamic radius of the models to the experimental value. The small discrepancy can be explained by some degree of flexibility of the protein also in the organic solvent phase, as flexibility is known to decrease the observed hydrodynamic size.
Figure 6. The This can be expected to be accompanied by compaction of inset shows a Kratky plot of q2I q versus q. The approach presented here whereby an alternative medium is used to confine Figure 7.
Guide to the Papers of William A. Fowler, 1917-1994
The model is the average of 10 runs. The approach yields novel insight into the nature of the dynamic and flexible structures of proteins in carbon tails, only the head groups, hydrating water and pro- the set of natively unfolded or intrinsically unstructured pro- tein are revealed. An average three-dimensional 3D structural teins recently identified in the literature.
It also suggests that model is obtained Figure 7 with a prolate shape of about a range of methods employing encapsulation in water-in-oil 10 nm in length and a radius of 1. The samples were also investigated by DLS to obtain addi- Experimental Section tional information on the sizes. All reagents were of analytical degree of flexibility. The mass-average hydrodynamic radius is grade and used as received. The solu- has a thickness of 1. The transparent supernatant was used for namic radius. Thus, the slightly smaller hydrodynamic radius further studies.
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OPN was measured at concentrations of 1. The spectra published. Measurements were performed at The software de- a spherical search space filled with small spheres, the program em- livered with the system was used for deriving the particle distribu- ploys a simulated annealing optimisation that determines the set tion as a function of the hydrodynamic radius by using the of spheres that provides the best fit of the experimental data.
Number distributions and mass distributions, as Since the obtained models are not unique several runs give slight- well as average hydrodynamic radii, were calculated. It employs a rotating-anode X-ray source CuKa, 0. The instrument has an integrated vacuum tistics or an approach that generates native-like folded conforma- in order to reduce background. The instrument covers a range of tions for the protein.
For a chosen statistics, a subset of structures scattering vectors q between 0. Ensemble distributions of the radius of gyration and of length. All measurements were made at room temperature. The the maximum sizes are subsequently calculated. The hydrodynamic SAXS data were corrected for variations in detector efficiency and radius of the ensemble of each of conformations was calculated by spatial distortions, and azimuthally averaged.
This gives the aver- ware package. A typical acquisition time for the experiments was 2 h. The authors thank Prof.
The approach is similar to a classical Zimm analysis, except that it is expected to be applica- The financial support to the iNANO center from the Danish Re- ble at higher concentrations in the full SAXS q range, since it has search Councils, the Villum Kahn Rasmussen Foundation, the a more general form that can reproduce the behaviour found by Lundbeck Foundation and the Carlsberg Foundation is gratefully PRISM theory and simulations for more strongly interacting sys- acknowledged. This analysis gives the ensemble  M.
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