Solution Structure of the Detergent–Photosystem II Core Complex Investigated by Small-Angle Scattering Techniques

Abstract

Albeit achieving the X-ray diffraction structure of dimeric photosystem II core complexes (dPSIIcc) at the atomic resolution, the nature of the detergent belt surrounding dPSIIcc remains ambiguous. Therefore, the solution structure of the whole detergent−protein complex of dPSIIcc of Thermosynechococcus elongatus (T. elongatus) solubilized in n-dodecyl-ß-D-maltoside (ßDM) was investigated by a combination of small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS) with contrast variation. First, the structure of dPSIIcc was studied separately in SANS experiments using a contrast of 5% D 2 O. Guinier analysis reveals that the dPSIIcc solution is virtually free of aggregation in the studied concentration range of 2−10 mg/mL dPSIIcc, and characterized by a radius of gyration of 62 Å. A structure reconstitution shows that dPSIIcc in buffer solution widely retains the crystal structure reported by Xray free electron laser studies at room temperature with a slight expansion of the entire protein. Additional SANS experiments on dPSIIcc samples in a buffer solution containing 75% D 2 O provide information about the size and shape of the whole detergent− dPSIIcc. The maximum position of P(r) function increases to 68 Å, i.e., it is about 6 Å larger than that of dPSIIcc only, thus indicating the presence of an additional structure. Thus, it can be concluded that dPSIIcc is surrounded by a monomolecular belt of detergent molecules under appropriate solubilization conditions. The homogeneity of the ßDM−dPSIIcc solutions was also verified using dynamic light scattering. Complementary SAXS experiments indicate the presence of unbound detergent micelles by a separate peak consistent with a spherical shape possessing a radius of about 40 Å. The latter structure also contributes to the SANS data but rather broadens the SANS curve artificially. Without the simultaneous inspection of SANS and SAXS data, this effect may lead to an apparent underestimation of the size of the PS II−detergent complex. The formation of larger unbound detergent aggregates in solution prior to crystallization may have a significant effect on the crystal formation or quality of the ßDM−dPSIIcc.

“Invisible” Detergents Enable a Reliable Determination of Solution Structures of Native Photosystems by Small-Angle Neutron Scattering

“…In addition, the radii of gyration Rg determined from the Guinier plots and from the P ( R ) functions listed in Table are about 2 to 5 Å larger when using hDDM compared with dDDM. All SANS curves are roughly consistent with trimeric PSI, ,,, suggesting that the conventional approaches of contrast matching of protonated detergents lead to seemingly larger complex sizes, as indeed previously observed for PSI. ,,, …”

“…The latter may arise from detergent interacting with the protein itself and from free detergent micelles present in the buffer solution. In situ size exclusion chromatography (SEC) has proven to be an efficient way to remove free micelles from a sample solution, , whose scattering contribution may otherwise seriously complicate the SANS data analysis. ,, In addition, SANS combined with contrast variation is a highly promising approach to match out (and thus “hide”) the scattering of detergent molecules in both micelles and protein–detergent complexes. ,− However, due to the chemically heterogeneous nature of the hydrophilic and hydrophobic parts of protonated detergents, the elimination of their scattering contribution over the entire Q -range remains challenging. , This may be a reason for the very different solution structures reported previously in the case of PSI, ,,, and for their often overestimated sizes compared with the respective crystal structures. More recently, it was shown that matching out detergents can be significantly improved by using adequate deuteration to homogenize the scattering contribution of hydrophilic and hydrophobic parts of a detergent molecule …”

Investigation of Photosystem II Functional Size in Higher Plants under Physiological and Stress Conditions Using Radiation Target Analysis and Sucrose Gradient Ultracentrifugation

“…Crystallographic analysis of the isolated core preparations has established the dimeric nature of PSII as a homodimer [ 19 , 33 ]. The present study addresses the question about the functionality of PSII under normal and stress conditions, both in vitro and in vivo.…”

“…It is known that the PSII structure is maintained by some lipids [ 37 ], and that the detergent can substitute for the membrane lipids [ 38 ]. The dimeric core has been shown to be surrounded by a monomolecular belt of detergent molecules under appropriate solubilising conditions [ 33 ]. The structural and functional role of phosphatidylglycerol in PSII and in PSII dimer–monomer interconversion is known [ 39 , 40 , 41 , 42 , 43 ].…”

Dynamics of Apolipoprotein B-100 in Interaction with Detergent Probed by Incoherent Neutron Scattering

“…However, these techniques are not suitable for apo B-100, and the characterization of membrane proteins still often requires taking the detergent contribution into account . For the structure of protein–detergent complexes, it is usually examined using contrast-matching techniques. − With respect to their dynamics, first studies of bacteriorhodopsin did not separate the remaining detergent or lipids from the protein. − Then, because deuterium has a smaller incoherent cross-section compared to hydrogen, isotopic exchange can be performed on the buffer to reduce its signal. Deuterated detergents were therefore proposed for quasi-elastic neutron scattering (QENS) measurements on the bacterial photoreaction center .…”