The general possibility to align biological macromolecules in the magnetic field by the presence of orienting agents such as lipid bicelles has led to a wealth of new structural parameters which can be derived from residual tensorial interactions in high resolution NMR. Here, we report that alignment of water soluble biomacromolecules can be achieved in the presence of the naturally occurring two-dimensional crystals (purple membrane) of the membrane protein bacteriorhodopsin. The extent of the alignment is tunable by the concentration of purple membranes and by the addition of salt which reduces the strength of the electric interaction between solute macromolecule and the membrane. At very high salt concentrations the purple membrane suspension undergoes a phase transition to a very viscous state. In this state, rotation of the membranes is hindered such that the orientation of the membrane patches persists even in the absence of a magnetic field. The induced alignment of solute molecules is shown for the two proteins, ubiquitin and p53, with residual dipolar one-bond 1H−15N couplings in the order of 20 Hz. A concept for the description of the irreducible components of the alignment tensors as a linear vector space is presented. In the case of ubiquitin, the direction of the alignment tensor differs strongly from the alignment tensor observed in DMPC/DHPC bicelles. This offers the possibility of an accurate triangulation of the bond vector direction from a combination of the two alignment experiments.

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Biglycan, a ubiquitous proteoglycan, acts as a danger signal when released from the extracellular matrix. As such, biglycan triggers the synthesis and maturation of interleukin-1β (IL-1β) in a Toll-like receptor (TLR) 2-, TLR4-, and reactive oxygen species (ROS)-dependent manner. Here, we discovered that biglycan autonomously regulates the balance in IL-1β production in vitro and in vivo by modulating expression, activity and stability of NADPH oxidase (NOX) 1, 2 and 4 enzymes via different TLR pathways. In primary murine macrophages, biglycan triggered NOX1/4-mediated ROS generation, thereby enhancing IL-1β expression. Surprisingly, biglycan inhibited IL-1β due to enhancement of NOX2 synthesis and activation, by selectively interacting with TLR4. Synthesis of NOX2 was mediated by adaptor molecule Toll/IL-1R domain-containing adaptor inducing IFN-β (TRIF). Via myeloid differentiation primary response protein (MyD88) as well as Rac1 activation and Erk phosphorylation, biglycan triggered translocation of the cytosolic NOX2 subunit p47(phox) to the plasma membrane, an obligatory step for NOX2 activation. In contrast, by engaging TLR2, soluble biglycan stimulated the expression of heat shock protein (HSP) 70, which bound to NOX2, and consequently impaired the inhibitory function of NOX2 on IL-1β expression. Notably, a genetic background lacking biglycan reduced HSP70 expression, rescued the enhanced renal IL-1β production and improved kidney function of Nox2(-/y) mice in a model of renal ischemia reperfusion injury. Here, we provide a novel mechanism where the danger molecule biglycan influences NOX2 synthesis and activation via different TLR pathways, thereby regulating inflammation severity. Thus, selective inhibition of biglycan-TLR2 or biglycan-TLR4 signaling could be a novel therapeutic approach in ROS-mediated inflammatory diseases.

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