Electron transfer is used as a probe for angstrom-scale structural changes in single protein molecules. In a flavin reductase, the fluorescence of flavin is quenched by a nearby tyrosine residue by means of photo-induced electron transfer. By probing the fluorescence lifetime of the single flavin on a photon-by-photon basis, we were able to observe the variation of flavin-tyrosine distance over time. We could then determine the potential of mean force between the flavin and the tyrosine, and a correlation analysis revealed conformational fluctuation at multiple time scales spanning from hundreds of microseconds to seconds. This phenomenon suggests the existence of multiple interconverting conformers related to the fluctuating catalytic reactivity.

We report the results of a study, using digital video microscopy, of the melting transition in a quasi-two-dimensional suspension of uncharged silica spheres. This system was chosen to further test the dependence of the two-dimensional melting transition on the functional form of the colloid-colloid interaction. Our experimental data show that the solid phase undergoes a first order transition directly to the liquid phase. The system studied yields no evidence of the existence of a hexatic phase interpolating between the solid and liquid phases in the melting process.

Decay-accelerating factor (DAF, also known as CD55), a glycosylphosphatidylinositol-linked (GPI-linked) plasma membrane protein, protects autologous cells from complement-mediated damage by inhibiting complement component 3 (C3) activation. An important physical property of GPI-anchored complement regulatory proteins such as DAF is their ability to translate laterally in the plasma membrane. Here, we used singleparticle tracking and tether-pulling experiments to measure DAF lateral diffusion, lateral confinement, and membrane skeletal associations in human erythrocyte membranes. In native membranes, most DAF molecules exhibited Brownian lateral diffusion. Fluid-phase complement activation caused deposition of C3b, one of the products of C3 cleavage, onto erythrocyte glycophorin A (GPA). We then determined that DAF, C3b, GPA, and band 3 molecules were laterally immobilized in the membranes of complement-treated cells, and GPA was physically associated with the membrane skeleton. Mass spectrometry analysis further showed that band 3, α-spectrin, β-spectrin, and ankyrin were present in a complex with C3b and GPA in complement-treated cells. C3b deposition was also associated with a substantial increase in erythrocyte membrane stiffness and/or viscosity. We therefore suggest that complement activation stimulates the formation of a membrane skeletonlinked DAF-C3b-GPA-band 3 complex on the erythrocyte surface. This complex may promote the removal of senescent erythrocytes from the circulation. IntroductionThe complement system is a major effector component of the innate immune response (1). The complement cascade, which involves sequential activation of serum complement proteins, leads to diverse inflammatory effects and, in some cases, lysis of the target. Activation of complement can occur through the classical, alternative, and lectin pathways. All 3 pathways lead to the formation of complement component 3 (C3) convertase, a central enzymatic complement complex that cleaves serum C3 into C3a and C3b. C3b can dock covalently on a membrane surface via amide or ester linkages. Downstream of C3 activation, C3 convertase participates in the formation of C5 convertase, a membrane-bound complex that cleaves serum C5 into C5a and C5b. C5b induces sequential recruitment of complement proteins C6, C7, C8, and C9 to form C5b-9, the terminal complement complex, which creates a pore in the membrane (2).