Phosphorescence depolarization and the measurement of rotational motion of proteins in membranes

Nature invented molecular rotatory devices such as the f lagellar motor and ATP synthase. Photoselection techniques have been frequently used to detect the rotational random walk of proteins but only rarely for the rotational drift of subunits in proteins. Pertinent theories predict an oscillatory behavior of the polarization anisotropy, r, for unidirectional rotational drift, as opposed to a monotonic relaxation of r for bidirectional random walk. The underlying assumption of an angular continuum is questionable for intersubunit rotation in proteins. We developed a theory for stepped rotatory devices. It predicts the damped oscillation of r under unidirectional drift. Damping increases with decreasing number of steps. For only three steps a quasi-monotonic relaxation of r is predicted for both random walk and drift. In photoselection experiments with active F-ATPase we observed the relaxation of r when a spectroscopic probe was attached to the central ␥-subunit. This behavior is compatible with the expectation for a three-stepped rotatory device.In the flagellar motor and in ATP synthase (F-ATPase) protein subunits rotate against each other. The rotation of bacterial flagellae has been detected in the light microscope by the rotation of the cell body after tethering of the flagellae to a solid support (1). The detection of rotatory motion in objects of smaller size like subunits in ATP-synthase or the load-free flagellar motor requires less macroscopic techniques. Recently, we detected and time resolved the activity-linked intersubunit rotation in F-ATPase by PARAP (polarized absorption recovery after photobleaching) (2). This spectroscopic technique applies to a large ensemble of molecules and is based on orientational photoselection. Rotational motion causes a transient of the polarization anisotropy, r (see Eq. 1). In these studies we observed a monotonic relaxation of the polarization anisotropy of a probe which was attached to ␥-subunit of this enzyme. It occurred in about 100 ms, which conformed with the turnover time of the enzyme under the given experimental conditions (see Fig. 4A for data and Fig. 3 for the enzyme structure).Does the transient behavior of r allow to discriminate between rotational random walk which is bidirectional from rotational drift under a driving force which is unidirectional? For a true rotatory motor-i.e., for drift without superimposed random walk-one expects an oscillatory transient of the r-parameter, and for rotatory random walk a monotonic decay. Theories for rotatory random walk and drift have been published by various authors (3-7). A theory by Hoshikawa and Asai (7) supports the above-mentioned expectation. Applying their equation 23 to a situation where the stator of a molecular rotatory motor is immobilized and a spectroscopic probe is attached to the rotor, an undamped oscillation of r is predicted.Their approach, however, is based on Fick's phenomenological equation for diffusion in a continuous angular space. Considering the flagellar motor and even more ...

supporting

confidence: 55%

Stepped versus continuous rotatory motors at the molecular scale

Sabbert

Junge

1997

“…Fluorescence depolarization has been used for many years to study molecular motion in the nanosecond time range (5,6). The extension of this technique into the picosecond time range (7,8), together with the introduction of triplet probes for the microsecondmillisecond region (9)(10)(11)(12)(13), means that optical probes can now be used to measure molecular motion over the enormous range of 10-11-10-1 sec. Moreover, time-resolved measurements can be made over this whole range, leading to a detailed picture of the different motions present in a given system.…”

mentioning

confidence: 99%

Oligosaccharide motion in erythrocyte membranes investigated by picosecond fluorescence polarization and microsecond dichroism of an optical probe.

Cherry¹,

Nigg²,

Beddard³

1980

Oligosaccharide chains on the surface of human erythrocytes were labeled with the probe eosin 5-thiosemicarbazide. The probe was conjugated to aldehydes produced by oxidation of sialic acid and galactose residues. The probe is associated mostly with glycophorin A after sialic acid labeling, whereas multiple components, including band 3 and lipids, are labeled after galactose oxidation. Fast molecular motion was studied by measuring steady-state and picosecond time-resolved fluorescence depolarization. Slower motions were investigated by observing flash-induced transient dichroism. It was found that'both eosin-labeled sialic acid and galactose residues exhibit a rapid motion with correlation time of approximately 3 nsec. This motion is assigned to independent motion of the probe, possibly in conjunction with a short segment of the oligosaccharide chain. The order parameter of the fast motion is 0.8-0.9, demonstrating that its angular amplitude is highly restricted. For eosin-labeled sialic acid, the order parameter in the microsecond time range is 0.2-0.3. It is deduced that a second, slower rotational motion is present, which is assigned to a cooperative motion of the oligosaccharide'chains. The correlation time of this motion is in the range 10-7-10-5 sec. Some eosin-labeled galactose residues may have a similar slow motion, but most appear to be remarkably immobile over the time range 10-8-10-3 sec.'A variety of biologically important phenomena involve molecular interactions at the cell surface. The surface of eukaryotic cells is typically covered by oligosaccharide side chains of glycoproteins and glycolipids present in the plasma membrane. These oligosaccharide moieties have frequently been implicated in surface recognition events (1-3). Although the chemical composition of surface oligosaccharides has been studied in some detail (3, 4), there have been almost no investigations of their physical properties. It is likely that an understanding of their dynamic and conformational properties will be of considerable importance in understanding their functional role.As a result of techniques developed in recent years, optical probes now provide a powerful approach to studying the dynamic properties of complex biological structures. Fluorescence depolarization has been used for many years to study molecular motion in the nanosecond time range (5, 6). The extension of this technique into the picosecond time range (7,8), together with the introduction of triplet probes for the microsecondmillisecond region (9-13), means that optical probes can now be used to measure molecular motion over the enormous range of 10-11-10-1 sec. Moreover, time-resolved measurements can be made over this whole range, leading to a detailed picture of the different motions present in a given system.In the present studies, we have oxidized sialic acid and galactose residues on the surface of erythrocytes and conjugated eosin 5-thiosemicarbazide (eosin-TSC) to the resultant aldehydes. We have used time-resolved fluorescence depolariza...

“…e., for probing proximity relationships on the cell surface (24)(25)(26)(27); (ii) time-resolved polarized luminescence (22,28,29) associated with the triplet state (30) for studying the rotational mobility of membrane components; and (iii) fluorescence recovery after photobleaching (FRAP) for studying translational diffusion (21,31 (33). (The FITC-conjugated antibodies are designated F-H-100 5/28, 27/55, and 30/6, whereas the Me4RITC-conjugated antibodies are designated R-H-100 5/28, 27/55, and 30/ 6.)…”

mentioning

confidence: 99%

“…e., for probing proximity relationships on the cell surface (24-27); (ii) time-resolved polarized luminescence (22,28,29) associated with the triplet state (30) for studying the rotational mobility of membrane components; and (iii) fluorescence recovery after photobleaching (FRAP) for studying translational diffusion (21,31 …”

mentioning

confidence: 99%

See 1 more Smart Citation

Distribution and mobility of murine histocompatibility H-2Kk antigen in the cytoplasmic membrane.

Damjanovich¹,

Trón²,

Szöllősi³

et al. 1983

The topographical distributions and mobilities of the murine histocompatibility antigen H-2Kk and of concanavalin A (Con A) binding sites have been studied on a murine lymphoma cell line. The spatial distribution of H-2Kk antigens, the average distance between H-2Kk antigens and Con A binding sites, and the separation of different determinants on the H-2K antigen itself were determined by using fluorescence resonance energy-transfer measurements with a dual-laser flow sorter. From the lack of energy transfer between bound monoclonal anti-H-2Kk antibodies conjugated with fluorescein (donor) and rhodamine (acceptor), we conclude that the H-2Kk antigen exists without appreciable clustering on the cell surface. Substantial energy transfer between appropriately labeled Con A and antibodies bound to the H-2Kk antigen shows that the two populations are interspersed. Donor/ acceptor pairs of monoclonal antibodies binding to different determinants on the same H-2Kk antigen exhibited a degree of energy transfer indicative of a mean separation of 8.6 nm between the sites. Time-resolved phosphorescence anisotropy measurements with anti-H-2Kk antibodies labeled with eosin or erythrosin yielded rotational mobility information for the antigen-antibody complexes on the cell membrane. The rotational correlation time of 10-20 ,us and the finite residual anisotropy are compatible with an uniaxial mode of rotation of monomeric antigen around its transmembrane portion and, thus, provide additional evidence for an unclustered distribution. Capping by rabbit anti-mouse IgG immobilized the antigen-antibody complex. Fluorescence recovery after photobleaching was used to calculate an apparent lateral diffusion coefficient of 5 ± 3 X 10-10 cm2s-1 for the H-2Kk an-' tigen labeled with fluoresceinated IgG or its corresponding Fab fragment.The K and D regions of the H-2 major histocompatibility gene complex (MHC) of the mouse encode the class I antigens expressed on the surface of most somatic cells. The class I antigens consist of a highly polymorphic integral membrane glycoprotein of Mr 45,000 associated with an invariant f82-microglobulin, a Mr 11,600 protein not coded for by the MHC. These antigens and other MHC gene products guide thymic lymphocytes in distinguishing self from nonself. The H-2K and H-2D regions provide the alloantigens that control (i) allograft rejection (1-3), (ii) induction of lytic (killer) T cells (1-4), and (iii) differentiation of restricted T cells that lyse cells bearing both a foreign (e.g., viral) antigen and a H-2K or H-2D antigen presented by the initial stimulating cells (4). Models for the mechanism by which the latter process occurs provide for the recognition of a complex between MHC products and foreign antigen on the target cell (altered self; refs. 2, 5-7) or both antigens separately (dual recognition; refs. 8 and 9). These models require a finite mobility and redistribution of the class I antigens on the cell surface. Considerations of a similar nature apply to other cell-cell interactions ...