While still incomplete, the first data concerning the biochemistry of T cell receptor-ligand interactions in cell-free systems seem to have considerable predictive value regarding whether a T cell response is strong or weak or suppressive. This data will help considerably in elucidating the mechanisms behind T cell responsiveness. Also of great interest are the first structures of T cell receptor molecules and, particularly, TCR-ligand complexes. These appear to confirm earlier suggestions of a fixed orientation for TCR engagement with peptide/MHC and should form the basis for understanding higher oligomers, evidence for which has also just emerged. We conclude with an analysis of the highly diverse CDR3 loops found in all antigen receptor molecules and suggest that such regions form the core of both TCR and antibody specificity.

While much is known about intracellular signaling events in T cells when T cell receptors (TCRs) are engaged, the mechanism by which signaling is initiated is unclear. We have constructed defined oligomers of soluble antigen-major histocompatibility complex (MHC) molecules, the natural ligands for the TCR. Using these to stimulate specific T cells in vitro, we find that agonist peptide/MHC ligands are nonstimulatory as monomers and minimally stimulatory as dimers. Similarly, a partial-agonist ligand is very weakly active as a tetramer. In contrast, trimeric or tetrameric agonist ligands that engage multiple TCRs for a sustained duration are potent stimuli. Ligand-driven formation of TCR clusters seems required for effective activation and helps to explain the specificity and sensitivity of T cells.

The machinery that conducts the light-driven reactions of oxygenic photosynthesis is hosted within specialized paired membranes called thylakoids. In higher plants, the thylakoids are segregated into two morphological and functional domains called grana and stroma lamellae. A large fraction of the luminal volume of the granal thylakoids is occupied by the oxygen-evolving complex of photosystem II. Electron microscopy data we obtained on dark-and light-adapted Arabidopsis thylakoids indicate that the granal thylakoid lumen significantly expands in the light. Models generated for the organization of the oxygen-evolving complex within the granal lumen predict that the light-induced expansion greatly alleviates restrictions imposed on protein diffusion in this compartment in the dark. Experiments monitoring the redox kinetics of the luminal electron carrier plastocyanin support this prediction. The impact of the increase in protein mobility within the granal luminal compartment in the light on photosynthetic electron transport rates and processes associated with the repair of photodamaged photosystem II complexes is discussed.T he primary processes of photosynthesis in cyanobacteria, algae, and higher plants are carried out within flattened vesicles, called thylakoids, which host the molecular complexes that conduct the light-driven reactions of photosynthesis and provide a medium for energy transduction. In higher plants and some green algae, the thylakoids are differentiated into two distinct morphological domains: cylindrical stacked regions ranging between 300 and 600 nm in diameter, coined grana, and unstacked membrane regions that interconnect the grana called stroma lamellae. The photosynthetic protein complexes are unevenly distributed between the two domains: most of photosystem II (PSII) and the major light-harvesting antenna complex II (LHCII) are localized in the appressed regions of the grana, whereas photosystem I (PSI) and ATP synthase are confined to nonappressed membrane regions, which include the stroma lamellae and grana end membranes and margins (refs. 1-4 and references therein).The current study focuses on the thylakoid luminal compartment. This compartment forms a continuous aqueous space encased by the thylakoid membranes, which separate it from the chloroplast stroma. The major inhabitant of the thylakoid lumen in the granal membrane domains is the oxygen-evolving complex (OEC), which stabilizes the manganese catalytic center of PSII and optimizes the ionic environment for water oxidation (ref. 5 and references therein). The very high density of PSII in the granal membranes (6) implies that the space available for movement in the lumen of these thylakoids may be limited, depending on the thickness of this compartment. This impending constraint, in turn, raises the possibility that diffusion of proteins in the granal thylakoid lumen may be modulated by reversible changes in its thickness. Such changes may occur in response to alterations in the light environment. To test this possibility, we ...

The light-harvesting and energy-transducing functions of the chloroplast are performed within an intricate lamellar system of membranes, called thylakoid membranes, which are differentiated into granum and stroma lamellar domains. Using dualaxis electron microscope tomography, we determined the three-dimensional organization of the chloroplast thylakoid membranes within cryo-immobilized, freeze-substituted lettuce (Lactuca sativa) leaves. We found that the grana are built of repeating units that consist of paired layers formed by bifurcations of stroma lamellar sheets, which fuse within the granum body. These units are rotated relative to each other around the axis of the granum cylinder. One of the layers that makes up the pair bends upwards at its edge and fuses with the layer above it, whereas the other layer bends in the opposite direction and merges with the layer below. As a result, each unit in the granum is directly connected to its neighbors as well as to the surrounding stroma lamellae. This highly connected morphology has important consequences for the formation and function of the thylakoid membranes as well as for their stacking/unstacking response to variations in light conditions.

T cells initiate many immune responses through the interaction of their T-cell antigen receptors (TCR) with antigenic peptides bound to major histocompatibility complex (MHC) molecules. This interaction sends a biochemical signal into the T cell by a mechanism that is not clearly understood. We have used quasielastic light scattering (QELS) to show that, in the presence of MHC molecules bound to a full agonist peptide, TCR/peptide-MHC complexes oligomerize in solution to form supramolecular structures at concentrations near the dissociation constant of the binding reaction. The size of the oligomers is concentration dependent and is calculated to contain two to six ternary complexes for the concentrations tested here. This effect is specific as neither molecule forms oligomers by itself, nor were oligomers observed unless the correct peptide was bound to the MHC. These results provide direct evidence for models of T-cell signalling based on the specific assembly of multiple TCR/peptide-MHC complexes in which the degree of assembly determines the extent and qualitative nature of the transduced signal. They may also explain how T cells maintain sensitivity to antigens present in only low abundance on the antigen-presenting cell.

Adaptability of oxygenic photosynthetic organisms to fluctuations in light spectral composition and intensity is conferred by state transitions, short-term regulatory processes that enable the photosynthetic apparatus to rapidly adjust to variations in light quality. In green algae and higher plants, these processes are accompanied by reversible structural rearrangements in the thylakoid membranes. We studied these structural changes in the thylakoid membranes of Arabidopsis thaliana chloroplasts using atomic force microscopy, scanning and transmission electron microscopy, and confocal imaging. Based on our results and on the recently determined three-dimensional structure of higher-plant thylakoids trapped in one of the two major light-adapted states, we propose a model for the transitions in membrane architecture. The model suggests that reorganization of the membranes involves fission and fusion events that occur at the interface between the appressed (granal) and nonappressed (stroma lamellar) domains of the thylakoid membranes. Vertical and lateral displacements of the grana layers presumably follow these localized events, eventually leading to macroscopic rearrangements of the entire membrane network.

Antigen-dependent activation of T lymphocytes requires T cell receptor (TCR)-mediated recognition of specific peptides, together with the MHC molecules to which they are bound. To achieve this recognition in a reasonable time frame, the TCR must scan and discriminate rapidly between thousands of MHC molecules differing from each other only in their bound peptides. Kinetic analysis of the interaction between a TCR and its cognate peptide-MHC complex indicates that both association and dissociation depend heavily on the temperature, indicating the presence of large energy barriers in both phases. Thermodynamic analysis reveals changes in heat capacity and entropy that are characteristic of protein-ligand associations in which local folding is coupled to binding. Such an ''induced-fit'' mechanism is characteristic of sequence-specific DNA-binding proteins that must also recognize specific ligands in the presence of a high background of competing elements. Here, we propose that induced fit may endow the TCR with its requisite discriminatory capacity and suggest a model whereby the loosely structured antigen-binding loops of the TCR rapidly explore peptide-MHC complexes on the cell surface until some critical structural complementarity is achieved through localized folding transitions. We further suggest that conformational changes, implicit in this model, may also propagate beyond the TCR antigen-binding site and directly affect self-association of ligated TCRs or TCR-CD3 interactions required for signaling.

The energy landscape of proteins is thought to have an intricate, corrugated structure. Such roughness should have important consequences on the folding and binding kinetics of proteins, as well as on their equilibrium fluctuations. So far, no direct measurement of protein energy landscape roughness has been made. Here, we combined a recent theory with single-molecule dynamic force spectroscopy experiments to extract the overall energy scale of roughness e for a complex consisting of the small GTPase Ran and the nuclear transport receptor importin-b. The results gave e45k B T, indicating a bumpy energy surface, which is consistent with the ability of importin-b to accommodate multiple conformations and to interact with different, structurally distinct ligands.