Upon transfer from strongly denaturing to native conditions, proteins undergo a collapse that either precedes folding or occurs simultaneously with it. This collapse is similar to the well known coil-globule transition of polymers. Here we employ singlemolecule fluorescence methods to fully characterize the equilibrium coil-globule transition in the denatured state of the IgGbinding domain of protein L. By using FRET measurements on freely diffusing individual molecules, we determine the radius of gyration of the protein, which shows a gradual expansion as the concentration of the denaturant, guanidinium hydrochloride, is increased all the way up to 7 M. This expansion is observed also in fluorescence correlation spectroscopy measurements of the hydro- fluorescence correlation spectroscopy ͉ protein folding ͉ single-molecule fluorescence T he coil-globule (CG) transition is a hallmark of the physics of polymers in solution. When a polymer molecule is transferred from a good solvent to a bad one, it undergoes a collapse from an expanded coil-like conformation to a contracted, globule-like conformation. This collapse is typically a second-order phase transition and can be accounted well by mean-field theory (1, 2). Proteins are heteropolymers and therefore should exhibit a similar transition. Because proteins also undergo a first-order folding transition to form their native structure, it is of prime interest to deduce the relation between collapse and folding (3). If the CG transition precedes folding significantly, then the final rearrangements of the protein chain to form the native structure occur within a relatively limited configurational space, which might affect the efficiency and speed of the process. The CG transition of proteins is also important for the elucidation of the properties of their denatured states. Chain collapse can have an impact on secondary-structure formation in the denatured protein (4). Determining the energetics of the collapse, which involve changes in the solvation energy of the protein as the solution conditions are varied, might allow us to understand better the role of the denatured state in the folding transition (5).The occurrence of a collapse preceding protein folding was inferred from kinetic experiments (6-11) and also reported in equilibrium experiments, using small-angle x-ray scattering (SAXS) (12) and single-molecule fluorescence (13,14). However, the thermodynamics of the CG transition have not been characterized yet. In this work, we use two methods, singlemolecule FRET (smFRET) and fluorescence correlation spectroscopy (FCS), to measure the CG transition of a 64-amino acid protein exhibiting two-state folding, the IgG-binding domain of protein L (hereafter denoted simply as protein L) (15). The CG transition is driven by the chemical denaturant guanidinium hydrochloride (GuHCl). We then employ a version of the mean-field theory of Sanchez (16) to obtain a full thermodynamic characterization of the CG transition of a protein, identifying the transition point as well ...

Receptor-regulated cellular signaling often is mediated by formation of transient, heterogeneous protein complexes of undefined structure. We used single and two-color photoactivated-localization microscopy (PALM) to study complexes downstream of the T cell antigen receptor (TCR) in single molecule detail at the plasma membrane of intact T cells. The kinase ZAP-70 distributed completely with the TCRζ chain and both partially mixed with the adapter LAT in activated cells thus showing localized activation of LAT by TCR-coupled ZAP-70. In resting and activated cells LAT primarily resided in nanoscale clusters as small as dimers whose formation depended on protein-protein and protein-lipid interactions. Surprisingly, the adapter SLP-76 localized to the periphery of LAT clusters. This nanoscale structure depended on polymerized actin and its disruption affected TCR-dependent cell function. These results extend our understanding of the mechanism of T cell activation and the formation and organization of TCR-mediated signaling complexes, findings also relevant to other receptor systems.

The adapter molecule LAT is a nucleating site for multiprotein signaling complexes that are vital for the function and differentiation of T cells. Extensive investigation of LAT in multiple experimental systems has led to an integrated understanding of the formation, composition, regulation, dynamic movement, and function of LAT-nucleated signaling complexes. This review discusses interactions of signaling molecules that bind directly or indirectly to LAT and the role of cooperativity in stabilizing LAT-nucleated signaling complexes. In addition, it focuses on how imaging studies visualize signaling assemblies as signaling clusters and demonstrate their dynamic nature and cellular fate. Finally, this review explores the function of LAT based on the interpretation of mouse models using various LAT mutants.

Studies of protein-protein interactions, carried out in polymer solutions, are designed to mimic the crowded environment inside living cells. It was shown that crowding enhances oligomerization and polymerization of macromolecules. Conversely, we have shown that crowding has only a small effect on the rate of association of protein complexes. Here, we investigated the equilibrium effects of crowding on protein heterodimerization of TEM1-beta-lactamase with beta-lactamase inhibitor protein (BLIP) and barnase with barstar. We also contrasted these with the effect of crowding on the weak binding pair CyPet-YPet. We measured the association and dissociation rates as well as the affinities and thermodynamic parameters of these interactions in polyethylene glycol and dextran solutions. For TEM1-BLIP and for barnase-barstar, only a minor reduction in association rate constants compared to that expected based on solution viscosity was found. Dissociation rate constants showed similar levels of reduction. Overall, this resulted in a binding affinity that is quite similar to that in aqueous solutions. On the other hand, for the CyPet-YPet pair, aggregation, and not enhanced dimerization, was detected in polyethylene glycol solutions. The results suggest that typical crowding agents have only a small effect on specific protein-protein dimerization reactions. Although crowding in the cell results from proteins and other macromolecules, one may still speculate that binding in vivo is not very different from that measured in dilute solutions.

Bacteria display an array of contact-dependent interaction systems that have evolved to facilitate direct cell-to-cell communication. We have previously identified a mode of bacterial communication mediated by nanotubes bridging neighboring cells. Here, we elucidate nanotube architecture, dynamics, and molecular components. Utilizing Bacillus subtilis as a model organism, we found that at low cell density, nanotubes exhibit remarkable complexity, existing as both intercellular tubes and extending tubes, with the latter frequently surrounding the cells in a "root-like" fashion. Observing nanotube formation in real time showed that these structures are formed in the course of minutes, displaying rapid movements. Utilizing a combination of super-resolution, light, and electron microscopy, we revealed that nanotubes are composed of chains of membranous segments harboring a continuous lumen. Furthermore, we discovered that a conserved calcineurin-like protein, YmdB, presents in nanotubes and is required for both nanotube production and intercellular molecular trade.