Monolayers of molecules, which retain their function in the adsorbed state on solid surfaces, are important in materials science, analytical detection, and other technology approaching the nanoscale. Molecular monolayers, including layers of functional biological macromolecules, offer new insight in electronic properties and stochastic single-molecule features and can be probed by new methods which approach the single-molecule level. One of these is in situ scanning tunneling microscopy (STM) in which single-molecule electronic properties directly in aqueous solution are probed. In situ STM combined with physical electrochemistry, single-crystal electrodes, and spectroscopic methods is now a new dimension in interfacial bioelectrochemistry. We overview first some approaches to spectroscopic single-molecule imaging, including fluorescence spectroscopy, chemical reaction dynamics, atomic force microscopy, and electrochemical single-electron transfer. We then focus on in situ STM. In addition to high structural resolution, in situ STM offers a single-molecule spectroscopic perspective. This emerges most clearly when adsorbate molecules contain accessible redox levels, and the tunneling current decomposes into successive single-molecule interfacial electron transfer (ET) steps. Theories of electrochemical ET and in situ STM of redox molecules as well as specific cases are addressed. Two-step in situ STM represents different molecular mechanisms and even new ET phenomena, related to coherent many-electron transfer. A number of systems are noted to accord with these views. The discussion is concluded by attention to one of the still very few redox proteins addressed by in situ STM, the blue copper protein Pseudomonas aeruginosa azurin. Use of comprehensive electrochemical techniques has ascertained that well-defined protein monolayers in two opposite orientations can be formed and interfacial tunneling patterns disclosed. P. aeruginosa azurin emerges as by far the most convincing case where in situ STM of functional metalloproteins to single-molecule resolution has been achieved. This comprehensive approach holds promise for broader use of in situ STM as a single-molecule spectroscopy of metalloproteins and illuminates prerequisites and limitations of in situ STM of biological macromolecules.

Extracellular polymeric substances play important roles in microbial extracellular electron transfer processes.

Profilin, an essential G-actin-binding protein, has two opposite regulatory functions in actin filament assembly. It facilitates assembly at the barbed ends by lowering the critical concentration (Pantaloni, D., and Carlier, M.-F. (1993) Cell 75, 1007-1014); in contrast it contributes to the pool of unassembled actin when barbed ends are capped. We proposed that the first of these functions required an input of energy. How profilin uses the ATP hydrolysis that accompanies actin polymerization and whether the acceleration of nucleotide exchange on G-actin by profilin participates in its function in filament assembly are the issues addressed here. We show that 1) profilin increases the treadmilling rate of actin filaments in the presence of Mg 2؉ ions; 2) when filaments are assembled from CaATP-actin, which polymerizes in a quasireversible fashion, profilin does not promote assembly at the barbed ends and has only a Gactin-sequestering function; 3) plant profilins do not accelerate nucleotide exchange on G-actin, yet they promote assembly at the barbed end. The enhancement of nucleotide exchange by profilin is therefore not involved in its promotion of actin assembly, and the productive growth of filaments from profilin-actin complex requires the coupling of ATP hydrolysis to profilin-actin assembly, a condition fulfilled by Mg-actin, and not by Ca-actin.Living cells undergo changes in shape and motile behavior by spatially and temporally controlled rearrangements of the actin cytoskeleton. In the physiological ionic conditions, F-actin is assembled at steady state in the cell medium. Changes in the F-actin/G-actin ratio, which occur in response to stimuli, are made possible by shifts in steady state, i.e. changes in the critical concentration for filament assembly. These changes are elicited by capping proteins and profilin (1, 2) and amplified by G-actin-binding proteins (3). A high level of capping of barbed ends maintains the high critical concentration of pointed ends in the cytoplasm. A steep energetic gradient is therefore created between the cell medium and the loci where uncapped barbed ends are nucleated at the plasma membrane. We understand that in this way capping of barbed ends in the cytoplasm is required for a more efficient local actin assembly. In support of this view, recent evidence indeed indicates that the level of motility in fibroblasts (4) and Dictyostelium (5) correlates with the level of barbed end capping. Similarly, the actinbased propulsive movement of Listeria results from the local creation and maintenance of new uncapped barbed ends at the bacterium surface, while filaments are capped in the bulk cytoplasm (6).Profilin has unique properties among G-actin-binding proteins. Under physiological ionic conditions (Mg-actin, 0.1 M KCl), it binds G-actin tightly (K ϭ 10 7 M Ϫ1 for vertebrate profilin I) and participates in the establishment of the pool of unassembled actin when barbed ends are capped. In contrast, when barbed ends are uncapped, the participation of profilinactin complex in...

The background to the concept of the term "molten globule" as a description of intermediates observed in the folding of globular proteins is discussed. These compact intermediates are characterised by certain properties including the presence of secondary structure and considerable conformational mobility compared to the native, functional state. Those intermediates that are thermodynamically stable under mild denaturing conditions have many features in common with the transient intermediates that accumulate significantly during the process of folding. Attention is drawn to cases where the two types are however distinguished on grounds of their Stokes radius, in which cases there is currently no direct evidence for the involvement of the stable intermediates on the folding pathway. Experimental evidence relating to the early stages in folding is reviewed and compared, highlighting the temporal relationship between general collapse of the polypeptide chain and the formation of secondary structure. The continued use of the term "molten globule" is recommended where the minimum essential structural criteria for this state are met.