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.

The folding of the all-beta sheet protein, interleukin-1 beta, was studied with nuclear magnetic resonance (NMR) spectroscopy, circular dichroism, and fluorescence. Ninety percent of the beta structure present in the native protein, as monitored by far-ultraviolet circular dichroism, was attained within 25 milliseconds, correlating with the first kinetic phase determined by tryptophan and 1-anilinonaphthalene-8-sulfonate fluorescence. In contrast, formation of stable native secondary structure, as measured by quenched-flow deuterium-hydrogen exchange experiments, began after only 1 second. Results from the NMR experiments indicated the formation of at least two intermediates with half-lives of 0.7 to 1.5 and 15 to 25 seconds. The final stabilization of the secondary structure, however, occurs on a time scale much greater than 25 seconds. These results differ from previous results on mixed alpha helix-beta sheet proteins in which both the alpha helices and beta sheets were stabilized very rapidly (less than 10 to 20 milliseconds).

Profilin (PFN) is an ubiquitous, low-M r , actin-binding protein involved in the organization of the cytoskeleton of eukaryotes including higher plants. PFNs are encoded by a multigene family in Arabidopsis. We have analyzed in vivo functions of Arabidopsis PFN by generating transgenic plants carrying a 35S-PFN-1 or 35S-antisense PFN-1 transgene. Etiolated seedlings underexpressing PFN (PFN-U) displayed an overall dwarf phenotype with short hypocotyls whose lengths were 20% to 25% that of wild type (WT) at low temperatures. Light-grown PFN-U plants were smaller in stature and flowered early. Compared with equivalent cells in WT, most cells in PFN-U hypocotyls and roots were shorter, but more isodiametric, and microscopic observations of etiolated PFN-U hypocotyls revealed a rough epidermal surface. In contrast, light-grown seedlings overexpressing PFN had longer roots and root hair although etiolated seedlings overexpressing PFN were either the same size or slightly longer than WT seedlings. Transgenic seedlings harboring a PFN-1-GUS transgene directed expression in root and root hair and in a ring of cells at the elongating zone of the root tip. As the seedlings matured PFN-1-GUS was mainly expressed in the vascular bundles of cotyledons and leaves. Our results show that Arabidopsis PFNs play a role in cell elongation, cell shape maintenance, polarized growth of root hair, and unexpectedly, in determination of flowering time.

This first structure of the enzyme converting dopamine to norepinephrine provides new perspectives on numerous disorders.

A model describing conformational exchange of His 61 in plastocyanin from Anabaena variabilis is presented. A detailed picture of the exchange dynamics has been obtained using solution NMR relaxation measurements, chemical shift titrations, and structural information provided by a high-resolution crystal structure of the protein. A three-site model for chemical exchange that involves interconversion among the tautomeric and protonated forms of the histidine side chain with rates that are fast on the NMR chemical shift time scale can account for all of the data. In general, in the limit of fast exchange, it is not possible to obtain separate measures of chemical shift differences and populations of the participating states using NMR. However, we show here that when the data mentioned above are combined, it is possible to extract values of all of the parameters that characterize the exchange process, including rates, populations, and chemical shift changes, and to provide cross-validations that establish their accuracy. The methodology is generally applicable to the study of histidine side chain dynamics, which can play an important functional role in many protein systems.