Physicochemical code for quinary protein interactions in Escherichia coli

This study shows that the diffusive motions of proteins in live cells are by no means without control but follow simplistic physical−chemical rules that can be quantified and optimized through surface composition. Most strikingly, human proteins are observed to stick to the “foreign” environment of bacterial cells, whereas the bacterial analogue moves around freely. Even so, the human proteins can predictably be transformed to bacterial behavior with a few structurally benign surface mutations, and, conversely, the bacterial protein can be made to stick. The findings have not only fundamental implications for how protein function is controlled at the physical−chemical level but can also be used to adjust protein motion in Escherichia coli at will

Alternative Explanations for "Multistate" Kinetics in Protein Folding: Transient Aggregation and Changing Transition-State Ensembles

Deviations from classical two-state kinetics in protein folding need not always be explained by the presence of rapidly formed intermediates. In some cases, such deviations are caused by short-lived aggregates whereas in other cases they arise from changes of the position of the transition state. These are two new facets of the mechanism of two-state folding. The first part of this account describes the effect of aggregates which form transiently in the first few milliseconds of the refolding reaction. The aggregates show many similarities with folding intermediates, but may be identified by their disappearance at low concentrations of protein where the two-state conversion of monomeric protein becomes predominant. In the second part, the focus is directed to two-state folding and movements of the transition state ensemble. The movements are used to derive information about the shape of the free-energy profile for folding. It emerges from a comparison of the kinetic behaviour of several small model proteins that the free-energy barrier for folding could be generally broad and level. An attractive feature of broad barriers is that, depending on minor variations in the fine-structure of the free-energy profile, they account for a wide range of seemingly unrelated folding data, including deviations from classical two-state kinetics determined by free-energy extrapolations

High-energy channeling in protein folding

Recent controversy about the role of populated intermediates in protein folding emphasizes the need to better characterize other events on the folding pathway. A complication is that these involve high-energy states which are difficult to target experimentally since they do not accumulate kinetically. Here, we explore the energetics of high-energy states and map out the shape of the free-energy profile for folding of the two-state protein U1A. The analysis is based on nonlinearities in the GdnHCl dependence of the activation energy for unfolding, which we interpret in terms of structural changes of the protein-folding transition state. The result suggests that U1A folds by high-energy channeling where most of the conformational search takes place isoenergetically at transition-state level. This is manifested in a very broad and flat activation barrier, the top of which covers more than 60% of the reaction coordinate. The interpretation favors a folding mechanism where the pathway leading to the native protein is determined by the sequence's ability to stabilize productive transitio

On the osmotic pressure of cells

The chemical potential of water (//h2o) provides an essential thermodynamic characterization of the environment of living organisms, and it is of equal significance as the temperature. For cells, //H,0 is conventionally expressed in terms of the osmotic pressure. We have previously suggested that the main contribution to the intracellular osmotic pressure of the bacterium E. coli is from soluble negatively-charged proteins and their counter-ions (1). Here, we expand on this analysis by examining how evolutionary divergent cell types cope with the challenge of maintaining the osmotic pressure within viable values. Complex organisms, like mammals, maintain constant internal osmotic pressure of around 0.285 osmol, matching that of 0.154 M NaCl. For bacteria it appears that optimal growth conditions are found for similar or slightly higher osmotic pressures (0.25 - 0.4 osmol), despite that they represent a much earlier stage in evolution. We argue that this value reflects a general adaptation for optimising metabolic function under crowded intracellular conditions. Environmental osmotic pressures that differ from this optimum require therefore special measures, as exemplified with gram-positive and gram-negative bacteria. To handle such situations, their specific membrane encapsulations allow for a compensating turgor pressure that can take both positive and negative values. Moreover, the establishment of a positive turgor pressure allows increased frequency of metabolic events through increased intracellular protein concentrations. A remarkable exception to the rule of 0.25 - 0.4 osmol, is found for halophilic archaea with internal osmotic pressures around 15 osmol. Tlie internal organization of these archaea differs in that they utilize a repulsive electrostatic mechanism operating only in the ionic-liquid regime to avoid aggregation, and that they stand out from other organisms by having no turgor pressure

Transient aggregates in protein folding are easily mistaken for folding intermediates

It has been questioned recently whether populated intermediates are important for the protein folding process or are artefacts trapped in nonproductive pathways. We report here that the rapidly formed intermediate of the spliceosomal protein U1A is an off-pathway artefact caused by transient aggregation of denatured protein under native conditions. Transient aggregates are easily mistaken for structured monomers and could be a general problem in time-resolved folding studies