In-cell thermodynamics and a new role for protein surfaces.

There is abundant, physiologically relevant knowledge about protein cores; they are hydrophobic, exquisitely well packed, and nearly all hydrogen bonds are satisfied. An equivalent understanding of protein surfaces has remained elusive because proteins are almost exclusively studied in vitro in simple aqueous solutions. Here, we establish the essential physiological roles played by protein surfaces by measuring the equilibrium thermodynamics and kinetics of protein folding in the complex environment of living Escherichia coli cells, and under physiologically relevant in vitro conditions. Fluorine NMR data on the 7-kDa globular N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) show that charge-charge interactions are fundamental to protein stability and folding kinetics in cells. Our results contradict predictions from accepted theories of macromolecular crowding and show that cosolutes commonly used to mimic the cellular interior do not yield physiologically relevant information. As such, we provide the foundation for a complete picture of protein chemistry in cells.

Local chemistry of the surfactant's head groups determines protein stability in reverse micelles.

When comparing protein folding in vitro and in vivo significant differences have been found. This has been attributed to crowding and confinement effects. Using a combination of GHz- and THz-dielectric relaxation spectroscopy and MD simulations, we studied hydration dynamics and reviewed protein stability data inside sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and cetyltrimethylammonium bromide (CTAB) reverse micelles which are model systems for confinement. We find that water inside anionic AOT and cationic CTAB reverse micelles is characterized by a strong dielectric depolarization giving rise to a very low relative permittivity compared to an unconfined solution. Despite differences in the hydration dynamics of the surfactant's head groups, simulations using the two-phase thermodynamics method predict a similar reduction in water entropy for both reverse micelle systems compared to bulk water. When we compare the stability data of proteins in these reverse micelles we find that in contrast to our initial expectation, protein stability correlates rather with the local chemistry of the hydrated head groups than with the excluded volume effect or the low global permittivity.

The temperature dependence of the Hofmeister series: thermodynamic fingerprints of cosolute-protein interactions.

The Hofmeister series is a universal homologous series to rank ion-specific effects on biomolecular properties such as protein stability or aggregation propensity. Although this ranking is widely used, outliers and exceptions are discussed controversially and a molecular level understanding is still lacking. Studying the thermal unfolding equilibrium of RNase A, we here show that this ambiguity arises from the oversimplified approach to determine the ion rankings. Instead of measuring salt effects on a single point of the protein folding stability curve (e.g. the melting point Tm), we here consider the salt induced shifts of the entire protein 'stability curve' (the temperature dependence of the unfolding free energy change, ΔGu(T)). We found multiple intersections of these curves, pinpointing a widely ignored fact: the Hofmeister cation and anion rankings are temperature dependent. We further developed a novel classification scheme of cosolute effects based on their thermodynamic fingerprints, reaching beyond salt effects to non-electrolytes.

Circular trimers of gelatinase B/matrix metalloproteinase-9 constitute a distinct population of functional enzyme molecules differentially regulated by tissue inhibitor of metalloproteinases-1.

Gelatinase B/matrix metalloproteinase-9 (MMP-9) (EC 3.4.24.35) cleaves many substrates and is produced by most cell types as a zymogen, proMMP-9, in complex with the tissue inhibitor of metalloproteinases-1 (TIMP-1). Natural proMMP-9 occurs as monomers, homomultimers and heterocomplexes, but our knowledge about the overall structure of proMMP-9 monomers and multimers is limited. We investigated biochemical, biophysical and functional characteristics of zymogen and activated forms of MMP-9 monomers and multimers. In contrast with a conventional notion of a dimeric nature of MMP-9 homomultimers, we demonstrate that these are reduction-sensitive trimers. Based on the information from electrophoresis, AFM and TEM, we generated a 3D structure model of the proMMP-9 trimer. Remarkably, the proMMP-9 trimers possessed a 50-fold higher affinity for TIMP-1 than the monomers. In vivo, this finding was reflected in a higher extent of TIMP-1 inhibition of angiogenesis induced by trimers compared with monomers. Our results show that proMMP-9 trimers constitute a novel structural and functional entity that is differentially regulated by TIMP-1.

Protein Stability in Reverse Micelles.

The N-terminal SH3 domain of the Drosophila signal transduction protein drk was encapsulated in reverse micelles. Both the temperature of maximum stability and the melting temperature decreased on encapsulation. Dissecting the temperature-dependent stability into enthalpic and entropic contributions reveals a stabilizing enthalpic and a destabilizing entropic contribution. These results do not match the expectations of hard-core excluded volume theory, nor can they be wholly explained by interactions between the head groups in the reverse micelle and the test protein. We suggest that geometric constraints imposed by the reverse micelles need to be considered.

Protein stabilization by macromolecular crowding through enthalpy rather than entropy.

The interior of the cell is a densely crowded environment in which protein stability is affected differently than in dilute solution. Macromolecular crowding is commonly understood in terms of an entropic volume exclusion effect based on hardcore repulsions among the macromolecules. We studied the thermal unfolding of ubiquitin in the presence of different cosolutes (glucose, dextran, poly(ethylene glycol), KCl, urea). Our results show that for a correct dissection of the cosolute-induced changes of the free energy into its enthalpic and entropic contributions, the temperature dependence of the heat capacity change needs to be explicitly taken into account. In contrast to the prediction by the excluded volume theory, we observed an enthalpic stabilization and an entropic destabilization for glucose, dextran, and poly(ethylene glycol). The enthalpic stabilization mechanism induced by the macromolecular crowder dextran was similar to the enthalpic stabilization mechanism of its monomeric building block glucose. In the case of poly(ethylene glycol), entropy is dominating over enthalpy leading to an overall destabilization. We propose a new model to classify cosolute effects in terms of their enthalpic contributions to protein stability.

Templating effects in aristolochene synthase catalysis: elimination versus cyclisation.

Analysis of the products generated by mutants of aristolochene synthase from P. roqueforti (PR-AS) revealed the prominent structural role played by the aliphatic residue Leu 108 in maintaining the productive conformation of farnesyl diphosphate to ensure C1-C10 (σ-bond) ring-closure and hence (+)-aristolochene production.