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A topic that has attracted considerable interest in recent years is the possibility to perform thermodynamic studies of proteins directly in-cell or in complex environments which mimic the cellular interior. Nuclear magnetic resonance (NMR) could be an attractive technique for these studies but its applicability has so far been limited by technical issues. Here, we demonstrate that 2D NMR methods can be successfully applied to measure thermodynamic parameters provided that a suitable choice of the residues used for the calculation is made. We propose a new parameter, named RAD, which reflects the level of protection of a specific amide proton in the protein core and can guide through the selection of the resonances. We also suggest a way to calibrate the volumes to become independent of technical limitations. The methodology we propose leads to stability curves comparable to that calculated from CD data and provides a new tool for thermodynamic measurements in complex environments. Multidimensional NMR spectroscopy can provide insight into the unfolding of proteins in cells, but may be sensitive to the choice of residue used as a reference. Here 2D 1H-15N NMR is used to obtain thermodynamic parameters of unfolding of Yfh1, relying on an internal standard to normalise peak volumes.

To improve our mechanistic understanding of zinc metalloenzymes, we report a joint computational and experimental study of a minimal carbonic anhydrase (CA) mimic, a 22-residue Zn-finger hydrolase. We combine classical molecular dynamics (MD) simulations, quantum mechanics/molecular mechanics (QM/MM) geometry optimizations, and QM/MM free energy simulations with ambient and high-pressure kinetic measurements to investigate the mechanism of the hydrolysis of the substrate p-nitrophenylacetate (pNPA). The zinc center of the hydrolase prefers a pentacoordinated geometry, as found in most naturally occurring CAs and CA-like enzymes. Two possible mechanisms for the catalytic reaction are investigated. The first one is analogous to the commonly accepted mechanism for CA-like enzymes: a sequential pathway, in which a Zn2+-bound hydroxide acts as a nucleophile and the hydrolysis proceeds through a tetrahedral intermediate. The initial rate-limiting step of this reaction is the nucleophilic attack of the hydroxide...

Changes of the extracellular milieu could affect cellular crowding. To prevent detrimental effects, cells use adaptation mechanisms to react to such conditions. Using fluorescent crowding sensors, we show that the initial response to osmotic stress is fast but imperfect, while the slow response renders cells more tolerant to stress, particularly in the presence of osmolytes.

Here, we study the stabilities of different naturally fragile mutants of 3' phosphoadenosine 5' phosphosulfate (PAPS) synthases. The goal is to establish a relationship between the stability of the mutants in the cellular environment and their enzymatic activity. In vitro stability measurements have suggested that protein stability is a major factor for cellular PAPS availability [1]. We aim to specifically analyze different cellular factors for in-cell PAPSS stability. One crucial factor is the macromolecular crowding effect. The cell is filled up to a volume of 40 % with macromolecules. Often, artificial macromolecular crowding agents are used to mimic these conditions. We previously studied macromolecular crowding effects via a thermodynamic analysis of the thermal unfolding of ubiquitin [2]. We observed enthalpic stabilization and entropic destabilization forces for all tested crowders. Further, we tested how such artificial cosolutes reflect the physicochemical properties of the complex cellular environment. Therefore, we developed a FRET-based macromolecular crowding sensor to study the crowding effect in living cells [3]. We found that the in-cell crowding effect is distributed heterogeneously and can change significantly upon osmotic stress. In comparison, we now used Fast Relaxation Imaging [4] to study the stability of PAPSS within the cellular environment and compare the results to in vitro crowding studies.References:1. J. Boom, D. Heider, S.R. Martin, A. Pastore, and J.W. Mueller. PAPS synthases - naturally fragile enzymes specifically stabilized by nucleotide binding., J. Biol. Chem. 2012, 287(21):17645-55.2. M. Senske, L. Tork, B. Born, M. Havenith, C. Herrmann, S. Ebbinghaus, J. Am. Chem. Soc. 2014, 136, 9036-9041.3. D. Gnutt, M. Gao, O. Brylski, M. Heyden, S. Ebbinghaus, Angew. Chem Int. Ed. 2015, 54(8):2548-2551.4. S. Ebbinghaus, A. Dhar, J.D. McDonald and M. Gruebele. “Protein folding stability and dynamics imaged in a living cell.” Nature Methods 2010, 7:319-323.

PAPS synthases are bifunctional enzymes providing the cell with the sulfate donor PAPS (3'-phosphoadenosine-5′-phosphosulfate), which is further used by sulfotransferases for modification of several biomolecules (e.g. steroids). Isoform PAPS synthase 2 (PAPSS2) has been shown to be fragile within in vitro experiments, but is stabilized by binding of its endogenous ligands. Mutations affecting its activity lead to disease states like bone and cartilage malformation as well as metabolic diseases. Compared to in vitro conditions, the cellular milieu is crowded by large biopolymers resulting in intermolecular interactions and excluded-volume effects affecting each biopolymer inside the cell.To understand the stability of this large enzyme (70 kDa), we conducted biophysical studies on the individual domains, APS kinase and ATP sulfurylase, as well as the full length protein. Using Fast Relaxation Imaging we studied disease-relevant mutants of PAPSS2 directly within the cell. The data provide novel thermodynamic insights into PAPSS2 stability inside the cellular milieu and its influence on the naturally destabilized protein. These results also unravel new insights into disease mechanisms of PAPSS2 mutations.