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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.

Proteins and nucleic acids fold and behave in a highly occupied matrix of macromolecules, the cellular environment. Although this environment is filled up to a volume of 40% with macromolecules, the effect of crowding on biochemical reactions as well as biophysical properties of proteins has been rarely considered. Therefore, studies on macromolecular crowding are mainly conducted using artificially created polymer based substances as mimics of the in cell environment. Still, a common concept how the excluded volume effect in the cell affects protein folding, function and structure is lacking. Understanding these properties will be important to unravel the underlying mechanism of protein misfolding and aggregation as well as the behavior of intrinsically disordered proteins in a living cell.We introduce a FRET based random coil polymer to characterize crowding both in vitro and inside a living cell. We find different compression of the polymer in artificial crowding substances compared to protein crowders such as BSA or oocyte lysate. Injection of the probe inside cells confirms this result and reveals a heterogeneous environment which, on average, shows comparable polymer conformations as in diluted buffer. The polymer conformation is used to quantify crowding differences in the cytosol and the nucleus as well as to identify crowding in cells influenced by different extrinsic conditions. Severe osmotic stress is used to probe compression of the polymer in the highly concentrated cytosolic environment.We conclude that the FRET labelled polymer provides a new approach to investigate and characterize the cellular solvation properties with high spatio-temporal resolution in a variety of systems and identify crowding differences due to the architecture of the cellular matrix. Therefore, it will help to understand how the cell, as the native environment in which proteins evolved, might modulate and tune biomolecule properties and functions.