Subcellular modulation of protein VlsE stability and folding kinetics

Abstract

The interior of a cell interacts differently with proteins than a dilute buffer because of a wide variety of macromolecules, chaperones, and osmolytes that crowd and interact with polypeptide chains. We compare folding of fluorescent constructs of protein VlsE among three environments inside cells. The nucleus increases the stability of VlsE relative to the cytoplasm, but slows down folding kinetics. VlsE is also more stable in the endoplasmic reticulum, but unlike PGK, tends to aggregate there. Although fluorescent-tagged VlsE and PGK show opposite stability trends from in vitro to the cytoplasm, their trends from cytoplasm to nucleus are similar.Keywords: cellular compartments; folding kinetics; protein stability; quinary interactionThe physical environment surrounding a protein plays an important role in shaping its folding landscape [1][2][3]. Hence, temperature, crowding, denaturants, and osmolytes are often utilized to study the folding of proteins [2,4,5]. These factors can even perturb the landscape to yield alternative global free energy minima, thus causing some proteins to adopt more than one native structure [4,6].The native environment of many proteins, the interior of a cell, is very different from a dilute buffer. The cell promotes crowding and 'quinary ' (transient and weak) interactions [7]. Recent attempts to replicate the cellular environment have relied on the addition of inert macromolecules, including polyethylene glycol, ficoll, and dextran [4,8,9]. While the use of these crowders has revealed much about the stabilizing effects of crowding, it does not fully mimic the cellular environment [10,11]. Thus, there is still much to be learned about the balance between crowding (stabilizing) and quinary interactions (sometimes destabilizing) inside cells [12,13].Experiments in mammalian cells using two different F€ orster Resonance Energy Transfer (FRET)-labeled Abbreviations FRET, F€ orster resonance energy transfer; NLS, nuclear localization signal; DMEM, Dulbecco's modified Eagle's medium; ER, endoplasmic reticulum; FBS, fetal bovine serum; FReI, fast relaxation imaging.

Non‐Steric Interactions Predict the Trend and Steric Interactions the Offset of Protein Stability in Cells

“…The melting temperatures of VlsE and PGK in sodium phosphate buffer are 40±1 °C and 39±1 °C, respectively. These are consistent with past measurements of VlsE, 39±1 °C, and PGK, 38±1 °C . The change in the melting temperature as a function of cell lysate and Ficoll concentration is reported in Figure .…”

“…Non‐steric interactions can be stabilizing or destabilizing . Thus steric and non‐steric interactions can compete in a tug‐of‐war for in‐cell protein stability: phosphoglycerate kinase (PGK) is stabilized, lambda repressor is unchanged, and superoxide dismutase (SOD1), variable major protein‐like sequence expressed (VlsE),, B1 domain of protein G (GB1), and chymotrypsin inhibitor 2 (Cl2) are destabilized in a manner that depends on protein surface mutations, cell pH, and cell type.…”

“…This is compared to thermal stability in 10 mM potassium phosphate buffer +200 mM KCl pH 7 (K, ▴), lysis buffer (L, ▪ (black)), lysis buffer+300 mg/ml Ficoll PM 70 (▪ (blue)), and living U‐2 OS cells (U, ⧫). In cell VlsE data is from reference and in cell PGK data is from reference [15,24] . Melting temperatures (Tm) were extracted from a two‐state model (Equation S1) fit of the thermal unfolding curves.…”

An in vitro mimic of in‐cell solvation for protein folding studies

“…Yeast PGK (Figure 1a) and Borrelia burgdorferi VlsE (Figure 1b) were selected as model proteins for these studies because they have opposite stability trends when moved from in vitro into cells. In sodium phosphate buffer, PGK and VlsE have similar melting temperatures, T m = 39 ± 1°C and T m = 40 ± 1°C, respectively . However, inside U‐2 OS cells PGK is stabilized, T m = 44 ± 2°C, and VlsE is destabilized, T m = 35 ± 2°C .…”

Crowding in Cellular Environments at an Atomistic Level from Computer Simulations

“…17,22,23,73,92,93 Largely unaltered conformations in vivo vs in vitro are seen with in-cell solution NMR 40,44 and solid-state (SS) NMR, 54 but other evidence from experiments and simulations indicates that cellular environments may remodel the folded or unfolded ensemble or both 16,17,49,71,94−100 with potentially varying effects in different cellular compartments. 82 This suggests a more complicated picture where it is unclear whether remodeling of the ensemble is again driven mostly by entropic volume exclusion effects or by more enthalpic effects due to interactions with protein crowders and other components of the biological environment. 16,17 Significant crowding effects are also expected for intrinsically disordered peptides (IDPs) where extended and dynamic ensembles have to be accommodated under crowded environments.…”