All atom insights into the impact of crowded environments on protein stability by NMR spectroscopy

The high density of macromolecules affecting proteins due to volume exclusion has been discussed in theory but numerous in vivo experiments cannot be sufficiently understood taking only pure entropic stabilization into account. Here, we show that the thermodynamic stability of a beta barrel protein increases equally at all atomic levels comparing crowded environments with dilute conditions by applying multidimensional high-resolution NMR spectroscopy in a systematic manner. Different crowding agents evoke a pure stabilization cooperatively and do not disturb the surface or integrity of the protein fold. The here developed methodology provides a solid base that can be easily expanded to incorporate e.g. binding partners to recognize functional consequences of crowded conditions. Our results are relevant to research projects targeting soluble proteins in vivo as it can be anticipated that their thermodynamic stability increase comparably and has consequently to be taken into account to coherently understand intracellular processes.

reports on both the native state as well as the unfolded protein ensemble. Thus, the fraction of native protein, fn, can be reliably calculated as presented in Figure 2A, B and reported in Supplementary Table 1.        Determining overall thermodynamic stability of BsCspB monitored via following folding-to-unfolding transitions induced by urea using peak height or peak volume in two-dimensional NMR spectra focusing on 13 structurally well separated cross-peaks. Increase in the transition midpoint, CM, of amide protons observed in two-dimensional 1 H-15 N HSQC NMR spectra comparing dilute conditions with the presence of c = 120 g/L Dex20 analyzing peak heights (A) or peak volumes (B). Beta sheet regions comprising BsCspB according to PDB ID 1NMG are indicated by using a background colored in gray. The mean of CM is shown         Numerical results for this data fitting are provided in Supplementary Table 3.   Table 5.
Supplementary Table 1 Analysis of one-dimensional 1 H NMR spectroscopically detected folding-to-unfolding transitions shown in Method  Table 2 Results obtained for amide protons (NH) using linear extrapolation of the transition region ranging from c = 2 to 4 M urea in two-dimensional heteronuclear 1 H-15 N HSQC spectra. Folding-to-unfolding transitions have been acquired under three different conditions abbreviated using dil (dilute conditions), dex (c = 120 g/L Dex20) and peg (c = 120 g/L PEG1). Each single residue was fitted using a global value for the cooperativity of folding, m, applying all three different experimental conditions as discussed previously 5 . sc : side chain. Values for ΔCM are shown in Figure 2C, D and are graphically highlighted in Figure 5.  Table 3 Results obtained for methine group protons (CH, labeled as HA, HB, HG) using linear extrapolation of the transition region ranging from c = 2 to 4 M urea in two-dimensional heteronuclear 1 H-13 C HSQC spectra.
Folding-to-unfolding transitions have been acquired under three different conditions abbreviated using dil (dilute conditions), dex (c = for 120 g/L Dex20) and peg (c = for 120 g/L PEG1). Each proton group being present in a single residue was fitted using a global value for the cooperativity of folding, m, applying all three different experimental conditions as discussed previously 5 . Values for ΔCM are shown in Figure 3A, B and are graphically highlighted in Supplementary Figure 17.  Table 4 Results obtained for methylene group protons (CH2, labeled as HB, HB1, HB2, HA, HA1, HA2, HG1, HG2) using linear extrapolation of the transition region ranging from c = 2 to 4 M urea in two-dimensional heteronuclear 1 H-13 C HSQC spectra. Folding-to-unfolding transitions have been acquired under three different conditions abbreviated using dil (dilute conditions), dex (c = 120 g/L Dex20) and peg (c = 120 g/L PEG1). Each proton group being present in a single residue was fitted using a global value for the cooperativity of folding, m, applying all three different experimental conditions as discussed previously 5 .
Values for ΔCM are shown in Figure 3C, D and are graphically highlighted in Supplementary Figure 17. Results obtained for methyl group protons (CH3, labeled as HB, HD, HD1, HD2, HG, HG1, HG2) using linear extrapolation of the transition region ranging from c = 2 to 4 M urea in two-dimensional heteronuclear 1 H-13 C HSQC spectra. Folding-to-unfolding transitions have been acquired under three different conditions abbreviated using dil (dilute conditions), dex (c = for 120 g/L Dex20) and peg (c = for 120 g/L PEG1). Each proton group being present in a single residue was fitted using a global value for the cooperativity of folding, m, applying all three different experimental conditions as discussed previously 5 . Values for ΔCM are shown in Figure 3E, F and are graphically highlighted in Supplementary Figure 17.

Discussion of potential enthalpic and entropic contributions regarding crowding effects on the thermodynamic stability of proteins determined in presence of urea
Chemical unfolding of proteins as induced by urea occurs by a combined mechanism of direct interaction of urea molecules with the protein backbone and its side chains as well as by indirect changes in the hydrogen-bonding network of the hydration sphere of the protein 6  of the DNA-binding domain of lac repressor 9 . The enthalpic contribution due to preferential interaction of PEG with aromatic side chains could be quantified as a perturbation of the m value (cooperativity of unfolding) which corresponds to significant changes in the exposed surface area upon unfolding 9 . In the case of BsCspB here, we found that the m value is not perturbed in the presence of 120 g/L PEG1 compared to dilute solution (and also not for 120 g/L Dex20). The aromatic residues comprising BsCspB are not contributing to the hydrophobic core of this protein but they are exposed both in the native state as well as in the unfolded ensemble.
To sum up, we cannot exclude potential enthalpic interactions of PEG1 with BsCspB and corresponding aromatic side chains but we note that there is no change in m value detectable in presence of 120 g/L PEG1 which would be one prominent parameter of an existing enthalpic interaction. Note that we discuss other parameters for potential enthalpic interactions possibly existing between PEG1/Dex20 and BsCspB in the main part of our manuscript.
Thus, the gain in free energy of unfolding leads to a significant increase in thermodynamic stability of BsCspB upon addition of 120 g/L PEG1 or 120 g/L Dex20 which cannot be attributed to favorable aromatic interactions 8 . Further, the gain in thermodynamic stabilization seen for PEG1 is of same extent as in the presence of the macromolecular crowder Dex20 which equals the expected gain in stability due to excluded volume theory.