TMAO-Protein Preferential Interaction Profile Determines TMAO’s Conditional In Vivo Compatibility  Jiang Hong, Shangqin Xiong  Biophysical Journal  Volume 111, Issue 9, Pages 1866-1875 (November 2016) DOI: 10.1016/j.bpj.2016.09.035 Copyright © 2016 Biophysical Society Terms and Conditions

Figure 1 Determination of preferential interactions (μ23/RT) of TMAO with model compounds by osmometry. The excess osmolality, ΔOsm, is plotted versus the concentration product, m2m3. The error bar is propagated from the standard deviation of multiple readings of osmolality of corresponding solutions. Lines are linear fits to Eq. 3 with the slope equal to μ23/RT. The results are presented as subplots (A–D) for clarity. Biophysical Journal 2016 111, 1866-1875DOI: (10.1016/j.bpj.2016.09.035) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 2 Comparison of the preferential interaction potential ((10,000∗μ23/RT)/ASAi) of TMAO with that of urea (25). The error bar is propagated from the uncertainties in preferential interactions (Table 1) or reported for urea. Biophysical Journal 2016 111, 1866-1875DOI: (10.1016/j.bpj.2016.09.035) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 3 Comparison of the predicted and experimentally determined TMAO m-values or preferential interactions, μ23/RT. (A) μ23/RT of TMAO with model compounds (Table 1) (solid triangles) and with cyclodipeptides (c(GG), c(AG), and c(AA)), obtained by converting the determined transfer free energy at 1 M TMAO in the Lin (11) (solid circles) and Murphy (12) (open circles) groups, with M−1 to m−1 unit conversion using the molality value (1.08) for 1 M TMAO as a conversion factor. (B) TMAO m-values for unfolding a set of proteins and μ23/RT with native protein at specified T (Table S3). The line in both plots denotes the equality of the predicted and experimentally determined values. Biophysical Journal 2016 111, 1866-1875DOI: (10.1016/j.bpj.2016.09.035) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 4 Representative plot of the TMAO effect on Aβ42 oligomerization. Aggregation was initiated by diluting protein stock (60–80 μM) to a same final concentration (∼4−5 μM) for all samples being compared. Oligomerization was monitored by tracking the fluorescence self-quenching of TAMRA-Aβ42, compared among TMAO, buffer alone, and urea. All measurements were performed at 25°C without stirring. The 20 mM phosphate buffer (pH 6.5) also contains 150 mM NaCl, 1 mM EDTA, and 5 mM DTT. Biophysical Journal 2016 111, 1866-1875DOI: (10.1016/j.bpj.2016.09.035) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 5 TMAO effect on the stability of an average globular protein of 1000 Å2 of ΔASA is compared with that of urea (25) (A) and dissected to contributions from the individual functional groups (B). The error in (A) is propagated from the uncertainty in the preferential interaction potential. Refer to Table S4 for the detailed calculation. Biophysical Journal 2016 111, 1866-1875DOI: (10.1016/j.bpj.2016.09.035) Copyright © 2016 Biophysical Society Terms and Conditions