Supporting Information Monitoring Methanol-induced protein unfolding by fluorescence anisotropy measurements of covalently labelled rhodamine probe. Antonin Soleilhac,a) Franck Bertorelle,a) Philippe Dugourd,a) Marion Girod,b) and Rodolphe Antoine a)* a) Institut lumière matière, UMR5306 Université Claude Bernard Lyon1-CNRS, Université de Lyon 69622 Villeurbanne cedex, France. b)Univ Lyon, CNRS, Université Claude Bernard Lyon 1, Ens de Lyon, Institut des Sciences Analytiques, UMR 5280, 5 rue de la Doua, F-69100 VILLEURBANNE, France.

CorrespondingAuthor * Rodolphe Antoine address: Institut lumière matière, UMR5306 Université Claude Bernard Lyon1-CNRS, 5 rue de la Doua, 69100 Villeurbanne, France. Mail : rodolphe.antoine@univ-lyon1.fr

Eur. Phys. J. D (2017) 71: 142DOI: 10.1140/epjd/e2017-70760-3

Mass Spectrometry

Mass spectra of Myoblobin (Mb) and RhB labeled Mb have been recorded in different solvents using a linear quadrupole ion trap mass spectrometer (LTQ, Thermo Fisher Scientific, San Jose, CA). It is known that Holo-Mb is a Mb form where a Heme group is non-covalently bonded to the protein. Average molecular weight of the protein (apo-form) is 16,951.5 Da, whereas Heme mass is 616, 2 Da.* Figure S1a) shows the mass spectrum of RhB-labeled Mb solution in water (PBS). It is shown that the bond between the protein and its heme can be preserved through ESI-MS when the protein is closed to its native form. 9+, 8+ and 7+ charge states of holo-Mb are predominant, nevertheless, RhB-labeled (holo-) Mb are also visible (mass of rhodamine B isothiocianate : 500.6 Da).

Figure S1b) shows mass spectrum of Mb in different solvent conditions. When increasing methanol proportion, average Charge State Density is increasing and apo-Mb form appears (Mb without its Heme group).

Figure S1: Mass spectra of a) Rhodamine B labeled Myoglobin in Water (PBS) and b) Myoglobin in (black) Water (PBS) and (red) Water (PBS) / MeOH (70:30)

*Greg W. Kilby, Noncovalently Associated Complexes: Apomyoglobin and Myoglobin Studies Using the HPLC-Chip/MS System Coupled with ESI-TOF Mass Spectrometry, Applications Scientist Agilent Technologies, Little Falls, Delaware, USA

Circuclar Dichroism

Circular Dichroism spectra of Myoglobin and Rhodamine B labeled Myoglobin have been recorded in different solvents using a ChirascanqCD Spectrometer (Applied Photophysics). The secondary structures of the protein (and protein complex) – alpha helix, beta strands, turns and unordered structures - have been analyzed via an on-line analysis software for protein Circular Dichroism spectra: Dichroweb**. Average values of the secondary structure have been calculated using three different methods: SELCON3, CONTIN and CDSSTR. Results shown in Figure S2 confirm that the dye labeling doesn’t affect the secondary structure of the protein. Furthermore, analysis of Circular Dichroism spectra of RhB labeled Mb (see table S2) reveals a partial alteration of its secondary structure when increasing the methanol proportion.

Figure S2: Circular Dichroism spectra of Myoglobin and Rhodamine B labeled Myoglobin in Water (PBS)

**Whitmore, L. and Wallace, B.A. (2008) Biopolymers 89: 392-400.

**Whitmore, L. and Wallace, B.A. (2004) Nucleic Acids Research 32: W668-673.

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Myoglobin in Water (PBS) RhB labeled Myoglobin in Water (PBS)

Table S1: Average values of the secondary structure for Rhodamine B labeled Myoglobin in different solvents

Study of possible leaks of RhB induced by methanol?

In order to study if methanol can induced an “unlabeling” of RhB, we dialyzed in PBS (according the same protocol) two solutions at a given concentration, one contains RhB-labeled Mb in water (PBS), and the other one contains RhB-labeling Mb in water(PBS) / MeOH (70:30). After dialysis, both species are in the same solvent (water (PBS)) but have different history.

After absorption measurements, we can see that the intensity of the absorption band of RhB is the same in both cases, meaning that the concentration of RhB hasn’t changed after dialysis with and without the presence of methanol confirming that methanol doesn’t break the covalent bond between the dye and the protein. However, the intensity of the Soret band has decreased in presence of methanol meaning that some Mb lost their Heme, which is consistent with MS measurements.

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Figure S3: Absorption spectra of RhB-labeled Mb in water (PBS) with and without the presence of methanol.