Biophysical evidence for the secondary helical conformation adopted by Mic10

Following the optimization of the expression protocol for Mic10His (see section 2.1 of the Materials and Method section for more information), a screening of detergents and refolding methods (step wise dialysis (SW), fast dilution and on-column) gave similar results, thus we opted for using the dialysis method since there is less loss of material (Fig. 4.7A, DPC lines). Figure 4.7A shows the SDS-PAGE gel of the detergent refolding screening in sodium dodecyl sulfate (SDS), n-dodecyl phosphocholine (DPC), n-octyl-ß-D-glucopyranoside (OG) and n-dodecyl-β-D-maltopyranoside (DDM from Avanti). The SDS-PAGE gel of figure 4.7A shows a major band at ~15 kDa corresponding to Mic10His, however, other higher molecular weight bands are present in the gel. Due to the well known propensity of Mic10 to homo-oligomerize, we wondered if the higher molecular weight bands observed might correspond to Mic10 homo-oligomers. Thus, with the help of our collaborators (Dr. Daryna Tavarsenko University of Göttingen) the presence of Mic10 homo-oligomers was confirmed by western blot using a polyclonal anti Mic10 and anti-His antibody (Appendix 4.V). This finding is consistent with previously reported data, that support a strong Mic10 protein-protein interaction and a strong stability of the oligomeric state (194, 203).

Figure 4.7. Heterogeneous contribution from alpha and beta sheet secondary structure in refolded Mic10.

In A, Mic10 refolding in different detergents using 15 % SDS-PAGE and coomassie staining. In B, predicted model of Mic10His by the SWISS protein severs and C shows the helix position in the amino acid sequence from the predicted structure F. D and E are the CD spectra recorded for Mic10-His in different detergents SDS and DPC, respectively.

The secondary structure of the refolded Mic10His was inspected by CD. The CD spectra recorded from refolded Mic10His in both SDS and DPC show a negative absorption from 240 to 200 nm (Fig. 4.7D and 4.7E). Contrary to what we expected, a helical secondary structure, the CD spectra measured show a large heterogeneity regarding the secondary structures present in the samples. The CD spectrum in DPC micelles shows a higher contribution of beta conformation with a single minimum at 217 nm (Fig. 4.7E), while in the CD spectrum in SDS micelles, shows two minima at about 213 nm and 222 nm are observed (Fig. 4.7D) indicating a larger helical contribution in this environment. In both cases, DPC and SDS, the helical content predicted by the JASCO software is below 50%. Thus, these data suggest that Mic10His in detergent environments adopts secondary structures with a different contribution of beta and alpha-helix secondary structure that depends on the detergent used.

To obtain more detailed information of the secondary structure of Mic10His, the system was investigated using solution NMR spectroscopy. Table 4.12 summarizes the different detergents tested.

GVFTSVLFF⁸⁹K RRA⁹³FPVWLGI GFGVGRGYAE G¹¹¹DAIFRSSAG

LRSSK

205 210 215 220 225 230 235 240

SDS

205 210 215 220 225 230 235 240

DPC

Table 4.12. Detergent properties.


Figure 4.8 shows the ¹⁵N-HSQC spectrum recorded in the different detergent conditions. The sample temperature was increased from 30 to 37°C to decrease the correlation time (τc) of the particle in solution. Out of the four detergents tested, SDS gives the best sensitivity and spectral quality (Fig. 4.8A). DM (Fig. 4.8B) and DPC (Fig. 4.8D) have almost no detectable signal in the ¹⁵N-HSQC. The lack of sensitivity could be caused by the slow motion of the protein in the presence of detergent, fast exchange with the solvent, which will increase with temperature at 37ºC, and/or the coupling value of the covalent bond used during the magnetization transfer (¹JHN ~95 Hz). ¹³C-HSQC spectra of SDS and DM show sufficient spectral quality (Appendix 4.VI) suggesting that the lack of sensitivity in the ¹⁵N-HSQC is most likely due to exchange with the solvent. Although, this does not totally exclude the change of the HN coupling (¹JHN) which will considerably decrease the magnetization transfer efficiency.

The 2D ¹⁵N-HSQC NMR spectra recorded for Mic10His refolded in SDS (Fig. 4.8A) and LDAO (Fig. 4.8C) showed narrow amide proton (H^N ) chemical dispersion of about 1 ppm, from 7.5 to 8.5 ppm, suggesting that Mic10His adopts a helical conformation. This is consistent with the bioinformatics predictions and the CD data. Detergents such as SDS have been characterized as being harsh and promoting helical formation (224). In order to test the effect of SDS itself on the protein secondary structure, ¹³C-HSQC spectra in DM and SDS were acquired (Appendix 4.VI). The chemical shift changes in the spectra are due to the difference in the sample temperatures of 37ºC and 30ºC for SDS and DM, respectively. No other significant chemical shift differences were observed. Thus, large structural influence of SDS in Mic10 can be ruled out.

Detergent Critical micellar concentration

(mM) Molecular weight

(Da) Purchase

SDS 7 - 10 288 Merck

DPC 1.5 351 Avanti

OG 20 292 Sigma

DM 0.2 510 Avanti

LDAO 1.7 229 Sigma

Figure 4.8. Detergent screening by NMR.

Four different detergent conditions were tested for Mic10-His, SDS (A), DM (B), LDAO (C) and DPC (D). SDS turns out to be the optimal detergent in these conditions ~80 µM (note concentration before refolding) of Mic10 refolded in 20 mM Sodium phosphate buffer 150 mM NaCl at pH 7.4 using 1%. 15N-HSQC spectra were recorded on a 400 mHz Bruker spectrometer with a 3 channel TCI probe using 106 ms in the direct dimension (¹H) and 46 ms in the indirect dimension (¹⁵N) with spectral with of 12 and 24 ppm, respectively.


SDS was used to further investigate Mic10 structure since it provides both the best CD and NMR spectral quality. A ¹³C,¹⁵N-Mic10His enriched sample was prepared and refolded in a 1% SDS solution. The sample was used for acquiring assignment data in a 700 MHz spectrometer equipped with a 5 mm cryoprobe (Fig. 4.9). TROSY based experiments that give higher sensitivity and resolution for the slowly tumbling membrane proteins were used (225).

Using the automatic peak picking tools from CcpNmr (219), only about half of the expected HNpeaks (65 H^N peaks) were picked from the ¹⁵N-HSQC out of 120 H^N peaks expected (125 – 5 prolines – 1 N-terminus = 120 peaks). Two peaks at about 10 ppm in proton and 130 ppm in nitrogen are observed corresponding to ¹⁵HNε from the two tryptophan sidechains. The assignment of those peaks belonging to the tryptophan sidechain was confirmed by acquiring 3D HNCA. The ΝεHε peaks are absent from the HNCA spectrum because there is no NCA correlation in the tryptophan sidechain. During the course of acquisition, 72 hours, several peaks on the spectrum cease to be visible (Fig. 4.9, red spectra, peaks represented by blue arrows), suggesting that over time the Mic10His protein aggregates or/and degrades. Spectral

10 9 8 7

changes over such a short period of time limit the NMR applicability for assignment purposes since the acquisition of the complete set of spectra required for the assignment and the structure calculation need several weeks.

Figure 4.9. Poor stability of Mic10His in SDS detergent micelles conditions.

TROSY based ¹⁵N-HSQC (trosyetf3gpsi) using ~300 µM of Mic10His refolded in 20 mM Sodium phosphate buffer 150 mM NaCl at pH 7.4 using 1% of SDS. spectra were recorded on a 700 MHz Bruker spectrometer with a three channel TXI probe using 121 ms (2048 points) in the direct dimension (¹H) and 60 ms (256 points) in the indirect dimension (¹⁵N) with spectral with of 14 and 35 ppm, respectively. Processing was performed applying a square sine function in both dimensions with only 60 ms (1024) points in the direct dimension (¹H).

Although, we could not obtain all the data for this purpose, we attempted to obtain information on the conformation adopted by the glycine residues which might be involved in GxxxG motifs. NMR chemical shift provides qualitative information regarding the protein secondary structure in a residue-specific manner (226). For this purpose, a 3D HNCA TROSY spectrum was recorded to access Cα chemical shifts. Glycine residues have particular ¹⁵N and

13C chemical shifts: a high field carbon alpha chemical shift between 40 and 50 ppm and a nitrogen chemical shift between 100 and 115 ppm. Figure 4.10A, shows the CN projection of the 3D HNCA spectrum recorded on a 700 MHz Bruker spectrometer. Nine out of the thirteen expected glycine peaks are observed in the HNCA spectra (Fig. 4.10D, bold). Based on the models and prediction, the number of glycine residues excluding the TM regions can be estimated to be four (Fig. 4.5 and 4.6). Therefore, at least five out of these nine Cα glycine

10 9.5

peaks identified are expected to be part of the transmembrane domains. Figure 4.10B indicates the chemical shift expected from a beta-sheet (red), random coil (white) or alpha-helical (blue) structure for glycine. Three Cα glycine residues have their chemical shift around the values expected for a random coil region with an average chemical shift of ~45.4 ppm and the others show chemical shifts values above 46 ppm which suggest an alpha helical conformation. Furthermore, the HNCA spectrum shows Cα peaks shifted to lower field in the carbon dimension with chemical shifts above 60 ppm. These chemical shift values are a signature of Cα of Ile, Val, Phe, Pro, Ser and Tyr in a helical conformation which is consistent with the structural model of Mic10 adopting a helical conformation.

Figure 4.10. Part of the glycine content in Mic10His adopts a helical conformation in SDS micelles.

A, shows the 2D plane of a TROSY based HNCA spectrum acquired on a 700 MHz Burker spectrometer at 37ºC. B, shows an expansion of the glycine region of the 2D plane, with a color code indicating the expected

MSYYHHHHHHDYDIPTTENL YFQG^AMG^{ILMSEQAQTQQPA}

KSTPSKDSNK NGSSVSTILD TKWDIVLSNM LVKTAMG^FG^V GVFTSVLFFK RRAFPVWLG^IG^FG^VG^RG^YAE

glycine Cα chemical shift from helical (in blue), random coil (in white) or beta sheet (in red). In C, a table representing the chemical shift values of the nine glycine residues that have been observed (no assignment available so far). In D, the amino acid sequence of Mic10His with the glycine residues (in bold), the glycines that are expected to be outside the membrane region (orange) and the glycines that are expected to be in the TM regions (black and italic).


Altogether, the biophysical data recorded are consistent and support the model that Mic10 adopts a random-coil/alpha helical structure in detergent environments. With the available data, we could not obtain residue-specific assignment of the glycine peaks observed in the 3D NMR spectra, which shows the mostly alpha helical conformation based on their chemical shift. However, it is worth noting that all the data were obtained in detergent micelles which differ from the native membrane environment. This could lead to a looser protein fold and therefore a different protein folding from that in in vivo conditions. Even though all detergents tested here have been successfully used in other NMR studies with alpha-helical membrane proteins, one should be particularly cautious with the potential artefacts due to the detergent used. Therefore, we further investigated Mic10’s structure in a more a native-like environment, lipid bilayers, using solid-state NMR.

3.3 INHOMOGENEOUS NMR LINE SHAPEFROM MIC10 RECONSTITUTEDINNATIVE-LIKE