Tim23ims as intrinsically disordered monomer

Tim23 ^1-96 (or Tim23ims) eluted as a single peak from the SEC column and possess constant hydrodynamic radii as a function of increasing concentration (data not shown from DOSY-NMR) indicating that it exits as a monomer in solution. The CD

3.1 Intermembrane space domains (ims) of mitochondrial translocases 71

spectroscopy was used to measure the secondary structure content of Tim23ims. The obtained CD spectrum of Tim23ims showed a sharp negative band around 196 nm and very low ellipiticity above 210 nm, which is often seen in case of disordered proteins (Figure 18, A). Furthermore, ¹H-¹⁵N HSQC of Tim23ims exhibited poor amide proton chemical shift dispersion and sharp resonances which are characteristic of rapidly interconverting conformers of the disordered proteins (Figure 18, B).

Figure 18: Tim23ims is intrinsically disordered. (A) Near-UV circular dichorism spectrum in phosphate buffer at 10µM concentration. (B) ¹H-¹⁵N-HSQC spectra of Tim23ims show narrow chemical shift dispersion and sharp resonances in amide proton region.

Intrinsically disordered proteins are known to possess transient long range interactions and residual secondary structural elements that may populate differentially under different physiological conditions (Bertoncini et al. 2005; Mukrasch et al. 2009).

To investigate the presence of such residual secondary structural elements at residue level for Tim23 ^1-96 secondary carbon chemical shifts, ¹H-¹⁵N Het NOE and ¹H-¹⁵N RDCs were measured.

The residue specific secondary structure propensity of Tim23ims as exhibited from average secondary C^α and C’ chemical shift showed that most of the residues possess values within ±0.5ppm than that of the random coil chemical shifts (Figure 19, A). Smaller deviations in secondary chemical shift values in Tim23ims from that of

72 Results

random coil, indicate the lack of rigid secondary structural elements i.e. α-helices or β-sheets. The four or more consecutive positive values indicate the propensity for the helices. From Figure 19A, it is evident that residues 73-87, which have stretch of positive average values could transiently populate the helix.

The transient nature of secondary elements in terms of timescales of backbone motion could be understood by ¹H-¹⁵N HetNOE, which measures the amplitude of motion of HN bond vector at picosecond to nanosecond timescale. ¹H-¹⁵N HetNOE values are directly proportional to rigidity of backbone motion at previous said timescale i.e. higher positive value means more rigidity that would be expected for residues engaged in forming secondary structural elements. However, most of residues in Tim23imspossess average values ¹H-¹⁵N HetNOE below ±0.25 implying that backbone of Tim23 contain high degree of motion at picosecond to nanosecond timescale (Figure 19, B). The higher negative ¹H-¹⁵N HetNOE values are expected for highly flexible residues, which is interestingly observed for region A17-D22 in addition to expected sites of N and C- terminus.

1D (HN) RDCs that provide information about the average orientation of NH vector with respect to the external magnetic field showed small values along the sequence of Tim23ims (Figure 19,C). Thus, there exists small, yet, noteworthy conformational preference and dynamics in Tim23ims. Qualitatively, the sign of RDC for first 12 residues is opposite to the rest of polypeptide chain in Tim23ims. In addition, isolated positive values are seen for residues 7, 40, 53, 59 and 82 that are characteristic for turn formation (Mukrasch et al. 2009).

3.1 Intermembrane space domains (ims) of mitochondrial translocases 73

Figure 19: Structural properties of Tim23ims. (A) Averaged C^α/C’ secondary chemical shifts (CSI) observed in Tim23ims as a function of residue number. Negative (positive) secondary chemical shifts spanning several residues indicate propensity for beta-strand (alpha-helix).(B) Steady-state heteronuclear ¹⁵N-¹H-NOEs and (C) Profile of ¹D(HN) dipolar couplings in aligned in Pf2 bacteriophage of Tim23ims at 15°C. Different sign of residual dipolar coupling indicate different alignment of HN internuclear vector with respect to magnetic field. The error bars for dipolar couplings are based on Line width/Signal to Noise ratio.

74 Results

3.1.3.1 Long Range interactions in disordered Tim23ims

Paramagnetic relaxation enhancement (PREs) provides important information about the transient tertiary interactions in unfolded and partially folded proteins (Lietzow et al. 2002). To probe the transient long range interactions in Tim23ims, a cysteine was engineered at position 11 using site directed mutagenesis (Tim23ims T11C). Tim23ims T11C mutant was covalently attached to MTSL (a nitroxide containing paramagnetic tag). The nitroxide label causes paramagnetic dipolar relaxation of nuclear spins typically, amide protons and thus, the residues close to the site of paramagnetic tag will experience selective line broadening in the NMR spectrum.

The broadened H^N can be correlated to the average distance of residue from the site of paramagnetic tag up to 25Å. The paramagnetic relaxation enhancement (PRE) profile of Tim23-T11C tagged with MTSL is obtained from the ratio of intensity cross peaks in

1H- ¹⁵N-HSQC spectra of Tim23ims T11C in paramagnetic (with MTSL) and diamagnetic state (with MTSL+DTT) (I_p/I_d). The PRE profile for residues (±10 residues on either side of MTSL tag) close to T11 as expected shows the severe amide proton (H^N) broadening (Figure 20). The residues 25-40 and 40-56 have the average intensity broadening of 0.35 and 0.6 respectively, indicating presence of transient interaction in this region. Additionally, intensity broadening was observed for the C-terminal residues 90-94 suggesting that the Tim23ims in solution is not completely an extended polypeptide chain but possess the intramolecular long range contacts between its N and C-terminus. The residues such as N40, D54, L58 and L71 are well resolved in the spectra and show an average intensity attenuation value of 0.2. The residues 74-88 show intensity ratio (Ip/Id) value close to unity. This implies that these residues lack transient interactions with N-terminus T11C.

3.1 Intermembrane space domains (ims) of mitochondrial translocases 75

Figure 20: Intramolecular long range interactions in Tim23ims. PRE profile of Tim23ims T11C obtained with paramagnetic tag (MTSL) conjugated at position 11 (T11C) of Tim23ims. The intensity ratios are derived from signal intensity ¹H-¹⁵N HSQC spectra of Tim23ims T11C with MTSL paramagnetic (oxidized) and DTT added (reduced). The error bars are obtained based on the S/N values.

Deviations from intensity ratio of 1 beyond residue 25 are indicative of presence long range interactions.

Some resonances such as N40, D54, L58 and L71 that shows more severe broadening in comparison to its neighboring resonances are labeled.