*2.3. Discrepancies between SEC and Fluorescence Anisotropy Suggest a Contribution of Tags in Proteins Hydrodynamic Behavior*

The parameters *V* and *θexp* were derived for the various monomeric species (Figure 2C,D, and Table 1). The more the ratio *θexp*/*θcalc* differs from unit, the more asymmetric the protein is. The *θexp*/*θcalc* ratio of His6 VPg (2.41) and His6 eIF4E (1.62) indicated that their shape differed from a sphere. In addition, although the His6 VPg molecular weight was 4295 Da less than His6 eIF4E, its *V* value was 1.37 times larger, indicating that it was significantly less compact, a pre-molten globule feature. The untagged form of VPg displayed an expected decrease of its hydrodynamic molar volume with respect to its tagged form. Interestingly, the eIF4E\* untagged form showed a higher *V* value (46.5 L/mol) and hence, a significant decrease in compaction compared to His6 eIF4E\* (42.9 L/mol). This could be due to interactions between the His6 tag and the eIF4E N-ter IDR [23], an effect previously discussed [27]. Because of the intrinsic conformational entropy and the positive charges cluster of the disordered His6 tag, its interactions with other parts of the protein are more likely to occur. This could account for the more compact hydrodynamic behavior of His6 eIF4E. For most 3D structures solved from tagged proteins, the peptide tag is too disordered to be resolved. However, it is worth mentioning that, in most of the cases, comparisons of tagged with corresponding untagged structures determined from X ray diffraction data revealed only minor structural differences of the type that might be observed when comparing two identical sequences solved in different space groups [28]. This tends to show that these purification tags generally had no significant effect on the structure of the native protein. However, the importance of the proteins hydrodynamics properties cannot be understated as it accounts for the polypeptide chain dynamics, which drives most of biological functions. This is exemplified through the richness of the functional interpretations provided by NMR data related to disordered segments [29].

A comparison of *V* values between tagged and untagged species allows for the discussing of tag contribution to compaction. A decrease of the Perrin's plot slope was observed for His6 VPg\*-His6 eIF4E binary complex when compared to the slope of His6 VPg\* (Figure 2A). Upon analysis of this data, it was shown that the hydrodynamic molar volume *V* linked to the labeled VPg enlarged from 58 L/mol to 121 L/mol, (Table 1). The later, attributed to the complex, was 20% larger than could be expected by adding the *V* value of His6 VPg\* and His6 eIF4E monomers.

Hence, the anisotropy approach revealed a possible contribution of the two flexible His tags conformational entropy to the proteins hydrodynamic behavior. By contrast, the *V* value experimentally determined for the native untagged binary complex was close to that of the sum of the monomer values (Table 1). Compaction can be estimated by *ρ*, the molecular density value. Interestingly, the compaction within the binary complex suggested by SEC was not observed by anisotropy. In the SEC experiments, elution is concomitant to dilution, and hence to a modification of the species distribution. This could contribute to average the observed apparent molecular weight of each species. As stated in the experimental procedures section, anisotropy measurements on binary complexes as a function of various viscosity values were performed after the molecular species in presence have reached an equilibrium. The *ρ* value is directly obtained from *V* the experimental molecular volume, and thus, the anisotropy approach provides a likely more accurate way to estimate compaction than SEC does through the use of *Mapp* in the empirical linear Equation (4). More generally, *ρ* values provided by anisotropy were 1.5 time higher than those deduced from the SEC (Table 1).


**Table 1.** Physical parameters of the various molecular forms of VPg, eIF4E and of their binary complexes.

HV, His6 VPg; HE, His6 eIF4E; V, His6 VPg; E, eIF4E; EΔ(1–46), 1–46 residues deleted eIF4E; HVHE, His6 VPg-His6 eIF4E binary complex; VE, VPg-eIF4E binary complex. MW (molecular weight), *VR* Retention volume), *Mapp* (apparent molecular mass), *Rh* (hydrodynamic radius), *ρ* (apparent molecular density), *Vapp* (apparent molecular volume), *θexp* (experimental rotational correlation time), *θcalc* (calculated rotational correlation time), *h* (estimated hydration degree). <sup>1</sup> Determined for *η* = 0.01 P (water) at 298 K; *τ*, AEDANS fluorescence lifetime: 15.6 ns; <sup>2</sup> Calculated from pea eIF4E structure (2WMC) using the Hydropro software (http://leonardo.inf.um. es/macromol/programs/hydropro/hydropro.htm); <sup>3</sup> value obtained from His6 VPg\*-His6 eIF4E binary complex; † Mass spectrometry (MALDI-TOF); ‡ Calculated from amino acid sequence.
