In the past decade, a wealth of experimental data has demonstrated that a large fraction of proteins, while functional, are intrinsically disordered at physiological conditions. Many intrinsically disordered proteins (IDPs) undergo a disorder-to-order transition upon binding to their biological targets, a phenomenon known as induced folding. Induced folding may occur through two extreme mechanisms, namely conformational selection and folding after binding. Although the pre-existence of ordered structures in IDPs is a prerequisite for conformational selection, it does not necessarily commit to this latter mechanism, and kinetic studies are needed to discriminate between the two possible scenarios. So far, relatively few studies have addressed this issue from an experimental perspective. Here, we analyze the interaction kinetics between the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (NTAIL) and the X domain (XD) of the viral phosphoprotein. Data reveal that NTAIL recognizes XD by first forming a weak encounter complex in a disordered conformation, which is subsequently locked-in by a folding step; i.e., binding precedes folding. The implications of our kinetic results, in the context of previously reported equilibrium data, are discussed. These results contribute to enhancing our understanding of the molecular mechanisms by which IDPs recognize their partners and represent a paradigmatic example of the need of kinetic methods to discriminate between reaction mechanisms.

The hydrophobic effect is a major driving force in protein folding. A complete understanding of this effect requires the description of the conformational states of water and protein molecules at different temperatures. Towards this goal, we characterise the cold and hot denatured states of a protein by modelling NMR chemical shifts using restrained molecular dynamics simulations. A detailed analysis of the resulting structures reveals that water molecules in the bulk and at the protein interface form on average the same number of hydrogen bonds. Thus, even if proteins are ‘large’ particles (in terms of the hydrophobic effect, i.e. larger than 1 nm), because of the presence of complex surface patterns of polar and non-polar residues their behaviour can be compared to that of ‘small’ particles (i.e. smaller than 1 nm). We thus find that the hot denatured state is more compact and richer in secondary structure than the cold denatured state, since water at lower temperatures can form more hydrogen bonds than at high temperatures. Then, using Φ-value analysis we show that the structural differences between the hot and cold denatured states result in two alternative folding mechanisms. These findings thus illustrate how the analysis of water-protein hydrogen bonds can reveal the molecular origins of protein behaviours associated with the hydrophobic effect.

Despite the partial disorder-to-order transition that intrinsically disordered proteins often undergo upon binding to their partners, a considerable amount of residual disorder may be retained in the bound form, resulting in a fuzzy complex. Fuzzy regions flanking molecular recognition elements may enable partner fishing through non-specific, transient contacts, thereby facilitating binding, but may also disfavor binding through various mechanisms. So far, few computational or experimental studies have addressed the effect of fuzzy appendages on partner recognition by intrinsically disordered proteins. In order to shed light onto this issue, we used the interaction between the intrinsically disordered C-terminal domain of the measles virus (MeV) nucleoprotein (N TAIL ) and the X domain (XD) of the viral phosphoprotein as model system. After binding to XD, the N-terminal region of N TAIL remains conspicuously disordered, with a-helical folding taking place only within a short molecular recognition element. To study the effect of the N-terminal fuzzy region on N TAIL /XD binding, we generated N-terminal truncation variants of N TAIL , and assessed their binding abilities towards XD. The results revealed that binding increases with shortening of the N-terminal fuzzy region, with this also being observed with hsp70 (another MeV N TAIL binding partner), and for the homologous N TAIL /XD pairs from the Nipah and Hendra viruses. Finally, similar results were obtained when the MeV N TAIL fuzzy region was replaced with a highly dissimilar artificial disordered sequence, supporting a sequence-independent inhibitory effect of the fuzzy region.