Alice J Pettitt: An integrative characterisation of proline cis and trans conformers in a disordered peptide

Unlike folded proteins, disordered proteins are highly dynamic and they often exist as an ensemble of diverse heterogeneous conformations that lack a single threedimensional structure. Compared to folded proteins, the primary sequences of disordered proteins have a nearly 2-fold increase of proline residues (4), which are well-known to reduce the formation of secondary structure in proteins (5). In particular, proline residues in disordered proteins have been shown to play key roles in regulating protein-protein interactions (6), posttranslational modifications (7), and liquid-liquid phase separation (8).

Most peptide bonds within proteins exist exclusively in the energetically favourable trans conformation. However, for proline residues, the energy barrier between the cis and trans isomers is lower due to the cyclic structure of the amino acid, approximately 85 kJ·mol-1 (9). Thus, isomerisation is generally a slow process, occurring at a rate of 10-3 - 10-2 s -1 at room temperature, depending on the adjacent residues(10,11). The cis proline population typically varies between 5% and 10% in disordered proteins (12), suggesting that the presence of multiple cis proline conformations is possible within polyproline disordered protein ensembles. These ensembles thus sample a vast conformational space of very slowly exchanging conformers, which increases the complexity (13). 

Molecular dynamics (MD) simulations are often used to characterise the ensemble of disordered proteins as they can resolve individual conformations within an ensemble at atomic resolution, which is a challenge for many experimental techniques. Significant progress has been made over the last decade to optimise force fields for modelling disordered proteins (14–16), as well as advances in the integration of MD simulations and experimental data to improve their accuracy (17,18). Despite these advances, sampling the full configurational energy landscape of disordered protein ensembles in all-atom explicit solvent MD simulations is extremely computationally expensive. 

Proline cis/trans isomerisation presents an additional challenge due to the slow timescales of this process (10,11), which are generally not accessible in brute-force MD simulations alone, even on today’s most powerful computers. However, when suitable collective variables (CVs) can be identified, metadynamics, an enhanced sampling approach, offers an effective method for sampling slow motions (19,20). Indeed, metadynamics has been used to encourage exploration of the full configurational space of disordered proteins (21) and cis/trans proline isomerisation in simulations of dipeptides and folded systems (22,23). In the latter cases, the ζ angle (Cα i-1, Oi-1, Cδ i, Cα i, where i = proline) was employed as one CV for the isomerisation and pyramidalisation of the amide nitrogen (N1) and the ψ angle (Ni, Cα i, C’i, Ni+1) was employed as an additional CV to control the amide orientation, which may affect the rate of transition between the cis and trans proline conformations. Both CVs are required to enhance proline cis/trans sampling as they compensate for each other.

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