Computational Studies On The Folding Mechanisms And Stabilities Of Tubulin Active Peptides And Some Model Peptides

The αβ-tubulin dimer is the structural subunit of microtubules(MTs),the cytoskeletal elements crucial to a variety of cellular processes,such as plant cell division,intracellular transport,the maintenance of cell walls,stomatal movement,signal transaction and plant gravitropism in the organisms.The αβ-tubulin dimer is also known to bind with a variety of substrates:e.g.,colchicine,vinblastine and taxol and so on.The αβ-tubulin dimer has a high dynamic instability,which disassemblizes at the minus end(α-subunits)of microtubule and reassemblize at the plus end(β-subunits)in the plant cell.Meanwhile,the tubulin active peptide segments also undergo multiple conformational transitions during the folding process.Their folded conf-igurations are closely associated with the depolymerization and polymerization of the αβ-tubulin dimer,playing a significant role in the biological functions of MTs.We have studied the conformations and stabilities of the large ap-tubulin dimer under different environments and its active peptide segments.With the aid of temperature-controlled molecular dynamics(TCMD)simulations,the folding mechanism of the tubulin active peptide(Pep1-28)was studied.It was found that the clear folding pathway can be displayed by gradually decreasing the temperatures.The noticeable folding was observed at about 550 K,and the folding and unfolding reversible mechanisms were determined as U(>1200 K)(?)11(1200-1000 K)(?)12(800 K)(?)13(600 K)(?)14(450 K)(?)F1(400 K)(?)F2(300 K),where U is an unfolded conformation,I1,12,13 and 14 are four important intermediates during the folding process,and F1 and F2 are two folded conformations with close structures,respectively.It can be seen that the TCMD method also presents the important intermediate conformations,providing direct and reliable proofs for the folding and unfolding mechanism of Pep1-28.The active peptide segment of the αβ-tubulin dimer exhibits only a short helical structure without typical tertiary strcture and folding kinetics,whereas the Trp-cage is a small fast－folding model peptide with a well-packed folding core and typical tertiary contacts.Besides,it seems to be similar to the misfolded conformation of Pep1-25.Based on the advantage of TCMD,the folding pathway of the Trp-cage in the absence and presence of water solvents were obtained.It is decomposed into nine steps,and shown as U(?)I1(?)I2(?)I3(?)I4(?)F1(?)F2(?)MSOL(?)TS(?)FSOL.In addition,it was found that the salt bridge will not play a significant role in the folding kinetics,in good agreement with the experimental results.The presence of water solvents induces the packing of the hydrophobic residues as the whole cage comparatively closes.In the water solvents,the Trp-cage folding begins to contract into the metal-stable state(MSOL),traverses the transition state(TS)and then arrives at the folded native-like structure(FSOL),which closely resembles the experimental native structure.The folding mechanism of the Trp-cage provides us a theoretical basis for further understanding the mis-folding process of Pep1-25 and the changes in its function caused by the abnormal conformational transitions.Molecular dynamics combined with single-site mutations were used to study the effects of the mutations on the Trp-cage stability.It was found that mutations W6G,L7G and P19G undergo frequent conformational transitions and cause significant stability decreases to the Trp-cage.Their hydrophobic centers are fully hydrated,with secondary and tertiary structure being destroyed,especially in the case of W6G.Instead,only slight influences are induced by mutations N1G,L2G,Q5G,K8G,D9G,P12G and S20G.Albeit involved in the hydrophobic cluster,P12 is not so crucial as the three C-terminal prolines and supports that its Pro/Trp interaction is not essential for the core formation.The other seven mutations(Y3G,I4G,S13G,S14G,R16G,P17G and P18G)also exert observable influences,although not so great as W6G,L7G and P19G,to the Trp-cage stability.The hydrophobic and helix collapses are concomitant in the Trp-cage.Noticeable stability decreases are resulted by destroying the hydrophobic center,or D9/R16 salt bridge or buried H-bonding interactions.Nonetheless,the D9/R16 salt-bridge is intrinsic in the Trp-cage and not readily affected,even with mutations.According to the above results,it can be seen that the mutations result in larger destabilizing effects to the Trp-cage structure by decreasing the hydrophobic interactions,tertiary contacts and native buried H-bonds as well as the ion-pairing interaction.These destabilizing effects depend on the orientations of the mutated residues and the chemical properties of their sidechains.The roles of each residue(actually its sidechain)and their cooperative interactions were thus evaluated for the Trp-cage stability and dynamics,which further our understanding towards the stability and dynamics of the αβ-tubulin dimer as well as its functions through the conformational transitions at the molecular and atomic levels.