Protein folding is one of the most fundamental problems in modern molecular biology. signal fields are 3 to 4 4 orders of magnitude weaker than nonchiral 2DIR signals the cross peaks in the CI 2D signals are explicitly coordinate-dependent and are therefore particularly sensitive to structural changes. CI 2DIR and CI 2D ultraviolet spectra have been predicted for proteins using QM/MM simulations 2 3 33 In this computational study we demonstrate how 2DIR spectroscopy may be used GSK-923295 to monitor the ultrafast folding process of the 20-residue Trp-cage peptide (Asn1-Leu2-Tyr3-Ile4-Gln5-Trp6-Leu7-Lys8-Asp9-Gly10-Gly11-Pro12-Ser13-Ser14-Gly15-Arg16-Pro17-Pro18-Pro18-Ser20) which is one of the fastest folding mini-proteins. Although Trp-cage is small and relatively simple the mechanism of its folding remains elusive. Some studies 38 39 have suggested that it follows a simple two-state folding mechanism. On the other hand recent UV-resonance raman experiments 40 show that Trp-cage is not a simple two-state miniprotein. Additionally the folding time determined by tryptophan fluorescence and recent 2D 1H NMR spectra experiment suggests downhill GSK-923295 folding mechanism 27. It is very interesting that even for such a small system we still have conflicting views of its folding mechanism. 2DIR spectra may provide a detailed picture of the structure and dynamics of the peptide along the pathway and the folding mechanism. Methods Molecular dynamics (MD) simulations All MD simulations were carried out using the AMBER 10 software package 41 with AMBER ff99SB protein force field 42. It has been GSK-923295 reported that the folding temperature for Trp-cage is in the range 313-317K 27. The constant temperature of 315K was maintained in our simulations by assigning atom velocities from a Gaussian distribution for the different trajectories 43. 50 200-ns trajectories were simulated. The initial structure is GSK-923295 given by a extended conformation. An implicit solvation model 44 with the collision frequency of 1 1 ps?1 was applied in the MD simulations. The SHAKE algorithm 45 was used to constrain covalent bonds involving hydrogen atoms. A timestep of 2 fs was used. These 50 trajectories covering total 10 μs simulations of peptide folding provide enough data for constructing the FEL. Several locations were harvested along the dominant folding pathway from the GSK-923295 unfolded to the folded state to calculate the IR signals. Calculation of 2DIR spectra Using the bosonic creation and annihilation operator of a vibrational exciton and and ωλ is the λth eigenvalue. The projected density of states shows that the higher frequency band in the isotope-labeled region originates from Pro18 while the lower frequency band originates from Trp6 as shown in Fig. 4(b)-(f). The absorptive 2DIR spectra are displayed in Fig. 5. All spectra are dominated by an inhomogeneously (diagonally) broadened peak centered near (?1640 1640 cm?1. The peak shape is largely determined by the inhomogeneous distribution and the homogeneous dephasing of 5.5 cm?1 which were used to compute the spectra. The diagonal L100 peak is red-shifted by ≈10 cm?1 compared to L1 consistent with the above linear absorption spectrum and the previous study . The similarity of the 2DIR spectra of the unlabeled amide groups indicates that the signals are not very sensitive to protein secondary structure motifs without the use of site-specific isotope-labeling. Figure 5 Isotope-labeled nonchiral (spectra in the region Rabbit Polyclonal to SCNN1D. of the isotope labeled residues shows some interesting features during folding (Fig. 5). Starting at L50 two isotope-labeled bands clearly begin to emerge at approximately (?1570 1570 cm?1 and (?1590 1590 cm?1. The band around (?1570 1570 cm?1 gradually increases from L50 to L100 and the intensities of the band around 1590 cm?1 are almost unchanged from L50 to L100. After the two bands appear at L50 the cross peak at (?1570 cm?1 1590 cm?1) emerges. At L1 GSK-923295 and L25 this cross peak is extremely weak and the coupling between the two isotope-labeling residues is nearly zero as shown in Fig. 6. At L50 the magnitude of the coupling increases by nearly an order of magnitude while the cross peak intensity also.