Exploring protein folding of Beta3s with two-dimensional infrared (2DIR) spectroscopy

Zaizhi Lai , Nicholas K. Preketes , Jun Jiang $ , Shaul Mukamel , Jin Wang

Department of Chemistry,

Department of Physics and Department of Applied Mathematics & Statistics, State University of New York at Stony Brook, Stony Brook, NY 11794 Department of Chemistry, University of California, Irvine, Irvine, CA 92697
$

School of Chemistry and Materials Science, University of

Science and Technology of China, Hefei, Anhui 230026, P. R. China. State Key Laboratory of Electroanalytical Chemistry, Changchun, Jilin 130021, China

Changchun Institute of Applied Chemistry, Chinese Academy of Sciences

Corresponding Author

E-mail: jin.wang.1@stonybrook.edu

Abstract Probing the underlying free energy landscape, pathways, and mechanism is the key for understanding protein folding in theory and experiment. Recently time-resolved two-dimensional infrared (2DIR) with femtosecond laser pulses, has emerged as a promising tool to investigate the dynamical process of protein folding on faster timescales than possible by NMR. We combined 2DIR spectroscopy and free energy prole generated by molecular dynamics to investigate the folding process of a small protein named Beta3s. Non-chiral and chiral 2DIR signals illustrate the variation of the spectrographic patterns when the protein evolves on the underlying free energy landscape. The non-chiral signals indicates the lobes of the imaginary part of the spectra tend to extend to the opposite directions when the beta-sheet elements grows in the folding process, and the chiral signals show the topological chirality of the protein gradually decreases when the native structure of the protein forms. This work provides a protocol for applying multidimensional IR spectroscopy to study protein folding.

The energy landscape theory provides an essential framework for understanding protein folding and has widely used to interpret the folding process[1, 2, 3]. The theory assumes a funnel-like shape of the surface which is suciently biased to lead the folding so that the native state can be reached on the experimental time scale. Monitering conformational changes of proteins is important to understand the details of energy landscape and thus uncover the mechanism of folding process. Unfortunately, there are very limited eective probes to detect structural evolution on the

energy landscape on the time scales of folding dynamics. Commonly used spectroscopic techniques such as NMR, due to the insuciency of well-resolved spectral traits for dierent structures and limited time resolution, may not directly trace the dynamical process. Recently, as one of the ultrafast vibrational spectroscopy that probes the transient molecular structural changes, the two-dimensional infrared (2DIR)spectroscopy provides a potential promising avenue to investigate protein folding dynamic[?, 5, 6], In 2DIR experiments, The amide I band, primarily associated with the peptide bond carbonyl stretch, is by far the most studied because it is sensitive to the hydrogen bonding, dipole-dipole interactions, and geometry of the peptide backbone, providing a good indicator of the secondary structures changes. The crosspeaks (o-diagonal peaks in 2DIR plots) of amide bands carry signatures of intraand intermolecular couplings, providing additional structural information. Here we report a theoretical 2DIR study of folding behaviors of Beta3s on the free energy landscape. Beta3s includes 20 amino acids (Thr1-Trp2-Ile3-Gln4-Asn5-Gly6-Ser7-Thr8Lys9-Trp10-Tyr11-Gln12-Asn13-Gly14-Ser15-Thr16-Lys17-Ile18-Tyr19-Thr20), and its solution conformation has been studied by NMR [7]. Data of Nuclear Overhauser Enhancement (NOE) and chemical shift shows that these 20 amino acid can fold into a single structured compact form, the three-stranded antiparallel -sheet conformation with turns at Gly14-Ser15 and Gly6-Ser7, in equilibrium solution[7].

In this study, molecular dynamics (MD) was applied to simulate the folding dynamics of the peptide. We used CHARMM c35b5 package [8] with a parameter set TOPH19/PARAM19[9] to perform all MD simulations and part of the analysis of the trajectories. The folding simulation were performed with an implicit model for the solvent[10]. This approximation was justied in a recent study which shows that the solvent does not inuence signicantly the results of the folding process of a a synthetic three-stranded antiparallel -sheet mini-protein, Betanova[11]. The nonbonded interaction list was extended to cut o as 7.5 A, and the covalent bond length including hydrogen atoms were xed with the SHAKE algorithm. From the native structure, all 20 trajectories were simulated at constant temperature 330K, and the simulation time for each trajectory was set to 500 ns. The total 10-s folding simulation times provided the ecient data to build up the free energy landscape which is a function of RMSD (root mean square deviation) and Q (fraction of native contacts of C atoms). The native contacts were listed in Table 1, and a contact was dened as formed when its pair distance falls within a range rnative 2 A, where rnative is the distance of that contact in the native structure. Along the folding dominant pathway from the unfolded state to the folded state, 100 locations were chosen on the energy landscape. The distance between any two adjacent locations was moderate, so that these locations can characterize the whole pathway. 150 conformations for each

No. 1 2 3 4 5 6 7 8 9 10 11

1st residue Thr1 Thr1 Trp2 Trp2 Ile3 Ile3 Gln4 Gln4 Thr8 Lys9 Trp10

2nd residue Tyr11 Gln12 Lys9 Tyr11 Lys9 Trp10 Thr8 Lys9 Thr20 Thr20 Tyr19

No. 12 13 14 15 16 17 18 19 20 21 22

1st residue Tyr11 Gln12 Asn13 Gly14 Lys17 Trp2 Ile3 Trp10 Tyr11 Ile18 Thr16

2nd residue Ile18 Lys17 Thr16 Thr16 Tyr19 Gln4 Asn5 Gln12 Asn13 Thr20 Ile18

Table 1: List of native contacts for peptide Beta3s location were chosen to calculate the excitation Hamiltonian and multidimensional ultraviolet spectroscopy signals. The protocol of Hamiltonian and signal calculations have been illustrated in the other papers[12]. Briey, The 2DIR signal was generated by three impulsive coherent short laser pulses with wavevectors k1 , k2 , and k3 , respectively. One can detect the coherent signal eld along the phase-matching direction:k4 = k1 + k2 + k3 . Calculations were performed for the non-chiral (xxxx) and chirality-induced (xxxy) polarization congurations. xxxx represents that all optical pulses were polarized along x direction, and xxxy represents that three optical pulses were polarized along 5

x direction while the other one y polarized. Fig.?? showed the free energy landscape of Beta3s folding. The free energy landscape was plotted as a function of the RMSD and Q and obtained by F = log (P ), where P was the statistic population obtained from all the 10-s MD simulated data. Five locations were chosen to illustrate the folding process along which L1 had extended strand structure, and the peptide traped into a state that the N-terminal and C-terminal of peptide were random-coil structures, yet the middle part of the peptide (8-13 residues) formed short -helix structure at L25. When the peptide arrived at L50, the short -helix structure reopened and the orientations of three strands formed. Basing on the three-strand frame ,two beta strands gradually grew from L50 to L75. L100 was the folded state which was consisted of three antiparallel sheets. This folding scenario is consistant with the previous study [13]. To demonstrate the structural changes that occur during folding, we further display the transition dipole couplings in Fig.??. At L1, the coupling is weak and is dominated by nearest-neighbor couplings. This is indicative of the random coil. At L25, a short -helix structure forms from residues 8 to 13 has formed as seen by the strong positive nearest neighbor coupling and the strong negative 1-3 coupling in this region [12]. ......... Fig.1(a) shows the infrared absorption spectra of the ve locations on the free

L1

L25

L50

L75

L100

Figure 1: Linear absorption and Non-chiral 2DIR spectra. (a)Linear absorption spectra of the locations L1, L25, L50, L75, and L100. (b-f)Non-chiral(xxxx) 2DIR signals of the ve locations.

energy landscape. They all have one major peak, which red-shifts from 1645 cm1 (the featured spectra region of random coil structure) at L1, to 1630cm1 (the featured spectra region of -sheets structure) at L100. Besides, the intensity of the peaks basically decrease during folding since the formation of the hydrogen bonds of the peptide backbone weakens the amide I bond. Fig.1(b-f) shows the absorptive 2D xxxx spectra. We present the imaginary part of the spectra which give better comparison for the dierent locations. From L1 to L100,

although they all have two lobes which locate beside the diagonal lines, the position and shape of the lobes vary during the folding process. Before the orientations of three strands form and the beta-sheet structures emerge at L75, the signals have similar gaussian shape at L1, L25, and L50. When the -sheets structure emerges and grows up at L75, and nally reaches the folded state L100, the two lobes tend to separate and the positive lobe(lower lobe) shrinks from [1620cm1 , 1660cm1 ] to [1620cm1 , 1640cm1 ], and apparently the upper part of this lobe tends to extend to the right direction. This variation of the 2D xxxx spectra can be understood by the symmetry of the structure and the short-range couplings between the amide I vibrations. When the antiparallel sheets gradually form, the amide I vibrational units (primarily C=O bonds) between dierent sheets interfere with each other due to the sheets getting closer and are stabilized by the inter-strand hydrogen bonds. The vibrational interaction for these sheets can be modeled as the coupled an-harmonic oscillators, and the couplings between these oscillators inuence signicantly the 2DIR spectra [15]. The formation of the sheets strengths this interaction and leads the lobes of 2DIR xxxx spectra separate by the + and resonances [15]. The chirality-induced signals depend explicitly on coordinate-dependent which may amplify or diminish the cross-peaks of 2DIR spectra, and this property make it very sensitive to the structural changes. There are two types of chirality in the

L1

L25

L50

L75

L100

Figure 2: (a-e)Chiral 2DIR spectra of the locations L1, L25, L50, L75, and L100 on the folding pathways. (f-h)Dierence of the chiral(xxxx) 2DIR signals of L25 and L1, L50 and L25, L100 and L75, respectively.

molecular systems. One is the local chirality originated from the local individual residues and the other is associated with the global structure of the protein. Here we focus on the second type because it may dominate the response in extended system [?]. Since the signals are inuenced by the chirality of the protein, one can expect that the additional chirality introduced by the hierarchical folding should be changed with respect to the chirality of the constituent elements. The vibrational circular dichroism (VCD) and CI 2DIR spectra are shown in Fig.

2. Fig.2(a) shows that the VCD intensity increases from L1 to L25 which has helix structure, and then decreases to L100 when the sheet structures gradually form during folding. In the xxxy spectra, the intensities of the diagonal lobes increase from L1 to L25, and then decrease to L100, similar to VCD spectra. Also, the ranges of the diagonal lobes shrink when the sheet structures form, especially the lower positive lobe. The cross peak of the xxxy spectra also has similar changes. From the L25 to the L75. The spatial antiparallel orientation gradually form and the preliminary antiparallel sheets emerge in these two strands. During this process, the cross peak locating on (-1620cm1 , 1645cm1 ) decreases, indicating the formation of the antiparallel orientations decreases the chirality of the protein and thus cancels the signals of the side bands. The intensity of the cross peak continues decreasing as the protein evolves to L100, implying the topological chirality of the protein further recedes as the -sheets grow up. The variation of the CI signals of the protein folding can be a good potential indicator to track the process of the protein folding. Studies showed [16, 17] the topological chirality of protein is very important in the folding process, yet there are still limited experimental tools to detect that. Although circular dichroism (CD) is a chiral signal, because of the inhomogeneous broadening of spectra due to geometric uctuations, the CD signatures of dierent structural features are still not well resolved. Coherent chirality-induced 2D signals, with its advantages

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of background-free and sensitivity to the structural change, oer a powerful tool to investigate the protein folding and thus shed an additional light on the general principles of protein folding. These results have demonstrated how conformational evolution on the folding free energy landscape can be monitered by the multidimensional IR spectroscopy. The linear absorption spectra, non-chiral and chirality-induced 2DIR spectra were simulated to illustrate the conformational changes during the foding process. The non-chiral signals indicates the lobes of the imaginary spectra tend to separate when the beta-sheet elements develops in the folding process, and the chiral signals show the topological chirality of the protein gradually decreases when the native structure of the protein forms, suggesting 2DIR spectra is good proble to detect protein folding dynamic.

Acknowledgement
The authors thank the National Science Foundation and National Institute of Health for support.

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