N I S T Center for Neutron Research

Accomplishments and Opportunities 2001

Native and Partially Unfolded Proteins: Neutron Inelastic Scattering and Simulations

Proteins are long chain-like molecules that are intricately folded. As they function they unfold in specific ways. How proteins can quickly refold without tangling remains a central mystery in molecular biology. Partially folded proteins provide an approach to this problem, and are also important in cell functioning. The approach is that stable partially folded states have been shown to resemble the kinetic folding intermediate states along the protein-folding pathway. A molten globule (M G) is a compact partially folded protein, with a backbone resembling the completely folded protein, but lacking the extensive specific side-chain packing interactions of the native-like (i.e., properly folded) structure. A full understanding of the mechanism of protein folding requires the knowledge of the structures, relative energetics, and dynamics of the species populating the folding pathway. Incoherent inelastic neutron scattering (I N S), which makes use of the large incoherent cross section of hydrogen nuclei, is a technique well suited to the study of protein internal molecular motion on the picosecond time scale. Molecular dynamics (M D) simulations, on the other hand, are a potentially valuable tool for interpreting neutron data on proteins (Refer to References 1 and 2), and may provide atomic level description of the motions taking place.

In this study, we have explored the dynamics of bovine α - lactalbumin, a calcium-binding protein, in the native and molten globule states, using both techniques. I N S data were collected on the Disk-Chopper Spectrometer at ≈ 32 µeV resolution for native and molten globular bovine α - lactalbumin. The simulations results were generated from ≈ 1 ns constant temperature and pressure M D trajectories. The model systems consisted each of a single protein monomer, immersed in a large water box (60 x 50 x 80 ). For the native state, the α - lactalbumin configuration was initiated from the crystal structure. The model molten globule state was generated by partially unfolding the protein at 500 K at atmospheric pressure until the radius of gyration reached the experimental value determined for the molten globule state (10 % expansion). A 1 n s trajectory was then generated at 300 K for data analysis and comparison with experiment.

Graphics Caption FIGURE 1. α - lactalbumin structure in the native (top) and molten globule (bottom) state from M D simulations. Note the loss of the β - domain structure, with most the α domain (helix) being conserved.

Graphics Caption FIGURE 2. Incoherent structure factor S(Q,ω) at selected wavevectors Q, measured and calculated from the simulation for the native (top) and molten globule (bottom) states.

Graphics Caption FIGURE 3. 100 p s mean squared fluctuations of the backbone atoms computed from the M D simulations of the native state (black) and the molten globule state (red).

Figure 1 shows the structure of the α - lactalbumin in the native and molten globule states obtained from the M D simulations in solution at 300 K. The overall structural change, manifested by the conservation of the helical α - domain and the loss of the β sheets in the β - domain, is consistent with N M R data (Refer to Reference 3). We therefore regard this structure as a reasonable model of a member of the M G ensemble for comparison with the I N S data.

In Figure 2 we report the incoherent structure factors S (Q,ω) as a functions of energy transfer h ω for selected momentum transfer Q, measured and calculated from the M D simulations trajectories. In both systems the agreement with experiment is remarkable, assessing the ability of the simulations to reproduce the picosecond dynamics of the protein. In agreement with previous INS data collected at lower resolution (Refer to Reference 4), the molten globule has a broader quasi-elastic spectra compared to the native state, which indicates that the protein atoms are more mobile in the M G state. The additional motion is large enough to be detected at the length scale probed by the D C S spectrometer, i.e., in the range 3 to 60 .

To investigate the relationship between the structure of the protein and the motion of its side chains, we have used the M D trajectories to follow the motion of these side chains quantitatively. Figure 3 reports the fluctuations per residue along the backbone for the native and the molten globule states, as a function of residue. These correspond to the 100p s root-mean-square amplitudes of motion of the backbone atoms, averaged over the simulation trajectories. First for the native state, the backbone fluctuations appear to correlate with the secondary structure of the protein, showing much smaller amplitudes in the α - helix and β - sheet regions. For the molten globule state, a significant increase in the protein fluctuation amplitudes is observed, in agreement with the broadening of the I N S spectra.

Figure 3 shows clearly that most of the additional motion occurs in the region of the protein that unfolds, i.e., the β - domain. The results indicate also that the motion of the side chains is by no means homogeneous. Differences in amplitudes up to 10 folds are observed between parts of the proteins, regardless of exposure to the solvent.

The M D study of the dynamics of partially folded states of the protein should in principle be extended to explore a large portion of the conformational space, i.e., be extended to a large ensemble of non-native conformers. At any rate, the present results, though limited, combined with neutron experiments are believed to capture the essence of the structural and dynamical changes taking place as the protein partially unfolds. They demonstrate the utility of the M D simulations for qualitatively elucidating the dynamical behavior of native and non-native proteins at the atomic level.

References

[1] M. Tarek, G. J. Matryna, and D. J. Tobias, J. Amer. Chem. Soc. 122, 10450 (2000).

[2] M. Tarek and D. J. Tobias, Biophys. J. 79, 3244 (2000).

[3] J. Baum et al., Biochemistry, 28, 7 (1989).

[4] Z. Bu et al., J. Mol. Biol. 301, 525 (2000).

Authors

M. Tarek
N I S T Center for Neutron Research
National Institute of Standards and Technology
Gaithersburg, MD 20899-8562
 and
Center for Molecular Modeling and
Department of Chemistry, University of Pennsylvania
Philadelphia, PA 19103

D. J. Tobias
Department of Chemistry
University of California
Irvine, CA 92697-632

Z. Bu and D. A. Neumann
N I S T Center for Neutron Research
National Institute of Standards and Technology
Gaithersburg, MD 20899-8562

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