Global and Local Structural Changes of Protein Unfolding

(1)

Global and Local Structural Changes of Protein

Unfolding

Ying-Jen Shiu ,*1_{U-Ser Jeng,}2_{Sheng-Hsien Lin}1,3

Summery: In the mean-field Ising model, the folding-unfolding behavior of a protein is regarded as an ensemble of units reduced from, presumably, peptide bonds or amino acid residues. Units of similar thermodynamic properties are further classified into groups that are related to, for instance, a-helices or b-sheets. Units of the same group are assumed to unfold collectively, whereas those of different groups may undergo either sequential or coupled unfolding. The introduction of unfolding groups facilitates the description of non-collective local structural changes experimentally observed via a multi-group unfolding. We incorporate denaturants and temperature effects into the free energy expression of the protein upon dissolution in a specific environment at thermal equilibrium, and a model protein, cytochrome c, was examined. Results indicate that there are at least four unfolding groups induced unfolding of cytochrome c: two are related to the prosthetic heme group, whereas another two groups are a-helices and global nearly group, which largely account for global changes in protein morphology. The extracted thermodynamic parameters, on the basis of the Ising model, can closely predict unfolding behaviors of the proteins in compounded denaturing environments. Keywords: cytochrome c; ising model; mean filed approximation; multi-group unfolding

Introduction

The Ising model with a mean field approx-imation developed by Lin’s group can describe both kinetics and thermodynamics of protein folding-unfolding influenced by denaturant effects[1–3] or calorimetric change of enthalpy, as well as the unfolding in atomic force measurements,[4,5] _{via a}

microscopic point of view. The model displays the unfolded fraction as f‘

u¼ 1þf

exp½2"‘þ4J‘ð12fu‘Þ

kBT g

1

, where the unfolding free energy DG‘_ðC m;TÞ ¼ 2"‘is determined by "‘_D"‘ mCmþ D"‘TðT T1=2‘ Þ. [6,7] The Ising model provides a simple yet coherent

description on the multi-group unfolding behaviors of the protein not only at global but also local scales via considering multi-groups unfolding as a summation behaviors

of the individual correlated groups

fu¼P‘g‘fu‘. In this study, the unfolded

cytochrome cwas introduced by presence of urea (0 to 10 M) as well as changes in temperature (295 to 363 K). The global morphology of cytochrome c was observed via small-angle X-ray scattering (SAXS), the local behaviors such as heme group and the secondary structure were observed by UV-Vis absorption and circular dichroism (CD).

Results and Discussion

To consistently extract parametric values 2DeT, 2Dem, and T1/2, we have jointly fitted

the three urea-dependent fuprofiles together

with the three temperature-dependent fu

profiles, as listed in Table 1.

The results show unfolding of a-helices is closely related to the global morphology

Macromol. Symp.2009, 279, 63–64 DOI: 10.1002/masy.200950510 63

1_{Institute of Atomic and Molecular Sciences,} Acade-mia Sinica, P. O. Box 23-166, Taipei 106, Taiwan Fax: (þ886) 2-23624929;

E-mail: [email protected]

2_{National Synchrotron Radiation Research Center,} Hsinchu 300, Taiwan

3_{Department Applied Chem. and Inst. Molecular} Science, National Chiao Tung University, Hsinchu, 30010, Taiwan

(2)

change in the unfolding process. In contrast, the fu,abs profile exhibits two-step features,

characteristic of a two-group unfolding at lower temperatures. The g values suggest that the global structural change with minor contributions from Heme and a-helices, and dominant contribution from the global-nearly group observed by SAXS. We have further extracted the thermal unfolding free energy DGo(T)¼ 2DeT(T T1/2) for each

group. With the denaturant and temperature effects incorporated in the free energy expression, we may predict the unfolded fraction of cytochrome c at arbitrary tem-perature and/or urea concentrations. The calculated fu profile using thermodynamic

parameters given in Table 1 matches well with the independent SAXS observation

of thermally induced unfolding of

cytochrome c in 6 M urea, as shown in Figure 1. This urea concentration is expected to result in decreased DG and hence a shift of the unfolded fraction profile

by DT¼ DemCm/DeT¼ 47 K from the

dena-turant-free counterpart.

Conclusions

Combining SAXS and spectroscopic tech-niques, we have demonstrated that a recently developed mean-field Ising model provides an adequate basis for the quanti-tative description of the unfolding beha-viors of cytochrome c induced by changes in temperature and urea concentration. A multi-group unfolding behavior of cyto-chrome c was observed. Thermodynamic parameters extracted from simple denatur-ing processes, on the basis of the Isdenatur-ing model, can be used to predict unfolding behaviors of the proteins in compounded denaturing environments. Integrated local and global structure information improves the understanding of the protein folding-unfolding mechanism.

[1] K. K. Liang, M. Hayashi, Y. J. Shiu, Y. Mo, J. Shao, J. Yan, S. H. Lin, J. Chin. Chem. Soc.2003, 50, 335–338. [2] K. K. Liang, M. Hayashi, Y. J. Shiu, Y. Mo, J. Shao, Y. Yan, S. H. Lin, Phys. Chem. Chem. Phys.2003, 5, 5300– 5308.

[3] Y. J. Shiu, C. Su, Y. L. Yeh, K. K. Liang, M. Hayashi, Y. Mo, Y. Yan, S. H. Lin, J. Chin. Chem. Soc.2004, 51, 1161– 1173.

[4] Y. L. Yeh, K. K. Liang, C. H. Chang, Y. J. Shiu, C. Su, M. Hayashi, G. Yang, J. M. Yuan, C. L. Chyan, S. Jang, F. Y. Li, S. H. Lin, Trends in Phys. Chem.2004, 40, 169– 205.

[5] Y. L. Yeh, C. H. Chang, K. K. Liang, Y. J. Shiu, C. Su, M. Hayashi, C. L. Chyan, G. Yang, Y. Mo, Y. J. Yan, S. H. Lin, Chem. Phys. Lett.2004, 399, 440–445.

[6] Y. J. Shiu, U. Jeng, C. Su, Y.-S. Huang, M. Hayashi, K.-K. Liang, Y.-L. Yeh, S. H. Lin, J. Appl. Cryst.2007, 40, s195–s199.

[7] Y. J. Shiu, U. Jeng, Y. S. Huang, Y. H. Lai, H. F. Lu, C. T. Liang, I. J. Hsu, C. H. Su, C. Su, I. Chao, A. C. Su, S. H. Lin, Biophys. J.2008, 94, 1–9.

Table 1.

Fitted thermodynamic parameters of cytochrome c in urea- and temperature-induced unfolding.

Group g 2Dem(kcal mol1M1) 2DeT(kcal mol1K1) T1/2(K) DGo(293K) (kcal mol1)

Heme 0.011 0.92 0.2 0.138 0.015 333 11 5.9 0.6

Heme 0.029 1.03 0.10 0.138 0.015 346 5 7.3 0.8

a-helices 0.12 1.30 0.21 0.167 0.026 354 1 10.2 0.8

Global-nearly 0.84 1.30 0.21 0.167 0.026 359 5 10.9 0.9

Figure 1.

Two temperature dependent profiles (solid curves) displayed together with the SAXS results (scattered points) demonstrate the unfolding fraction without and with 6 M urea of cytochrome c. Both calculated unfolding fraction profiles share the same thermo-dynamic parameters in Table 1. The profile of 6 M urea indicates a nice prediction for the SAXS observation.

Macromol. Symp.2009, 279, 63–64 64