擁擠效應是體內細胞質主要能穩定蛋白質的原因，經由在溶液中的排除體積效應，使蛋白質在擁擠環境中能變得更加穩定。本研究中以兩種擁擠試劑Ficoll 70和Dextran 70做為體外模擬擁擠環境的高分子聚醣類，以HP36代表α螺旋；WW-domain代表β摺疊；膠原蛋白代表三股螺旋，分別觀察三種不同結構的胜肽在兩種擁擠環境中的變化，利用CD光譜量測在擁擠環境下結構的強度與藉由熱變性與化學變性的實驗結果來探討其穩定性的變化。
在大部分的體外擁擠環境模擬中，擁擠試劑通常為高分子，其穩定度的結果會因為Flory–Huggins solution effect造成穩定程度呈現階梯狀的上升，並且擁擠效應內擁擠試劑所具有的軟效應在排除體積效應較不明顯時，時常主導了整個環境，導致擁擠效應的結果在擁擠試劑濃度較低時反而使胜肽變得更加不穩定。直到擁擠試劑的濃度上升至ㄧ定比例，排除體積效應才逐漸與軟效應相互抵消，且溶液中的胜肽結構趨向穩定。在化學變性的過程中，觀察到胜肽結構在解摺疊狀態的壓縮，代表著在擁擠試劑與化學變性試劑兩者的交互作用下，將會使胜肽的解摺疊結構改變，隨著擁擠試劑濃度上升至30% 時，摺疊態與解摺疊態間的能階差將會下降。最後將擁擠試劑的形狀與胜肽的構形作為探討目標，發現在溶液中形狀相似、大小接近的分子將會導致更強且更平滑的排除體積效應。
藉由實驗結果歸納出以大分子模擬擁擠環境的過程中所會面對到的困難，提供出ㄧ個更為完備的方式去模擬擁擠環境，在未來選用擁擠試劑與決定溶液中擁擠試劑的重量百分濃度時，將可以針對其溶液中的作用力做出更全面的考量，期望此結果可對於蛋白質舒適的擁擠環境所具有的作用力引發更多的探討，進而更加了解蛋白質結構在擁擠環境中對於環境的容忍力提升的原因，對於穩定蛋白質提供另一種簡單且不具破壞性的途徑。

The proteins in cells can be stabilized by the macromolecular crowding effects in cytoplasm due to excluded volumn effects. Here we used two different carbohydrate polymers, semi-rigid spherical Ficoll 70 and rod-like Dextran 70, to mimic the enviroment in cytoplasm. In our study, three different peptides, HP36 for an α-helix; WW-domain for a β-sheet; collagen for a triple helix, were chosen to study the crowding effects on their conformational stability in vitro.
Using polymers to be a crowder will have Flory–Huggins solution effect, and thus the peptide will be significantly stabilized as the concentration of crowders increases to approximately 15%. In this case, the crowding effect is not obvious at first and the soft effect may actually destabilize the peptide at low concentrations of crowders. After increasing the concentration of crowders, the crowding effect can gradually compensate the destabilization due to the soft effect and make the peptide more stable. In chemical denaturation, the interactions including crowders and denaturants are involved in the unfolded state, which will make the unfolded state more compact. As the crowder concentration increases up to 30%, the free energy gap between the folded state and the unfolded state will decrease. Furthermore, we will discuss the relationship between peptide conformations and crowder shapes. Our finding indicates that the crowding effect will be more significant if the shape and size of crowders are similar to those of peptides.
To sum up the results, our study attempted to provide a more accurate method to mimic a crowding enviroment. Hopefully, the results can initiate more attention to the interactions between solvents and proteins, and provide a simple physical way to stabilize protein.

1.Vardar, D.; Chishti, A. H.; Frank, B. H.; Luna, E. J.; Noegel, A. A.; Oh, S.W.; Schleicher, M.; McKnight, C. J., Villin‐Type Headpiece Domains Show a Wide Range of F‐actin‐binding Affinities. Cell Motility. Cytoskeleton 2002, 52, 9-21.
2.Bazari, W. L.; Matsudaira, P.; Wallek, M.; Smeal, T.; Jakes, R.; Ahmed, Y., Villin Sequence and Peptide Map Identify Six Homologous Domains. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 4986-90.
3.McKnight, J. C.; Doering, D. S.; Matsudaira, P. T.; Kim, P. S., A Thermostable 35-Residue Subdomain within Villin Headpiece. J. Mol. Biol. 1996, 260, 126-134.
4.Xiao, S.; Bi, Y.; Shan, B.; Raleigh, D. P., Analysis of Core Packing in a Cooperatively Folded Miniature Protein: The Ultrafast Folding Villin Headpiece Helical Subdomain. Biochemistry 2009, 48, 4607-4616.
5.Sudol, M., Structure and Function of the WW domain. Prog. Biophys. Mol. Biol. 1996, 65, 113-132.
6.Ranganathan, R.; Lu, K. P.; Hunter, T.; Noel, J. P., Structural and Functional Analysis of the Mitotic Rotamase Pin1 Suggests Substrate Recognition Is Phosphorylation Dependent. Cell 1997, 89, 875-886.
7.A., K. J.; Kai, L.; W., K. J., NMR Solution Structure of the Isolated Apo Pin1 WW domain: Comparison to the X‐ray Crystal Structures of Pin1. Biopolymers 2002, 63, 111-121.
8.Zarrinpar, A.; Lim, W. A., Converging on proline: the mechanism of WW domain peptide recognition. Nat. Struct. Biol. 2000, 7, 611-613.
9.陳柏翰、劉中行, 科學發展期刊 2004, 380期, 4-35.
10.黃彥富、湯正明、徐善慧, 科學發展期刊 2003, 362期, 44-47.
11.Cowan, P. M.; McGavin, S.; North, A. C. T., The Polypeptide Chain Configuration of Collagen. Nature 1955, 176, 1062.
12.Rich, A.; Crick, F. H. C., The Molecular Structure of Collagen. J. Mol. Biol. 1961, 3, 483-IN4.
13.Sakakibara, S.; Inouye, K.; Shudo, K.; Kishida, Y.; Kobayashi, Y.; Prockop, D. J., Synthesis of (Pro-Hyp-Gly)n of Defined Molecular Weights Evidence for the Stabilization of Collagen Triple Helix by Hydroxyproline. BBA.- Protein Structure 1973, 303, 198-202.
14.Brodsky, B.; Ramshaw, J. A. M., The Collagen Triple-Helix Structure. Matrix Biol. 1997, 15, 545-554.
15.Kuznetsova, I. M.; Turoverov, K. K.; Uversky, V. N., What Macromolecular Crowding Can Do to a Protein. Int. J. Mol. Sci. 2014, 15, 23090-23140.
16.Zimmerman, S. B.; Minton, A. P., Macromolecular Crowding: Biochemical, Biophysical, and Physiological Consequences. Annu. Rev. Biophys. Biomol. Struct. 1993, 22, 27-65.
17.Kim, J. S.; Yethiraj, A., Crowding Effects on Protein Association: Effect of Interactions between Crowding Agents. J. Phys. Chem. B. 2011, 115, 347-353.
18.Sharma, G. S.; Mittal, S.; Singh, L. R., Effect of Dextran 70 on the Thermodynamic and Structural Properties of Proteins. Int. J. Biol. Macromol. 2015, 79, 86-94.
19.Guseman, A. J.; Pielak, G. J., Cosolute and Crowding Effects on a Side-By-Side Protein Dimer. Biochemistry 2017, 56, 971-976.
20.Bhakuni, K.; Venkatesu, P., Crowded Milieu Tuning the Stability and Activity of Stem Bromelain. Int. J. Biol. Macromol 2018, 109, 114-123.
21.D., C. R.; J., G. A.; J., P. G., Intracellular pH Modulates Quinary Structure. Protein Sci. 2015, 24, 1748-1755.
22.Benton, L. A.; Smith, A. E.; Young, G. B.; Pielak, G. J., Unexpected Effects of Macromolecular Crowding on Protein Stability. Biochemistry 2012, 51, 9773-9775.
23.Sarkar, M.; Lu, J.; Pielak, G. J., Protein Crowder Charge and Protein Stability. Biochemistry 2014, 53, 1601-1606.
24.Rubinstein, M.; Colby, R. H., Polymer Physics, OUP Oxford, 2003.
25.Huggins, M. L., Solutions of Long Chain Compounds. J. Chem. Phys. 1941, 9, 440-440.
26.Flory, P. J., Thermodynamics of High Polymer Solutions. J. Chem. Phys. 1942, 10, 51-61.
27.Sigma-Aldrich Co. Basic steps in solid peptide synthesis using Fmoc-chemistry. http://www.sigmaaldrich.com/life-science/custom-oligos/custom-peptides/learning-center/solid-phase-synthesis.html (accessed June 28, 2018).
28.Merrifield, B., Solid Phase Synthesis. Science 1986, 232, 341-347.
29.Chan, W. C.; White, P. D. Fmoc Solid Phase Peptide Synthesis: A Practical Approach. Oxford University Press, 2000.
30. Skoog, D. A. H.; Crouch, S. R., Principles of Instrumental Analysis. Cengage
Learning. 2007.
31.Purdie, N.; Fasman G.D. Circular Dichroism and the Conformational Analysis of Biomolecules. Springer Science & Business Media, 2013
32.Webb, R. L., Nina Berova, Koji Nakanishi, and Robert W., Circular Dichroism. Principles and Applications, Wiley-VCH, 2000
33.Zaki, A.; Dave, N.; Liu, J., Amplifying the Macromolecular Crowding Effect Using Nanoparticles. J. Am. Chem. Soc. 2012, 134, 35-38.
34.Kumar, S.; Sharma, D.; Kumar, R., Role of Macromolecular Crowding on Stability and Iron Release Kinetics of Serum Transferrin. J. Phys. Chem. B. 2017, 121, 8669-8683.
35.N ozaki, Y., The Preparation of Guanidine Hydrochloride. Methods Enzymol. 1972, 26, 43-50.
36.Kaul, R.; Angeles, A. R.; Jäger, M.; Powers, E. T.; Kelly, J. W., Incorporating β-Turns and a Turn Mimetic out of Context in Loop 1 of the WW Domain Affords Cooperatively Folded β-Sheets. J. Am. Chem. Soc. 2001, 123, 5206-5212.
37.Zhu, T.-T.; Zhang, Y.; Luo, X.-A.; Wang, S.-Z.; Jia, M.-Q.; Chen, Z.-X., Difference in Binding of Long- and Medium-Chain Fatty Acids with Serum Albumin: The Role of Macromolecular Crowding Effect. J. Agric. Food. Chem. 2018, 66, 1242-1250.
38.Somendra, M. B.; Achille, G.; Amos, M., Flory Theory for Polymers. J. Phys.: Condens. Matter 2013, 25, 503101
39.Hong, J.; Gierasch, L. M., Macromolecular Crowding Remodels the Energy Landscape of a Protein by Favoring a More Compact Unfolded State. J. Am. Chem. Soc. 2010, 132, 10445-10452
40.Jäger, M.; Dendle, M.; Fuller, A. A.; Kelly, J. W., A Cross-Strand Trp–Trp Pair Stabilizes the hPin1 WW domain at the Expense of Function. Protein Sci. 2007, 16, 2306-2313.