臺灣博碩士論文加值系統

　　電荷胺基酸提供蛋白質靜電作用力(electrostatics)，而靜電作用力對於蛋白質結構的穩定性有重要的影響。β摺板(β-sheet)是蛋白質二級結構的其中一種並與許多蛋白質錯誤折疊的疾病相關。不同胺基酸對於形成β摺板有不同程度的傾向(propensity)，因此研究帶電荷胺基酸形成β摺板的傾向很重要。本研究著重在精胺酸(Arg)及其側鏈更長和更短的精胺酸衍生物(Agh, Agb, and Agp) 對於形成β摺板的傾向，以及序列位置的改變對其傾向的影響。β髮夾(β-hairpin)是β摺板最基本的構造。我們利用Fmoc主鏈保護的固相胜肽合成技術來合成不同序列位置帶有精胺酸及其衍生物的胜肽。
　　膠原蛋白三重螺旋(collagen triple helix)是由三條polyproline II螺旋(polyproline II helices)所組成。此研究中，我們將不同數目的POG三肽(POG triplets)插入三條具有異三聚體(heterotrimeric)性質且會形成polyproline II螺旋的胜肽序列中間。我們利用圓二色光譜儀做變溫實驗(thermal denaturation experiment)得出膠原蛋白三重螺旋的熔點(melting temperature)。此研究中，我們將不同數目的POG三肽(POG triplets)插入三條具有異三聚體(heterotrimeric)性質且會形成polyproline II螺旋的胜肽序列中間。我們利用圓二色光譜儀做變溫實驗(thermal denaturation experiment)得出膠原蛋白三重螺旋的熔點(melting temperature)。在變溫實驗中，熱平衡時間相當久，甚至接近熔點時的平衡時間需要2000分鐘之久。我們假設膠原蛋白三重螺旋在三聚體(trimer)與單體(monomer)間做平衡，並以膠原蛋白三重螺旋的加熱曲線來做fitting，得出膠原蛋白三重螺旋的熔點。在此實驗中，我們發現POG三肽對於polyproline II螺旋構造具有穩定的功效，然而隨著POG插入的數目增加膠原蛋白三重螺旋的特異性(specificity)會變差。本研究探討POG三肽的數目多寡對於膠原蛋白三重螺旋的穩定性與特異性之間的影響。

Electrostatics is important for protein structure stability. Charged amino acids contribute to these electrostatic interactions. β-Sheet is one of the protein secondary structures and is involved in various protein mis-folding diseases. Different amino acids have different propensities for β-sheet formation. Therefore, it is important to study the propensity of charged amino acids for β-sheet formation. This research focuses on the propensity of arginine (Arg) and arginine analogs with longer and shorter side chains (Agh, Agb, and Agp), and the position dependence of the corresponding sheet propensity. β-Hairpins are the simplest β-sheet structure. Hairpin peptides with arginine and arginine analogs incorporated at different guest sites were synthesized by Fmoc-based solid phase peptide synthesis.
Collagen triple helix consists of three polyproline II helices. In this project, different numbers of POG triplets were inserted in the middle of all three heterotrimeric collagen triple helix forming peptides. Thermal denaturation experiments were used to determine the melting temperatures of the collagen triple helices. Thermal equilibration was found to be extremely long at temperatures around the melting transition, and up to 2000 minutes were required for reversible denaturation and renaturation. The melting temperatures were derived by fitting with the thermal denaturation curve, assuming a two-state trimer to monomer equilibrium. The POG triplet was found to stabilize the polyproline II structure in a collagen triple helix. Specificity was gradually decreased with the addition of more POG triplets. This study provides insights into how to use POG triplets to tune the stability and specificity of a collagen triple helix.

Table of Contents
誌謝................................................i
中文摘要............................................iii
Abstract............................................v
Table of Contents...................................vii
List of Figures.....................................ix
List of Tables......................................xii
List of Schemes.....................................xii
Abbreviations.......................................xiii
Chapter 1 Introduction..............................1
1-1. The Central Dogma of Molecular Biology.........3
1-2. Proteins.......................................4
1-3. Hierarchy of Protein Structures................5
1-3-1. Primary Structure..........................5
1-3-2. Secondary Structure........................6
1-3-3. Tertiary Structure.........................10
1-3-4. Quaternary Structure.......................11
1-4. Driving Forces of Protein Folding..............11
1-5. Collagen Triple Helix..........................14
1-6. Thesis Overview................................14
1-7. Reference......................................15
Chapter 2 Effect of Arginine Side Chain Length on β-Hairpin Stability...................................21
2-1. Introduction...................................23
2-1-1. β-Sheet....................................23
2-1-2. β-Hairpins.................................24
2-1-3. Solid Phase Peptide Synthesis of Difficult Sequences...........................................26
2-1-4. Charged Amino Acids and Arginine Analogs...27
2-2. Results and Discussion.........................29
2-2-1. Peptide Design.............................29
2-2-2. Synthesis of Peptides with Agh/Arg at the Guest Site................................................32
2-2-3. Synthesis of Peptides with Agb at the Guest Site................................................38
2-2-4. Synthesis of Peptides with Agp at the Guest Site................................................51
2-3. Conclusion.....................................54
2-4. Future Aspects.................................54
2-5. Acknowledgement................................55
2-6. Experimental Section...........................56
2-6-1. General Materials and Methods..............56
2-6-2. Fmoc-(S)-2-amino-di-boc-6-guanidinohexanoic acid................................................57
2-6-3. Fmoc-(S)-2-amino-di-boc-6-guanidinopropanoic acid................................................58
2-6-4. Peptide Synthesis..........................59
2-6-5. Nuclear Magnetic Resonance Spectroscopy....62
2-7. Reference......................................83
Chapter 3 Effect of the Number of POG Triplets on Heterotrimeric Collagen Triple Helix Stability and Specificity.........................................91
3-1. Introduction...................................93
3-1-1. Collagen...................................93
3-1-2. Collagen Triple Helix Stability............93
3-1-3. Collagen Triple Helix Specificity..........96
3-1-4. Thermal Denaturation Experiment............96
3-2. Results and Discussion.........................98
3-2-1. Collagen Triple Helix Peptide Design.......98
3-2-2. Thermal Denaturation Experiments (Irreversible) ....................................................100
3-2-3. Determining Equilibration Times for Reversible Folding/Unfolding...................................113
3-2-4. Reversible Thermal Denaturation............126
3-3. Conclusion.....................................147
3-4. Future Aspect..................................147
3-5. Acknowledgement................................148
3-6. Experimental Section...........................149
3-6-1. General Materials and Methods..............149
3-6-2. Peptide Synthesis..........................150
3-6-3. UV-vis Spectroscopy........................152
3-6-4. Circular Dichroism Spectroscopy............153
3-7. Reference......................................167

Chapter 1
1.Sumner, J. B. The isolation and crystallization of the enzyme urease preliminary paper. J. Biol. Chem. 1926, 69, 435-441.
2.Milligan, G. Constitutive activity and inverse agonists of G protein-coupled receptors: a current perspective. Mol. Pharmacol. 2003, 64, 1271-1276.
3.Krishnan, J.; Selvarajoo, K.; Tsuchiya, M.; Lee, G.; Choi, S. Toll-like receptor signal transduction. Exp. Mol. Med. 2007, 39, 421-438.
4.Gosline, J.; Lillie, M.; Carrington, E.; Guerette, P.; Ortlepp, C.; Savage, K. Elastic proteins: biological roles and mechanical properties. Philos. Trans. R. Soc. B-Biol. Sci. 2002, 357, 121-132.
5.Crick, F. Central dogma of molecular biology. Nature 1970, 227, 561-563.
6.Martin, R. B. Free energies and equilibria of peptide bond hydrolysis and formation. Biopolymers 1998, 45, 351-353.
7.Kahne, D.; Still, W. C. Hydrolysis of a peptide bond in neutral water. J. Am. Chem. Soc. 1988, 110, 7529-7534.
8.Smith, R. M.; Hansen, D. E. The pH-rate profile for the hydrolysis of a peptide bond. J. Am. Chem. Soc. 1998, 120, 8910-8913.
9.Pauling, L.; Corey, R. B. The Planarity of the amide group in polypeptides. J. Am. Chem. Soc. 1952, 74, 3964-3964.
10.Weiss, M. S.; Jabs, A.; Hilgenfeld, R. Peptide bonds revisited. Nat. Struct. Mol. Biol. 1998, 5, 676-676.
11.Jabs, A.; Weiss, M. S.; Hilgenfeld, R. Non-proline cis peptide bonds in proteins. J. Mol. Biol. 1999, 286, 291-304.
12.Sanger, F.; Tuppy, H. The amino-acid sequence in the phenylalanyl chain of insulin. 2. The investigation of peptides from enzymic hydrolysates. Biochem. J. 1951, 49, 481-490.
13.Sanger, F.; Thompson, E. O. The amino-acid sequence in the glycyl chain of insulin. II. The investigation of peptides from enzymic hydrolysates. Biochem. J. 1953, 53, 366-374.
14.Pauling, L.; Corey, R. B.; Branson, H. R. The structure of proteins: two hydrogen-bonded helical configurations of the polypeptide chain. Proc. Natl. Acad. Sci. U. S. A. 1951, 37, 205-211.
15.Pauling, L.; Corey, R. B. The pleated sheet, a new layer configuration of polypeptide chains. Proc. Natl. Acad. Sci. U. S. A. 1951, 37, 251-256.
16.Venkatachalam, C. Stereochemical criteria for polypeptides and proteins. V. Conformation of a system of three linked peptide units. Biopolymers 1968, 6, 1425-1436.
17.Ramachandran, G.; Kartha, G. Structure of collagen. Nature 1954, 174, 269-270.
18.Ramachandran, G.; Kartha, G. Structure of collagen. Nature 1955, 176, 593-595.
19.Donohue, J. Hydrogen bonded helical configurations of the polypeptide chain. Proc. Natl. Acad. Sci. U. S. A. 1953, 39, 470-478.
20.Ramachandran, G. N.; Ramakrishnan, C.; Sasisekharan, V. Stereochemistry of polypeptide chain configurations. J. Mol. Biol. 1963, 7, 95-99.
21.Edsall, J. T.; Flory, P. J.; Kendrew, J. C.; Liquori, A.; Nemethy, G.; Ramachandran, G.; Scheraga, H. A proposal of standard conventions and nomenclature for the description of polypeptide conformation. J. Biol. Chem. 1966, 241, 1004-1008.
22.Kendrew, J. C.; Bodo, G.; Dintzis, H. M.; Parrish, R.; Wyckoff, H.; Phillips, D. C. A three-dimensional model of the myoglobin molecule obtained by x-ray analysis. Nature 1958, 181, 662-666.
23.Arnott, S.; Wonacott, A. J. Atomic co-ordinates for an α-helix: refinement of the crystal structure of α-poly-l-alanine. J. Mol. Biol. 1966, 21, 371-383.
24.Barlow, D.; Thornton, J. Helix geometry in proteins. J. Mol. Biol. 1988, 201, 601-619.
25.Hol, W. G.; Van Duijnen, P. T.; Berendsen, H. The α-helix dipole and the properties of proteins. Nature 1978, 273, 443-446.
26.Muñoz, V.; Serrano, L. Helix design, prediction and stability. Curr. Opin. Biotechnol. 1995, 6, 382-386.
27.Blake, C.; Koenig, D.; Mair, G.; North, A.; Phillips, D.; Sarma, V. Structure of hen egg-white lysozyme: a three-dimensional Fourier synthesis at 2 Å resolution. Nature 1965, 206, 757-761.
28.Hovmöller, S.; Zhou, T.; Ohlson, T. Conformations of amino acids in proteins. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2002, 58, 768-776.
29.Lim, Y.-b.; Lee, M. Nanostructures of β-sheet peptides: Steps towards bioactive functional materials. J. Mater. Chem. 2008, 18, 723-727.
30.Chothia, C. Conformation of twisted β-pleated sheets in proteins. J. Mol. Biol. 1973, 75, 295-302.
31.Ho, B. K.; Curmi, P. M. Twist and shear in β-sheets and β-ribbons. J. Mol. Biol. 2002, 317, 291-308.
32.Weatherford, D.; Salemme, F. R. Conformations of twisted parallel β-sheets and the origin of chirality in protein structures. Proc. Natl. Acad. Sci. U. S. A. 1979, 76, 19-23.
33.Citron, M. Alzheimer''s disease: strategies for disease modification. Nat. Rev. Drug Discov. 2010, 9, 387-398.
34.Feany, M. B.; Bender, W. W. A Drosophila model of Parkinson''s disease. Nature 2000, 404, 394-398.
35.Palmer, M. S.; Dryden, A. J.; Hughes, J. T.; Collinge, J. Homozygous prion protein genotype predisposes to sporadic Creutzfeldt-Jakob disease. Nature 1991, 352, 340-342.
36.Cowan, P. M.; McGavin, S. Structure of poly-L-proline. Nature 1955, 176, 501-503.
37.Hopfinger, A. Conformational properties of macromolecules. Elsevier: 2012.
38.Kumar, P.; Bansal, M. Structural and functional analyses of PolyProline-II helices in globular proteins. J. Struct. Biol. 2016, 196, 414-425.
39.Kelly, M. A.; Chellgren, B. W.; Rucker, A. L.; Troutman, J. M.; Fried, M. G.; Miller, A.-F.; Creamer, T. P. Host-guest study of left-handed polyproline II helix formation. Biochemistry 2001, 40, 14376-14383.
40.Adzhubei, A.; Eisenmenger, F.; Tumanyan, V.; Zinke, M.; Brodzinski, S.; Esipova, N. Approaching a complete classification of protein secondary structure. J. Biomol. Struct. Dyn. 1987, 5, 689-704.
41.Sreerama, N.; Woody, R. W. Molecular dynamics simulations of polypeptide conformations in water: A comparison of α, β, and poly (pro) II conformations. Proteins 1999, 36, 400-406.
42.Adzhubei, A. A.; Sternberg, M. J.; Makarov, A. A. Polyproline-II helix in proteins: structure and function. J. Mol. Biol. 2013, 425, 2100-2132.
43.Adzhubei, A. A.; Sternberg, M. J. Left-handed polyproline II helices commonly occur in globular proteins. J. Mol. Biol. 1993, 229, 472-493.
44.Stapley, B. J.; Creamer, T. P. A survey of left-handed polyproline II helices. Protein Sci. 1999, 8, 587-595.
45.Sreerama, N.; Woody, R. W. Poly (Pro) II helixes in globular proteins: Identification and circular dichroic analysis. Biochemistry 1994, 33, 10022-10025.
46.Shoulders, M. D.; Raines, R. T. Collagen structure and stability. Annu. Rev. Biochem. 2009, 78, 929-958.
47.Cubellis, M.; Caillez, F.; Blundell, T.; Lovell, S. Properties of polyproline II, a secondary structure element implicated in protein–protein interactions. Proteins 2005, 58, 880-892.
48.Shi, Z.; Woody, R. W.; Kallenbach, N. R. Is polyproline II a major backbone conformation in unfolded proteins? Adv. Protein Chem. 2002, 62, 163-240.
49.Rath, A.; Davidson, A. R.; Deber, C. M. The structure of “unstructured” regions in peptides and proteins: role of the polyproline II helix in protein folding and recognition. Biopolymers 2005, 80, 179-185.
50.Shi, Z.; Chen, K.; Liu, Z.; Kallenbach, N. R. Conformation of the backbone in unfolded proteins. Chem. Rev. 2006, 106, 1877-1897.
51.Pace, C. N.; Shirley, B. A.; McNutt, M.; Gajiwala, K. Forces contributing to the conformational stability of proteins. FASEB J. 1996, 10, 75-83.
52.Betz, S. F. Disulfide bonds and the stability of globular proteins. Protein Sci. 1993, 2, 1551-1558.
53.Klotz, I. M.; Langerman, N. R.; Darnall, D. W. Quaternary structure of proteins. Annu. Rev. Biochem. 1970, 39, 25-62.
54.Mihailescu, M.-R.; Russu, I. M. A signature of the T→ R transition in human hemoglobin. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 3773-3777.
55.Dill, K. A. Dominant forces in protein folding. Biochemistry 1990, 29, 7133-7155.
56.Stevens, M. J. Simple simulations of DNA condensation. Biophys. J. 2001, 80, 130-139.
57.Simonson, T.; Brooks, C. L. Charge screening and the dielectric constant of proteins: insights from molecular dynamics. J. Am. Chem. Soc. 1996, 118, 8452-8458.
58.Gilson, M. K.; Honig, B. H. The dielectric constant of a folded protein. Biopolymers 1986, 25, 2097-2119.
59.Kumar, S.; Tsai, C.-J.; Ma, B.; Nussinov, R. Contribution of salt bridges toward protein thermostability. J. Biomol. Struct. Dyn. 2000, 17, 79-85.
60.Wendler, K.; Thar, J.; Zahn, S.; Kirchner, B. Estimating the hydrogen bond energy. J. Phys. Chem. A 2010, 114, 9529-9536.
61.Baker, E.; Hubbard, R. Hydrogen bonding in globular proteins. Prog. Biophys. Mol. Biol. 1984, 44, 97-179.
62.Makhatadze, G. I.; Privalov, P. L. Energetics of protein structure. Adv. Protein Chem. 1995, 47, 307-425.
63.Klapper, M. H. On the nature of the protein interior. Biochim. Biophys. Acta-Protein Struct. Molec. Enzym. 1971, 229, 557-566.
64.Head-Gordon, T. Is water structure around hydrophobic groups clathrate-like? Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 8308-8312.
65.Frank, H. S.; Evans, M. W. Free volume and entropy in condensed systems III. Entropy in binary liquid mixtures; partial molal entropy in dilute solutions; structure and thermodynamics in aqueous electrolytes. J. Chem. Phys. 1945, 13, 507-532.
66.Koide, T.; Nagata, K. Collagen biosynthesis. Top. Curr. Chem. 2005, 247, 85-114.
67.Engel, J. Versatile collagens in invertebrates. Science 1997, 277, 1785-1786.
68.Royce, P. M.; Steinmann, B. Connective tissue and its heritable disorders: molecular, genetic, and medical aspects. John Wiley & Sons: 2003.
69.Palmer, M. S.; Dryden, A. J. Homozygous prion protein genotype predisposes to sporadic Creutzfeldt-Jakob disease. Nature 1991, 352, 340-342.
70.Ramshaw, J. A.; Shah, N. K.; Brodsky, B. Gly-XY tripeptide frequencies in collagen: a context for host–guest triple-helical peptides. J. Struct. Biol. 1998, 122, 86-91.
71.Salem, G.; Traub, W. Conformational implications of amino acid sequence regularities in collagen. FEBS Lett. 1975, 51, 94-99.
72.Holmgren, S. K.; Taylor, K. M.; Bretscher, L. E.; Raines, R. T. Code for collagen''s stability deciphered. Nature 1998, 392, 666-667.

Chapter 2
1.Pauling, L.; Corey, R. B. The pleated sheet, a new layer configuration of polypeptide chains. Proc. Natl. Acad. Sci. U. S. A. 1951, 37, 251-256.
2.Hobohm, U.; Scharf, M.; Schneider, R.; Sander, C. Selection of representative protein data sets. Protein Sci. 1992, 1, 409-417.
3.Hobohm, U.; Sander, C. Enlarged representative set of protein structures. Protein Sci. 1994, 3, 522-524.
4.Griep, S.; Hobohm, U. PDBselect 1992–2009 and PDBfilter-select. Nucleic Acids Res. 2009, 38, D318-D319.
5.Citron, M. Alzheimer''s disease: strategies for disease modification. Nat. Rev. Drug Discov. 2010, 9, 387-398.
6.Feany, M. B.; Bender, W. W. A Drosophila model of Parkinson''s disease. Nature 2000, 404, 394-398.
7.Palmer, M. S.; Dryden, A. J.; Hughes, J. T.; Collinge, J. Homozygous prion protein genotype predisposes to sporadic Creutzfeldt-Jakob disease. Nature 1991, 352, 340-342.
8.Minor, D. L.; Kim, P. S. Context is a major determinant of β-sheet propensity. Nature 1994, 371, 264-267.
9.Chou, P. Y.; Fasman, G. D. Conformational parameters for amino acids in helical, β-sheet, and random coil regions calculated from proteins. Biochemistry 1974, 13, 211-222.
10.Chou, P. Y.; Fasman, G. D. Prediction of the secondary structure of proteins from their amino acid sequence. Adv. Enzymol. Relat. Areas Mol. Biol. 1978, 47, 45-148.
11.Kim, C. A.; Berg, J. M. Thermodynamic β-sheet propensities measured using a zinc-finger host peptide. Nature 1993, 362, 267-270.
12.Minor Jr, D. L.; Kim, P. S. Measurement of the β-sheet-forming propensities of amino acids. Nature 1994, 367, 660-663.
13.Smith, C. K.; Withka, J. M.; Regan, L. A thermodynamic scale for the β-sheet forming tendencies of the amino acids. Biochemistry 1994, 33, 5510-5517.
14.Avbelj, F.; Baldwin, R. L. Role of backbone solvation in determining thermodynamic β propensities of the amino acids. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 1309-1313.
15.Otzen, D. E.; Fersht, A. R. Side-chain determinants of β-sheet stability. Biochemistry 1995, 34, 5718-5724.
16.Bai, Y.; Englander, S. W. Hydrogen bond strength and β‐sheet propensities: The role of a side chain blocking effect. Proteins: Struct., Funct., Bioinf. 1994, 18, 262-266.
17.Street, A. G.; Mayo, S. L. Intrinsic β-sheet propensities result from van der Waals interactions between side chains and the local backbone. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 9074-9076.
18.Yang, A.-S.; Honig, B. Free energy determinants of secondary structure formation: II. Antiparallel β-sheets. J. Mol. Biol. 1995, 252, 366-376.
19.Koehl, P.; Levitt, M. Structure-based conformational preferences of amino acids. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 12524-12529.
20.Syud, F. A.; Stanger, H. E.; Gellman, S. H. Interstrand side chain− side chain interactions in a designed β-Hairpin: significance of both lateral and diagonal pairings. J. Am. Chem. Soc. 2001, 123, 8667-8677.
21.Kabsch, W.; Sander, C. Dictionary of protein secondary structure: pattern recognition of hydrogen‐bonded and geometrical features. Biopolymers 1983, 22, 2577-2637.
22.Venkatachalam, C. Stereochemical criteria for polypeptides and proteins. V. Conformation of a system of three linked peptide units. Biopolymers 1968, 6, 1425-1436.
23.Richardson, J. S. The anatomy and taxonomy of protein structure. Adv. Protein Chem. 1981, 34, 167-339.
24.Wilmot, C.; Thornton, J. Analysis and prediction of the different types of β-turn in proteins. J. Mol. Biol. 1988, 203, 221-232.
25.Lewis, P. N.; Momany, F. A.; Scheraga, H. A. Chain reversals in proteins. Biochim. Biophys. Acta, Protein Struct. 1973, 303, 211-229.
26.Hutchinson, E. G.; Thornton, J. M. A revised set of potentials for β‐turn formation in proteins. Protein Sci. 1994, 3, 2207-2216.
27.Haque, T. S.; Gellman, S. H. Insights on β-hairpin stability in aqueous solution from peptides with enforced type I'' and type II'' β-turns. J. Am. Chem. Soc. 1997, 119, 2303-2304.
28.Sibanda, B.; Thornton, J. β-Hairpin families in globular proteins. Nature 1985, 316, 170-174.
29.Haque, T. S.; Little, J. C.; Gellman, S. H. " Mirror image" reverse turns promote β-hairpin formation. J. Am. Chem. Soc. 1994, 116, 4105-4106.
30.Haque, T. S.; Little, J. C.; Gellman, S. H. Stereochemical requirements for β-hairpin formation: model studies with four-residue peptides and depsipeptides. J. Am. Chem. Soc. 1996, 118, 6975-6985.
31.Stanger, H. E.; Gellman, S. H. Rules for antiparallel β-sheet design: D-Pro-Gly is superior to L-Asn-Gly for β-hairpin nucleation1. J. Am. Chem. Soc. 1998, 120, 4236-4237.
32.Kent, S. B. Chemical synthesis of peptides and proteins. Annu. Rev. Biochem. 1988, 57, 957-989.
33.Milton, R. d. L.; Milton, S. C.; Adams, P. A. Prediction of difficult sequences in solid-phase peptide synthesis. J. Am. Chem. Soc. 1990, 112, 6039-6046.
34.Krchnak, V.; Flegelova, Z.; Vagner, J. Aggregation of resin‐bound peptides during solid‐phase peptide synthesis. Int. J. Pept. Protein Res. 1993, 42, 450-454.
35.Fields, G. B.; Fields, C. G. Solvation effects in solid-phase peptide synthesis. J. Am. Chem. Soc. 1991, 113, 4202-4207.
36.Kent, S.; Alewood, D.; Alewood, P.; Baca, M.; Jones, A.; Schnolzer, M.; Epton, R. Innovations and Perspectives in Solid Phase Synthesis. Intercept. Ltd., Andover, UK 1992.
37.Tam, J. P.; Lu, Y.-A. Coupling difficulty associated with interchain clustering and phase transition in solid phase peptide synthesis. J. Am. Chem. Soc. 1995, 117, 12058-12063.
38.Beyermann, M.; Bienert, M. Synthesis of difficult peptide sequences: a comparison of Fmoc-and Boc-technique. Tetrahedron Lett. 1992, 33, 3745-3748.
39.Johnson, T.; Quibell, M.; Sheppard, R. C. N, O‐bisfmoc derivatives of N‐(2‐hydroxy‐4‐methoxybenzyl)‐amino acids: Useful intermediates in peptide synthesis. J. Pept. Sci. 1995, 1, 11-25.
40.Varanda, L. M.; Miranda, M. T. M. Solid‐phase peptide synthesis at elevated temperatures: a search for an optimized synthesis condition of unsulfated cholecystokinin‐12. J. Pept. Res. 1997, 50, 102-108.
41.Johnson, T.; Quibell, M.; Owen, D.; Sheppard, R. A reversible protecting group for the amide bond in peptides. Use in the synthesis of ‘difficult sequences’. J. Chem. Soc., Chem. Commun. 1993, 369-372.
42.Bedford, J.; Hyde, C.; Johnson, T.; Jun, W.; Owen, D.; Quibell, M.; Sheppard, R. Amino acid structure and “difficult sequences” in solid phase peptide synthesis. Chem. Biol. Drug Des. 1992, 40, 300-307.
43.Hyde, C.; Johnson, T.; Owen, D.; Quibell, M.; Sheppard, R. Some ‘difficult sequences’ made easy. Int. J. Pept. Protein Res. 1994, 43, 431-440.
44.Zhang, L.; Goldammer, C.; Henkel, B.; Zühl, F.; Panhaus, G.; Jung, G.; Bayer, E. In Magic mixture, a powerful solvent system for solid-phase synthesis of “difficult sequences”. In: Epton E (ed) Innovation and perspectives in solid phase synthesis: peptides, proteins and nucleic acids—biological and biomedical applications, 3rd International Symposium. Mayflower Worldwide Ltd., Birmingham, UK, 1994; 1994; pp 711-716.
45.Palasek, S. A.; Cox, Z. J.; Collins, J. M. Limiting racemization and aspartimide formation in microwave‐enhanced Fmoc solid phase peptide synthesis. J. Pept. Sci. 2007, 13, 143-148.
46.Chhabra, S. R.; Hothi, B.; Evans, D. J.; White, P. D.; Bycroft, B. W.; Chan, W. C. An appraisal of new variants of Dde amine protecting group for solid phase peptide synthesis. Tetrahedron Lett. 1998, 39, 1603-1606.
47.Pillai, R.; Marinelli, E. R.; Swenson, R. E. A flexible method for preparation of peptide homo‐and heterodimers functionalized with affinity probes, chelating ligands, and latent conjugating groups. Pept. Sci. 2006, 84, 576-585.
48.Izabela, R.; Jarosław, R.; Magdalena, A.; Piotr, R.; Ivan, K. Transportan 10 improves the anticancer activity of cisplatin. Naunyn-Schmiedeberg''s Arch. Pharmacol. 2016, 389, 485-497.
49.Barge, A.; Cappelletti, E.; Cravotto, G.; Ferrigato, A.; Lattuada, L.; Marinoni, F.; Tei, L. Synthesis of functionalised HP-DO3A chelating agents for conjugation to biomolecules. Org. Biomol. Chem. 2009, 7, 3810-3816.
50.Ligeti, M.; Gündüz, Ö.; Magyar, A.; Kató, E.; Rónai, A. Z.; Vita, C.; Varga, I.; Hudecz, F.; Tóth, G.; Borsodi, A. Synthesis and biological studies of nociceptin derivatives containing the DTPA chelating group for further labeling with therapeutic radionuclides. Peptides 2005, 26, 1159-1166.
51.Kumar, S.; Tsai, C.-J.; Ma, B.; Nussinov, R. Contribution of salt bridges toward protein thermostability. J. Biomol. Struct. Dyn. 2000, 17, 79-85.
52.Wu, C.-H.; Weng, M.-H.; Chang, H.-C.; Li, J.-H.; Cheng, R. P. Effect of each guanidinium group on the RNA recognition and cellular uptake of Tat-derived peptides. Biorg. Med. Chem. 2014, 22, 3016-3020.
53.Mitchell, D. J.; Steinman, L.; Kim, D.; Fathman, C.; Rothbard, J. Polyarginine enters cells more efficiently than other polycationic homopolymers. Chem. Biol. Drug Des. 2000, 56, 318-325.
54.Schmidt, N.; Mishra, A.; Lai, G. H.; Wong, G. C. Arginine‐rich cell‐penetrating peptides. FEBS Lett. 2010, 584, 1806-1813.
55.Luscombe, N. M.; Laskowski, R. A.; Thornton, J. M. Amino acid–base interactions: a three-dimensional analysis of protein–DNA interactions at an atomic level. Nucleic Acids Res. 2001, 29, 2860-2874.
56.Kuo, L.-H.; Li, J.-H.; Kuo, H.-T.; Hung, C.-Y.; Tsai, H.-Y.; Chiu, W.-C.; Wu, C.-H.; Wang, W.-R.; Yang, P.-A.; Yao, Y.-C. Effect of charged amino acid side chain length at non-hydrogen bonded strand positions on β-hairpin stability. Biochemistry 2013, 52, 7785-7797.
57.Syud, F. A.; Espinosa, J. F.; Gellman, S. H. NMR-based quantification of β-sheet populations in aqueous solution through use of reference peptides for the folded and unfolded states. J. Am. Chem. Soc. 1999, 121, 11577-11578.
58.Fields, G. B.; Noble, R. L. Solid phase peptide synthesis utilizing 9‐fluorenylmethoxycarbonyl amino acids. Int. J. Pept. Protein Res. 1990, 35, 161-214.
59.Cheng, R. P.; Weng, Y.-J.; Wang, W.-R.; Koyack, M. J.; Suzuki, Y.; Wu, C.-H.; Yang, P.-A.; Hsu, H.-C.; Kuo, H.-T.; Girinath, P. Helix formation and capping energetics of arginine analogs with varying side chain length. Amino Acids 2012, 43, 195-206.
60.Volkmer‐Engert, R.; Landgraf, C.; Schneider‐Mergener, J. Charcoal surface‐assisted catalysis of intramolecular disulfide bond formation in peptides. J. Pept. Res. 1998, 51, 365-369.
61.Fields, G. B. Methods for removing the Fmoc group. Pept. Synth. Protoc. 1995, 17-27.
62.Atherton, E.; Fox, H.; Harkiss, D.; Logan, C.; Sheppard, R.; Williams, B. A mild procedure for solid phase peptide synthesis: use of fluorenylmethoxycarbonylamino-acids. J. Chem. Soc., Chem. Commun. 1978, 537-539.
63.Atherton, E.; Fox, H.; Harkiss, D.; Sheppard, R. Application of polyamide resins to polypeptide synthesis: an improved synthesis of β-endorphin using fluorenylmethoxycarbonylamino-acids. J. Chem. Soc., Chem. Commun. 1978, 539-540.
64.Albericio, F.; Kneib-Cordonier, N.; Biancalana, S.; Gera, L.; Masada, R. I.; Hudson, D.; Barany, G. Preparation and application of the 5-(4-(9-fluorenylmethyloxycarbonyl) aminomethyl-3, 5-dimethoxyphenoxy)-valeric acid (PAL) handle for the solid-phase synthesis of C-terminal peptide amides under mild conditions. J. Org. Chem. 1990, 55, 3730-3743.
65.Harrison, J.; Petrie, G.; Noble, R.; Beilan, H.; McCurdy, S.; Culwell, A. Fmoc chemtstry’synthesis, kinetics, cleavage, and deprotection of arginine-containing peptides. Tech. Protein Chem. 1989, 506-516.
66.Wade, J.; Bedford, J.; Sheppard, R.; Tregear, G. DBU as an N alpha-deprotecting reagent for the fluorenylmethoxycarbonyl group in continuous flow solid-phase peptide synthesis. Pept. Res. 1991, 4, 194-199.
67.Dettin, M.; Pegoraro, S.; Rovero, P.; Bicciato, S.; Bagno, A.; Bello, C. SPPS of difficult sequences. J. Pept. Res. 1997, 49, 103-111.
68.Fields, C. G.; Mickelson, D.; Drake, S.; McCarthy, J.; Fields, G. Melanoma cell adhesion and spreading activities of a synthetic 124-residue triple-helical" mini-collagen". J. Biol. Chem. 1993, 268, 14153-14160.
69.Kates, S. A.; Solé, N. A.; Beyermann, M.; Barany, G.; Albericio, F. Optimized preparation of deca (L-alanyl)-L-valinamide by 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase synthesis on polyethylene glycol-polystyrene (PEG-PS) graft supports, with 1, 8-diazobicyclo [5.4. 0]-undec-7-ene (DBU) deprotection. Pept. Res. 1995, 9, 106-113.
70.Feichtinger, K.; Sings, H. L.; Baker, T. J.; Matthews, K.; Goodman, M. Triurethane-protected guanidines and triflyldiurethane-protected guanidines: new reagents for guanidinylation reactions. J. Org. Chem. 1998, 63, 8432-8439.
71.Haack, T.; Mutter, M. Serine derived oxazolidines as secondary structure disrupting, solubilizing building blocks in peptide synthesis. Tetrahedron Lett. 1992, 33, 1589-1592.
72.Mutter, M.; Nefzi, A.; Sato, T.; Sun, X.; Wahl, F.; Wöhr, T. Pseudo-prolines (psi Pro) for accessing" inaccessible" peptides. Pept. Res. 1994, 8, 145-153.
73.Wöhr, T.; Wahl, F.; Nefzi, A.; Rohwedder, B.; Sato, T.; Sun, X.; Mutter, M. Pseudo-prolines as a solubilizing, structure-disrupting protection technique in peptide synthesis. J. Am. Chem. Soc. 1996, 118, 9218-9227.
74.Wöhr, T.; Mutter, M. Pseudo-prolines in peptide synthesis: direct insertion of serine and threonine derived oxazolidines in dipeptides. Tetrahedron Lett. 1995, 36, 3847-3848.
75.Bax, A.; Davis, D. G. MLEV-17-based two-dimensional homonuclear magnetization transfer spectroscopy. J. Magn. Reson. (1969-1992) 1985, 65, 355-360.
76.Aue, W.; Bartholdi, E.; Ernst, R. R. Two‐dimensional spectroscopy. Application to nuclear magnetic resonance. J. Chem. Phys. 1976, 64, 2229-2246.
77.Bothner-By, A. A.; Stephens, R.; Lee, J.; Warren, C. D.; Jeanloz, R. Structure determination of a tetrasaccharide: transient nuclear Overhauser effects in the rotating frame. J. Am. Chem. Soc. 1984, 106, 811-813.
78.Piotto, M.; Saudek, V.; Sklenář, V. Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J. Biomol. NMR 1992, 2, 661-665.

Chapter 3
1.Shoulders, M. D.; Raines, R. T. Collagen structure and stability. Annu. Rev. Biochem. 2009, 78, 929-958.
2.Gelse, K.; Pöschl, E.; Aigner, T. Collagens—structure, function, and biosynthesis. Adv. Drug Del. Rev. 2003, 55, 1531-1546.
3.Koide, T.; Nagata, K. Collagen biosynthesis. Top. Curr. Chem. 2005, 247, 85-114.
4.Prockop, D. J.; Kivirikko, K. I.; Tuderman, L.; Guzman, N. A. The biosynthesis of collagen and its disorders. New Engl. J. Med. 1979, 301, 77-85.
5.Schofield, J. D.; Uitto, J.; Prockop, D. J. Formation of interchain disulfide bonds and helical structure during biosynthesis of procollagen by embryonic tendon cells. Biochemistry 1974, 13, 1801-1806.
6.Olsen, B. R.; Hoffmann, H.-P.; Prockop, D. J. Interchain disulfide bonds at the COOH-terminal end of procollagen synthesized by matrix-free cells from chick embryonic tendon and cartilage. Arch. Biochem. Biophys. 1976, 175, 341-350.
7.Bulleid, N. J.; Dalley, J. A.; Lees, J. F. The C‐propeptide domain of procollagen can be replaced with a transmembrane domain without affecting trimer formation or collagen triple helix folding during biosynthesis. EMBO J. 1997, 16, 6694-6701.
8.Bulleid, N. J.; Wilson, R. Type-III procollagen assembly in semi-intact cells: chain association, nucleation and triple-helix folding do not require formation of inter-chain disulphide bonds but triple-helix nucleation does require hydroxylation. Biochem. J. 1996, 317, 195-202.
9.Kirk, T.; Evans, J.; Veis, A. Biosynthesis of type I procollagen. Characterization of the distribution of chain sizes and extent of hydroxylation of polysome-associated pro-alpha-chains. J. Biol. Chem. 1987, 262, 5540-5545.
10.Greenspan, D. S. Biosynthetic processing of collagen molecules. Top. Curr. Chem. 2005, 247, 149-183.
11.Royce, P. M.; Steinmann, B. Connective tissue and its heritable disorders: molecular, genetic, and medical aspects. John Wiley & Sons: 2003.
12.Beck, K.; Brodsky, B. Supercoiled protein motifs: the collagen triple-helix and the α-helical coiled coil. J. Struct. Biol. 1998, 122, 17-29.
13.Kramer, R. Z.; Bella, J.; Mayville, P.; Brodsky, B.; Berman, H. M. Sequence dependent conformational variations of collagen triple-helical structure. Nat. Struct. Mol. Biol. 1999, 6, 454-457.
14.Engel, J.; Bächinger, H. P. Structure, stability and folding of the collagen triple helix. Top. Curr. Chem. 2005, 247, 7-33.
15.Rich, A.; Crick, F. The molecular structure of collagen. J. Mol. Biol. 1961, 3, 483-506.
16.Bella, J.; Eaton, M.; Brodsky, B.; Berman, H. M. Crystal and molecular structure of a collagen-like peptide at 1.9 angstrom resolution. Science 1994, 266, 75-82.
17.Nagarajan, V.; Kamitori, S.; Okuyama, K. Structure analysis of a collagen-model peptide with a (Pro-Hyp-Gly) sequence repeat. J. Biochem. 1999, 125, 310-318.
18.Ramachandran, G. Molecular structure of collagen. Int. Rev. Connect. Tissue Res. 1963, 1, 127-182.
19.Engel, J.; Chen, H. T.; Prockop, D. J.; Klump, H. The triple helix⇌coil conversion of collagen‐like polytripeptides in aqueous and nonaqueous solvents. Comparison of the thermodynamic parameters and the binding of water to (L‐Pro‐L‐Pro‐Gly) n and (L‐Pro‐L‐Hyp‐Gly) n. Biopolymers 1977, 16, 601-622.
20.Ramshaw, J. A.; Shah, N. K.; Brodsky, B. Gly-XY tripeptide frequencies in collagen: a context for host–guest triple-helical peptides. J. Struct. Biol. 1998, 122, 86-91.
21.Fietzek, P. P.; Kühn, K. Information contained in the amino acid sequence of theα1 (I)-chain of collagen and its consequences upon the formation of the triple helix, of fibrils and crosslinks. Mol. Cell. Biochem. 1975, 8, 141-157.
22.Salem, G.; Traub, W. Conformational implications of amino acid sequence regularities in collagen. FEBS Lett. 1975, 51, 94-99.
23.Hollingsworth, S. A.; Karplus, P. A. A fresh look at the Ramachandran plot and the occurrence of standard structures in proteins. Biomol. Concepts 2010, 1, 271-283.
24.Cram, D. J. The design of molecular hosts, guests, and their complexes. J. Incl. Phenom. Macrocycl. Chem. 1988, 6, 397-413.
25.Bretscher, L. E.; Jenkins, C. L.; Taylor, K. M.; DeRider, M. L.; Raines, R. T. Conformational stability of collagen relies on a stereoelectronic effect. J. Am. Chem. Soc. 2001, 123, 777-778.
26.Jenkins, C. L.; Vasbinder, M. M.; Miller, S. J.; Raines, R. T. Peptide bond isosteres: Ester or (E)-alkene in the backbone of the collagen triple helix. Org. Lett. 2005, 7, 2619-2622.
27.Persikov, A. V.; Ramshaw, J. A.; Brodsky, B. Prediction of collagen stability from amino acid sequence. J. Biol. Chem. 2005, 280, 19343-19349.
28.Gauba, V.; Hartgerink, J. D. Surprisingly high stability of collagen ABC heterotrimer: evaluation of side chain charge pairs. J. Am. Chem. Soc. 2007, 129, 15034-15041.
29.Gauba, V.; Hartgerink, J. D. Self-assembled heterotrimeric collagen triple helices directed through electrostatic interactions. J. Am. Chem. Soc. 2007, 129, 2683-2690.
30.Gauba, V.; Hartgerink, J. D. Synthetic collagen heterotrimers: structural mimics of wild-type and mutant collagen type I. J. Am. Chem. Soc. 2008, 130, 7509-7515.
31.Xu, F.; Zhang, L.; Koder, R. L.; Nanda, V. De novo self-assembling collagen heterotrimers using explicit positive and negative design. Biochemistry 2010, 49, 2307-2316.
32.Xu, F.; Zahid, S.; Silva, T.; Nanda, V. Computational design of a collagen A: B: C-type heterotrimer. J. Am. Chem. Soc. 2011, 133, 15260-15263.
33.Persikov, A. V.; Xu, Y.; Brodsky, B. Equilibrium thermal transitions of collagen model peptides. Protein Sci. 2004, 13, 893-902.
34.Edelhoch, H. Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 1967, 6, 1948-1954.
35.Pace, C. N.; Vajdos, F.; Fee, L.; Grimsley, G.; Gray, T. How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 1995, 4, 2411-2423.
36.Chakrabartty, A.; Kortemme, T.; Padmanabhan, S.; Baldwin, R. L. Aromatic side-chain contribution to far-ultraviolet circular dichroism of helical peptides and its effect on measurement of helix propensities. Biochemistry 1993, 32, 5560-5565.
37.Acevedo-Jake, A. M.; Clements, K. A.; Hartgerink, J. D. Synthetic, register-specific, AAB heterotrimers to investigate single point glycine mutations in Osteogenesis imperfecta. Biomacromolecules 2016, 17, 914-921.
38.Giddu, S.; Xu, F.; Nanda, V. Sequence recombination improves target specificity in a redesigned collagen peptide abc‐type heterotrimer. Proteins: Struct., Funct., Bioinf. 2013, 81, 386-393.
39.Parmar, A. S.; James, J. K.; Grisham, D. R.; Pike, D. H.; Nanda, V. Dissecting electrostatic contributions to folding and self-assembly using designed multicomponent peptide systems. J. Am. Chem. Soc. 2016, 138, 4362-4367.
40.Parmar, A. S.; Joshi, M.; Nosker, P. L.; Hasan, N. F.; Nanda, V. Control of collagen stability and heterotrimer specificity through repulsive electrostatic interactions. Biomolecules 2013, 3, 986-996.
41.Sarkar, B.; O’Leary, L. E.; Hartgerink, J. D. Self-assembly of fiber-forming collagen mimetic peptides controlled by triple-helical nucleation. J. Am. Chem. Soc. 2014, 136, 14417-14424.
42.Sarkar, S. K.; Young, P.; Sullivan, C.; Torchia, D. Detection of cis and trans X-Pro peptide bonds in proteins by 13C NMR: application to collagen. Proc. Natl. Acad. Sci. U. S. A. 1984, 81, 4800-4803.
43.Fischer, G. Chemical aspects of peptide bond isomerisation. Chem. Soc. Rev. 2000, 29, 119-127.
44.Grathwohl, C.; Wüthrich, K. The X‐Pro peptide bond as an nmr probe for conformational studies of flexible linear peptides. Biopolymers 1976, 15, 2025-2041.
45.Fallas, J. A.; Gauba, V.; Hartgerink, J. D. Solution structure of an ABC collagen heterotrimer reveals a single-register helix stabilized by electrostatic interactions. J. Biol. Chem. 2009, 284, 26851-26859.
46.Shah, N. K.; Ramshaw, J. A.; Kirkpatrick, A.; Shah, C.; Brodsky, B. A host− guest set of triple-helical peptides: stability of Gly-XY triplets containing common nonpolar residues. Biochemistry 1996, 35, 10262-10268.
47.Persikov, A. V.; Ramshaw, J. A.; Kirkpatrick, A.; Brodsky, B. Amino acid propensities for the collagen triple-helix. Biochemistry 2000, 39, 14960-14967.
48.Fields, G. B.; Noble, R. L. Solid phase peptide synthesis utilizing 9‐fluorenylmethoxycarbonyl amino acids. Int. J. Pept. Protein Res. 1990, 35, 161-214.
49.Cardona, V.; Eberle, I.; Barthelemy, S.; Beythien, J.; Doerner, B.; Schneeberger, P.; Keyte, J.; White, P. Application of Dmb-dipeptides in the Fmoc SPPS of difficult and aspartimide-prone sequences. Int. J. Pept. Res. Ther. 2008, 14, 285-292.