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Bosques et al. J Am Chem Soc. 2004, 14, 126(27): 8422- 8425 Presented by Anoop Mayampurath

Effects of Glycosylation on Peptide conformation: A Synergistic Experimental and Computational Study. Bosques et al. J Am Chem Soc. 2004, 14, 126(27): 8422- 8425 Presented by Anoop Mayampurath. Introduction. Oligosaccharides are large hydrophilic molecules (1000-2000 Da )

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Bosques et al. J Am Chem Soc. 2004, 14, 126(27): 8422- 8425 Presented by Anoop Mayampurath

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  1. Effects of Glycosylation on Peptide conformation: A Synergistic Experimental and Computational Study Bosques et al. J Am Chem Soc. 2004, 14, 126(27): 8422- 8425 Presented by AnoopMayampurath

  2. Introduction • Oligosaccharides are large hydrophilic molecules (1000-2000 Da) • Can affect structure of its conjugate in two ways • Co-translational, thus involves in folding • Carbohydrate can stabilize the mature protein [1]“Effect of N-linked glycosylation on glycopeptideand glycoprotein structure” B Imperiali and S E O’Connor Current Opinion in Chemical Biology 1999, 3:643–649

  3. Endo-glycosidase used to transfer carbohydrated derived for proteolytic glycoprotein digests to synthesized peptide. • The authors used a building-block approach to get three types of molecules • Non-glycosylated peptide 1 • 1-βglycopeptide • 1-αglycopeptide [2] Supplementary information: http://pubs.acs.org/doi/suppl/10.1021/ja0496266/suppl_file/ja0496266si20040121_063620.pdf?cookieSet=1 [3]“Conformational Switching by Asparagine-Linked Glycosylation” S E. O’Connor and B ImperialiJ. Am. Chem. Soc. 1997, 119, 2295-2296 [4]Bosques, C. J.; Tai, V. W.-F. Tetrahed. Lett. 2001, 42, 7207-7210.

  4. Based on AA 282-288 of hemaglutanin. • hcG – αβdimer[1] • 92 residue α subunit with two sites of glycosylation • Asn52 glycosylation contributes to signal transduction • Asn78 contributes to stability • In [3], the authors showed that glysosylation from a β-linked disaccharide induces a type-I β turn. • Was this unique?

  5. NMR • Every nucleus has an associated spin component • Nucleus of spin ½ align as shown in the presence of magnetic field • Now, when radiated with electromagnetic radiation, the lower energy spin flips up to align with other spin. This is when nuclei are in resonance. [5] http://www.chem.uic.edu/web1/ocol/spec/NMR.htm

  6. All nuclei do not shift at one frequency. The electron cloud plays a role. • This is called chemical shift (measured in ppm) • The more electronegativity, the lesser is the energy required to transition and lesser is the chemical shift [6] http://en.wikipedia.org/wiki/Carbon-13_NMR

  7. Interaction between proton field and binding electrons causes peak splitting • n protons = n+1 peaks • J (coupling constant in Hz) • Information about the dihedral angles

  8. Nuclear Overhauser Effect (NOE) • A small fraction of the nuclei do not achieve equilibrium • For nuclei close together ( ≈ 3.5 angstrom), energy is transferred from a saturated spin state to others, thereby enhancing the signal • Helps in structure elucidation • TOCSY • TOtal Correlation SpectroscopY • Correlation between all protons in a given spin system • Gives assignment of spin • ROESY • Rotating-frame Overhauser Effect SpectroscopY • detect NOE effects [7] http://www.cis.rit.edu/class/schp740/docu/avance/roesy.pdf

  9. Glycopeptide 1-α≈Unglycosylated peptide 1 vsGlycopeptide 1-β • Asx-turn (pseudo turn) vs. β turn • Asx turn • H bond between side chain of Asx (i) to neighboring (i+2 amide )backbone peptide group • β turn • H bond between carbonyl at I to amide at i+3 forming a closed ring • Backbone substitution of nitrogen with Cβ turn [8] “Secondary structures without backbone: an analysis of backbone mimicry by polar side-chains in proteins,  N Eswar and C Ramakrisnan, , Protein Eng.12 (1999), pp. 447–455

  10. Solution Structure solving using simlated annealing • Two classes of the ROESY spectrum • Weak (2-4 Å) • Strong (1-3Å) Parameters - VT coefficients - 70-80 degree limit for dihidral angles

  11. Molecular Dynamics Simulation • Starting structures based on 3 NOES and 3 JHNα • 1* different from 1, with a forced constraint of H-bonding for Asx turn. But after 2ns, it collapsed to 1.

  12. Plot of the φ backbone torsion angle for Ile2, Thr3, Asn5, and Thr7 (left to right) for the 10 ns MD simulation of 1, 1*, 1-α, and 1-β with 1 (black) and 1* (gray) superimposed • α-GlcNAc1–Thr7 and α-GlcNAc1–Trp8 H bonds in 1-α simulation • - α-GlcNAc1 and Asn5 causes the turn to destablize

  13. Conclusion • Only a β-linked glycan chain can affect the backbone through formation of a type-I β turn • Observations • 10 peptides (too short?) • Can we extrapolate and say something about hemaglutanin? • Lays down pipeline for studying/predicting influence • GLYCAM06 took these observations into account while updating their parameter set • Development of mimetics [9] “GLYCAM06: A GeneralizableBiomolecular Force Field” K N. Kirschner, A. B. Yongye, S. M. Tschampel, J. Gonza ´Lez-Outeirin,C. R. Daniels, B. L. Foley, R. J. Woods, Carbohydrates [10] “Asparaginesurrogates for the assembly of N-linked glycopeptidemimetics by chemoselectiveligation” S. Peluso and B. Imperial Tetrahedron LettersVolume 42, Issue 11, 11 March 2001, Pages 2085-2087

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