Effects of Glycosylation on Peptide conformation: A Synergistic Experimental and

Computational Study

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

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 glycopeptide and glycoprotein structure” B Imperiali and S E O’Connor Current Opinion in Chemical Biology 1999, 3:643–649

• 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-β glycopeptide– 1-α glycopeptide

[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 Imperiali J. Am. Chem. Soc. 1997, 119, 2295-2296[4] Bosques, C. J.; Tai, V. W.-F. Tetrahed. Lett. 2001, 42, 7207-7210.

• Based on AA 282-288 of hemaglutanin.• hcG – αβ dimer[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?

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

• 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

• 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

• 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

Glycopeptide 1-α≈Unglycosylated peptide 1 vs

Glycopeptide 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

• Solution Structure solving using simlated annealing

Two classes of the ROESY spectrum-Weak (2-4 Å)-Strong (1-3 Å)

Parameters - VT coefficients - 70-80 degree limit for dihidral angles

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.

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

• 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 Generalizable Biomolecular 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] “Asparagine surrogates for the assembly of N-linked glycopeptide mimetics by chemoselective ligation” S. Peluso and B. Imperial Tetrahedron Letters Volume 42, Issue 11, 11 March 2001, Pages 2085-2087