Recent Study on Carbohydrate Protein Interaction by SPR

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Recent Study on Carbohydrate Protein Interaction
by SPR

• Junfeng Xiao

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Outline of my talk

• Significance of studying carbohydrate protein
  interaction.
• Limitations of other Techniques currently used to
  study this area.
• Study done by SPR
• a. its advantages
• b. recent development in this area by SPR
• Future study

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Significance of studying carbohydrate protein
interaction

• Studying carbohydrate protein interaction can
• a. first help understanding biological
  function.
• b. then provide a prerequisite for
  developing new therapeutic agents.
• Carbohydrate protein interaction has biological
  and pathological importance.
• a. immune response
• b. protein folding
• c. tumor metastasis

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Heparin Protein Interaction
Robert J. Linhardt,etal Angew. Chem. Int. Ed.
2002, 41, 390 - 412
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Current techniques used to study the interaction

• Techniques used
• a. affinity chromatography
• b. isothermal titration calorimetry (ITC)
• c. X-ray crystallography and NMR
• d. Surface plasmon resonance (SPR)
• Types of interaction studied
• a. ionic interaction
• b. hydrogen bonding
• c. Hydrophobic interaction

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Limitations of first four methods

• affinity chromatography
• 1. inability in measuring hydrophobic
  interactions
• 2. difficulty in studying divalent cations
  based interaction
• isothermal titration calorimetry
• 1.capable of measuring only the
  association process
• 2. high concentrations required and
  precipitation resulted
• X-ray crystallography and NMR
• 1. difficulty of crystallizing unbound
  carbohydrates
• 2. high quantity needed and hard to get
  accurate association constant (Ka)

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Interaction study by SPR and recent method
improvement

• How SPR works
• Advantages of SPR in studying carbohydrate
  protein interaction
• One report on recent development done by SPR

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How SPR works

• SPR study on kinetics in biomolecular
  interactions.
• Components of SPR 1)Light source 2)Prism 3)
  Gold-coated sensor chip 4)Flow cell 5) Detector
• signal indicates correlation among
• Amount of analytes absorbed ?? change of
  reflection angle ?? refractive index change

http//chem.ch.huji.ac.il/eugeniik/spr.htm
Mattei, B., Borch, J., Roepstorff, P., Anal.
Chem., 2004 19A-25A
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Dissociation constant and association rate
constant determined through varying analyte
concentration
R.J Linhardt, etal FEBS Lett. 1999, 446, 327
-330.
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Advantages of SPR in studying carbohydrate
protein interaction

• SPR can provide fast and non-destructive
  monitoring on biomolecular interaction.
• No need for fluorescent, radioactive, or
  enzymatic reporter groups. Secondary binding
  event for detection also can be avoided.
• SPR detection for low affinity interaction can be
  done in the presence of excess of unbound
  proteins without background problem.

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Hows this SPR method been recently developed

• Many work have been done on modifying
  surface-based carbohydrate arrays as the
  microchip in SPR
• a. Various fabrication schemes for
  carbohydrate array have been developed.
• b. Different carbohydrate have been
  immobilized onto gold film surface and its
  interaction with its binding protein have been
  studied.

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One report on this array development

• Laura Kiessling in Univ. of Wisconsin worked
  out a new fabrication scheme to immobilize
  mannose and galactose onto gold surface and their
  interactions respectively with ConA and Jacalin
  were studies by SPR.
• Published in J. AM. CHEM. SOC. 2003. 125,
  6140-6148

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Highlights in this work

• mannose and galactose have been derivatized
  with free sulfhydryl group at anomeric linker
  end.
• 1. mannose
• 2. galactose

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SPR results show interactions of mannose with
ConA and galactose withJacalin
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Adsorption coefficients KADS and dissociation
constants KD are determined

• a. how Kabs determined?
• b. how KD determined?
• First ,plot of ? as a function
• of carbohydrate concentration
• Then, finding P in Frumkin isotherm equation.

Frumkin isotherm equation!
Making Plot of ? as a function Of protein
concentration first!
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Future study

• Developing more robust and more general
  fabrication process for carbohydrate array via
  disulfide attachment chemistry.
• Widening the study on pathologically related
  carbohydrate binding proteins.
• Combining with Mass Spect. technique, SPR-MS can
  provide high throughput screening ability.

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Thank You
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Limitations

• SPR is not well-suited to high-throughput assays,
  or the analysis of small molecules (Mr lt 1000).
• unsuitable for concentration measurements,
  because these require the analysis of many
  samples in parallel, including the standard
  curve.
• A second problem is that, for optimal
  sensitivity, concentration assays require long
  equilibration periods.
• at such high ligand densities accurate kinetic
  analysis is not possible because of
  mass-transport limitations and re-binding
  (Section 7.2).