Proteins And Ligands

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Proteins And Ligands

• Why we bother to study proteins at all

You may not feel Harassment
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• Proteins primary function is to interact with
  other molecules
• Receptors bind hormones.
• Antibodys bind antigens
• Membrane transport proteins move molecules across
  membranes
• Hemoglobin binds oxygen
• Hexokinase binds sugars and ATP

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Definitions

• Ligand the molecule a protein interacts with
• Can be another protein.
• May be covalently attached to protein
• Prosthetic group Ligand required for protein
  function generally tightly bound to protien.
• Holo protein A protein with its ligand bound
• Apo Protein A protein lacking its ligand
• Complex A protein and its ligand bound to one
  another. Another term for the holo protein.

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History of the Study of Ligand Binding

• First examined using old fashion thermodynamics
  and kinetics.
• Covalent modification of binding sites
• X-Ray Crystalography
• NMR Spectroscopy

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Structural Aspects of Ligand Binding

• X-ray Structures
• Gives incredible detail of the interaction
• Useful in determining specific interactions
• The importance of interactions must be confirmed
  via site directed mutagenesis or chemical
  modification
• Principle method is the difference Fourier map

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Structural Aspects of Ligand Binding

• Difference Fourier Maps
• Crystallize the protein with out the ligand and
  determine structure.
• Crystallize the protein with the ligand
• Soak crystals with ligand solution
• Co-crystallize with ligand
• Determine the Fourier map.

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Structural Aspects of Ligand Binding

• Difference Fourier Maps
• Subtract the Fourier map of the ligand -
  structure.
• Resulting Fourier map shows
• Ligand position
• Changes in the proteins structure.
• Ligands as small as O2 are readily apparent.

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Amzel et al. Proc. Nat. Acad. Sci. USAVol. 71,
No. 4, pp. 1427-1430, April 1974
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Structural Aspects of Ligand Binding

• Difference Fourier Maps
• Problems
• Often there are enough protein changes to cause
  great difficulties in locating ligand binding
  site

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Structural Aspects of Ligand Binding

• NMR
• Similar to the process used for apo protein
• Limited to smaller proteins
• Focus is on NOE interactions
• Free ligand structure should be determined before
  attempting complex
• Changes in ligand signals help in structure
  determination.
• Following hydrogen exchange of involved side
  chains quite effective in identification

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http//www.ncbi.nlm.nih.gov/pmc/articles/PMC224249
3/figure/f4/
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Structural Aspects of Ligand Binding

• Chemical Modification I
• Bind the ligand
• Treat protein with modifying reagent
• Ligand should protect side chains involved in
  binding
• Caveats.
• Residues protected by the fold of the protein
  might be identified as important.
• Ligand binding resulting in structural changes
  may skew results.

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Structural Aspects of Ligand Binding

• Chemical Modification II
• Treat the protein with limited modifying reagent
• Sort out the modified residues
• Determine ligand binding constants
• Identify residues that reduce the binding of
  ligand

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Structural Aspects of Ligand Binding

• Chemical Modification III Affinity labeling
• Incorporate a reactive group in the ligand
• Bind the ligand
• Determine the residues the reactive group
  interacts with
• Photo activated groups are useful
• Changing the chain length allows for distance
  measurements.

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Structural Aspects of Ligand Binding

• Chemical Modification III Affinity labeling
• Azides are useful groups
• Photo-activated
• Reacts with all side chains

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Structural Aspects of Ligand Binding

• Chemical Modification IV Bi-functional reagents
• Useful in identifying protein protein
  interactions
• Varying the length of the spacer between reactive
  groups provides a useful measuring tool.
• Excellent for establishing subunit nearest
  neighbor association

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Structural Aspects of Ligand Binding

• Chemical Modification IV Bi-functional reagents
• Hetro-bifunctional
• Useful for situations where you want to link to
  one group at a time.
• amine/carboxyl
• Amine photoreactive
• amine sulfhydryl
• protein nucleic acid
• sulfhydryl carboxyl
• sulfhydryl hydroxyl

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Just some of the 35 pages of Bi-functional
reagents available in the Pierce Catalog
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Properties of Ligand Binding

• For each distinct ligand there is usually a
  unique binding site in a peptide/domain.
• There may be more than on ligand binding site on
  a peptide/domain but they will bind different
  ligands.
• Often the binding of one ligand will affect the
  binding of the second in the domain.
• If a single peptide binds multiple ligands of the
  same type they are typically in different but
  simmilar domains in the protein
• There are exceptions
• Cytochromes Antenna proteins.

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Properties of Ligand Binding

• Most binding sites tend to be depressions on the
  surface of a basically spherical proteins.
• Prosthetic groups are usually found in the
  interior of proteins.
• Large ligands come in three types
• Protein protein interfaces tend to be flattened
  surfaces.
• Liner ligands that bind in a cleft in the
  basically spherical surface of the protein.
• DNA the protein tends to have fingers that clasp
  the DNA

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Properties of Ligand Binding

• Molecular interactions that aid in binding
• Salt Bridges
• Hydrogen bonds
• Dipole interactions
• Van der waals interactions
• Binding is a cumulative effect taking advantage
  of cooperative interactions as described in sec
  4.4.3
• Binding constants have a huge range from near 1
  to 1013.