Structure of proteins by X-ray crystallography

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Structure of proteins by X-ray crystallography

• George N. Phillips, Jr.
• ECE 532
• Spring 2005

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Points to be covered

• What are proteins?
• How they are organized?
• How do we know their three-dimensional
  structures?
• Why do you need a crystal and how does one get a
  crystal?

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Hierarchy in Protein Structure

• Protein structure is usually described in terms
  of an organizational hierarchy
• Primary sequence
• Secondary structure
• Tertiary structure
• Quaternary structure
• But remember it is the sequence that controls all
  that follows!

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Peptide Bond

• The peptide bond influences all aspects of
  protein structure and function.
• Key features
• 1. Planar
• 2. Fairly rigid, due to partial double bond
  character.
• 3. Almost always in trans configuration.
• 4. Polar. Can form at least two hydrogen bonds.
• 5. Places restrictions on the conformation of
  the polypeptide chain.

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?-helices

• This is the prototypical secondary structural
  element. It satisfies the hyrogen bonding
  requirements of the polypeptide chain (except for
  the ends).
• Properties
• It is compact and self contained.
• Right handed
• rise per residue, 1.5 Å
• Residue per turn, 3.5Å
• H-bond between CO(n).....H-N(n4)

How do you define the start and end of an a-helix?
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?-sheets
Antiparallel b-sheet
Parallel b-sheet

• ?-sheets fulfill the hydrogen bonding potential
  of the main-chain atoms, except at the edges.
  Adjacent strands are usually close in sequence.
• Properties
• Distance between Ca's is 3.6 Å in an extended
  strand
• Distance between strands 4.6 Å
• Strands are not flat. They have a characteristic
  right-handed twist
• How are sheets defined?

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Turns

• Turns are a major structural component of
  proteins. Without them there could be no globular
  proteins.
• They are characterized by an irregular series of
  conformational angles that fold the chain back on
  itself.
• Turns are often very compact and well ordered,
  though they are hot-spots for evolution.
  Sometimes they are sites of flexibility, at other
  times they are quite rigid. Need to look
  carefully at the structure.

Turns can be classified into a few well defined
arrangements.
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Major secondary structural elements of HIV-1
protease

• The secondary structure is mostly b-strand and
  random coil. There is one small helix and two
  b-turns.
• The b-sheets show their characteristic twist and
  associate into two layers, which is common in
  b-structures.
• The structure is compact, though this is not
  apparent in these ribbon representations.

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Space Filling Representation of HIV-1 Protease

• Ribbon representations are convenient for showing
  the path of the polypeptide chain but are
  misleading with respect to the compactness.
• What is not evident in this representation?
• Hydrogens? Water? Dynamic properties?

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Digression Packing of a-helices and b-sheets in
other proteins?

• Secondary structural elements segregate into
  layers of helices, strands, and coil to optimize
  the packing of the side chains. Proteins do not
  have large holes on their interior, except for
  functional reasons.

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Myoglobin and Hemoglobin Deliver Oxygen in
Organisms with Vascular Systems
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Light microscope
Electron microscope
X-ray Crystallography
1,000,000 x
100,000,000 X
2,000 X
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X-ray Laboratory
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Some crystallography basics
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X-ray infrastructure
Initial screening
Optimization
Identification
George Phillips Craig Bingman Ed Bitto Simon
Allard Jannelle Warrick Gary Wesenberg
Determination
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Crystals of molecules of a protein
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