Nuclear Magnetic Resonance

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Nuclear Magnetic Resonance
Atomic structure in solution High concentration
of protein 1 mM Basis - Atomic nuclei are
intrinsically magnetic Size limitation is 40 kDa
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Basis of NMR
Hydrogen has alpha and beta spin states Alpha can
be excited to beta by applying a pulse of
electromagnetic radiation
Energy difference is proportional to the strength
of the imposed magnetic field. The Resonance
spectrum change between alpha and beta states
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Chemical shifts reveal information about
structural changes
Degree of shielding of nucleus by electrons that
flow around nucleus is a measure of the electron
density Difference frequency chemical shifts
(ppm) relative to a reference sample scale
between 0-9 ppm
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Nuclear Overhauser Effect
Interaction of nuclei of two protons in close
proximity allows determination of distance
between them (Less than 5 angtroms apart)
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Converting a spectrum to 3-D structure
Distance constraints between proton pairs allows
determination of structure Spectrum generated on
a large number of molecules in multiple
conformations range of conformations a protein
adopts in solution
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Protein Crystallography
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Protein Structural Analysis
http//www.rcsb.org/pdb/holdings.htmlgrowth
Number PDB Entries
year

• High-Resolution Structural Techniques
• (determination of atom positions)
• Crystallography (solid-state)
• NMR (solution-state)

• limited by the ability of the molecule to
  crystallize

• limited to soluble peptide or protein molecules

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Small-angle X-ray Scattering

• Low-Resolution Techniques can be used to provide
  molecular envelope shapes
• Electron Microscopy with Image Reconstruction
• solid-state (often cryo-cooled)
• Limited to relatively large proteins and protein
  complexes such as viruses, membrane protein
  complexes, muscle proteins and the ribosome
• Small-angle Solution Scattering with
  Computational Modeling Methods
• solution-state
• useful range 10 - 1000 Å (15 500 kDa)

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Small-angle X-ray Scattering

• X-rays
• 1.54Å
• or neutrons

Q ? 4?sin?/?
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Probable Frequency Distribution of Inter-atomic
Vectors
Inverse Fourier Transform
46 Å

• P (r) - The probability of finding a vector of
  length r between scattering centers (atoms)
  within the scattering particle.

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Introduction to Protein Structure Building Blocks
of Proteins The 20 Amino Acids
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Introduction to Protein Structure Peptide Bonds
Connect the Building Blocks
side group
side group
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Introduction to Protein Structure Peptide Bonds
Connect the Building Blocks
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Introduction to Protein Structure Steric
Restrictions on PHI and PSI Angles
Ramachandran Plot for Typical Residue
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Introduction to Protein Structure Flexible
Backbone of a Protein
?1
?3
?3
?1
C-terminus
?2
?2
N-terminus
The set of ?i,?i angles define internal degrees
of freedom
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Three Dimensional Structure of Proteins
Secondary Structure Local regions of regular
folding helix, beta-sheet, random coil,
irregular folds Tertiary Structure Structure of
entire polypeptide chain Quanternary Structure
More than one polypeptide chain arranged in a
regular manner
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Secondary Structure

• Rules that protein structure must obey
• Little distortion of bond length and bond angles
  from those found in x-ray diffraction studies
• Van der Waals radii dictates how close to atoms
  can approach one another
• The amide group must remain in a trans
  configuration. Rotation is only possible about
  the two bonds adjacent to the ?-carbon.
• Noncovalent bonding is necessary to stabilize a
  regular folding. Hydrogen bonding between amide
  nitrogen and carbonyl oxygen.

psi
phi
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Alpha helix and Beta-sheets

• Each of these structures satisfied the 4 rules
• Peptide groups are planar, and each amide proton
  and carbonyl oxygen participates in hydrogen
  bonding
• Other possibilities not as common in proteins
  310 helix and ? helix (not been observed)

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Alpha-Helix

• -Repeats after 18 residues, 5 turns, c h x m
• -3.6 residues per turn, 0.15 nm/residue
• p nh 3.6 x 0.15 0.54 nm/turn
• -Each amido proton is hydrogen bonded to the
  carbonyl oxygen on the 4th residue up the helix
• -Hydrogen bonded loop (N) 13 atoms in
  alpha-helix

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Alpha Helix
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Beta-sheet

• Beta-sheet is equivalent to a n2 helix,
• Stabilized by hydrogen bonding between two stands
• Each residue is rotated 180 degrees to one
  another
• Linear hydrogen bonds can form between adjacent
  chains, allowing correct bond angles with minimal
  strain
• Parallel and anti-parallel beta-sheet

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Parallel and Antiparallel Beta-Sheets
Parallel
Anti-parallel
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Ramachandran Plots

• Examination of steric crowding of R-groups in the
  secondary structures
• Phi and Psi angles clockwise when looking in
  either direction form the ?-carbon (-180 to
  180)

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Ramachandran Plots

• Which structures are sterically possible
• Only a small area of possible angles exists
  because of van der Waals radii
• Right handed helix more favored than left all
  amino acids are in the L-form, and steric
  interference is less in a right handed helix.
  Synthesis of proteins from D-amino acids are left
  handed

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Ramachandran Plots

• Ramachandran plot of actual protein structure.
  Some difference from alpha-helix and beta-sheet.
  Glycine can adopt angles outside the allowable
  angles because of small side chain.

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Fibrous Proteins

• Major proteins of skin and connective tissue
  mechanical properties
• Alpha-keratins hair and finger nails
  intermediate filament proteins
• Predominantly alpha-helical
• Alpha-helices pair together in a left-handed coil
• Each 4th residue is nonpolar hydrophobic strip
  of hydrophobic residues on each alpha-helix
  allows two helices to stick together by
  hydrophobic interactions.
• The dimers then form into larger structures and
  are stabilized by disulfide cross-links within
  the fibers.

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Formation of keratin type intermediate filaments

• Monomers pair by coiled-coil
• Form 4-stranded protofilament
• 8-strand protofibril
• Disulfide cross-links stiffen fibers

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Fibroin

• Fibers spun by silkworms and spiders B-sheet
  utilized
• Repetitive sequence
• Allows the B-sheets to pack together by
  interdigitation of glycine and alanine or serine.
• Strong and flexible fiber (weak interactions
  between sheets, van der Waals).
• Small amounts of bulky amino acids disrupt
  beta-sheet. Increases stretchiness.

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Collagen

• Most abundant protein in vertebrates matrix
  that holds tissues together
• Composed of a triple helix of 3 polypeptide
  chains
• Individual chains are left-handed helices with
  3.3 residues per turn and the 3 chains wrap
  around one another in a right handed manner
  stabilized by hydrogen bonds between chains
• Each 3rd residue must be glycine which lies
  near the center of the triple helix, Gly Pro
  HydroxyProline, other residues are possible at
  2nd and 3rd position
• Hydroxylation of proline enzyme that catalyzes
  requires Vitamin C scurvy is Vit. C deficiency.
  Lesions on skin and gums
• Collagen Cross-linking between topocollagen
  molecules lysine oxidized to aldehyde react
  with another lysine via aldol condensation and
  dehydration.

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Elastin

• Mostly random coil with little 2ndary structure
• Cross-links between several chains
• Prevent over stretching and allows the fibers to
  snap back into place when tension is removed

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Globular Proteins

• Folded into compact structure
• Example myoglobin 70 alpha-helix, heme
  (prosthetic) group to carry oxygen
• Bovine pancreatic trypsin inhibitor helix,
  beta-sheet and nonregular folding

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Patterns in protein folding

• Domain compact locally folded region of
  tertiary structure, multiple domains occur in
  many larger proteins and perform specific
  functions
• Folding patterns
• Built on a packing of alpha-helices
• Built on a framework of Beta-sheets

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Rules of Tertiary Folding

• All globular proteins have a defined inside and
  out
• Hydrophobic amino acids are inside the protein
• Hydrophilic amino acids are outside
• Beta-sheets are usually twisted or wrapped into
  barrel structures
• Twist is usually left-handed, structure of silk
  is not completely planar but slightly twisted
• Not all parts of globular proteins are classified
  as helix, sheet or turn
• Random coil flexible regions not resolved by
  x-ray crystallography
• Irregularly structured regions not flexible

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Turns

• The polypeptide chain can turn corners in a
  number of ways to go from B-sheet to a-helix
• ??-turn, reversal of polypeptide chain direction
• 4 residues , i carbonyl is hydrogen bonded to the
  amide hydrogen of i 3
• ?-turn, the hydrogen bond is i and i 2.
• Proline usually plays a role and turns are
  usually at surface of protein

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Quanternary structure

• Two or more subunits stabilized by non-covalent
  forces and disulfide bonds
• Helical symmetry actin and tobacco mosiac virus

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