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Protein Methods; Fundamentals of Protein Structure

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Fundamentals of Protein Structure Andy Howard ... creates parallel -pleated sheet Bends around as it goes to create barrel 09/04/08 Biochemistry: ... – PowerPoint PPT presentation

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Title: Protein Methods; Fundamentals of Protein Structure


1
Protein MethodsFundamentals of Protein Structure
  • Andy HowardIntroductory Biochemistry, Fall
    20084 September 2008

2
Plans for Today
  • Levels of Structure
  • Primary
  • Secondary
  • Tertiary
  • Quaternary
  • Protein methods (Concluded)
  • Electrophoresis
  • Spectroscopy
  • Scattering
  • Why we care about structure

3
Electrophoresis
  • Separating analytes by charge by subjecting a
    mixture to a strong electric field
  • Gel electrophoresis field applied to a semisolid
    matrix
  • Can be used for charge (directly) or size
    (indirectly)

4
SDS-PAGE
  • Sodium dodecyl sulfate strong detergent, applied
    to protein
  • Charged species binds quantitatively
  • Denatures protein
  • Good because initial shape irrelevant
  • Bad because its no longer folded
  • Larger proteins move slower because they get
    tangled in the matrix
  • 1/Velocity ? vMW

5
SDS PAGE illustrated
6
Isoelectric focusing
  • Protein applied to gel without charged denaturant
  • Electric field set up over a pH gradient
    (typically pH 2 to 12)
  • Protein will travel until it reaches the pH
    wherecharge 0 (isoelectric point)
  • Sensitive to single changes in charge (e.g. asp
    -gt asn)
  • Readily used preparatively with samples that are
    already semi-pure

7
Ultraviolet spectroscopy
  • Tyr, trp absorb and fluoresce?abs 280-274 nm
    ?f 348 (trp), 303nm (tyr)
  • Reliable enough to use for estimating protein
    concentration via Beers law
  • UV absorption peaks for cofactors in various
    states are well-understood
  • More relevant for identification of moieties than
    for structure determination
  • Quenching of fluorescence sometimes provides
    structural information

8
X-ray spectroscopy
  • All atoms absorb UV or X-rays at characteristic
    wavelengths
  • Higher Z means higher energy, lower ??for a
    particular edge
  • Perturbation of absorption spectra at E Epeak
    ? yields neighbor information
  • Changes just below the peak yield oxidation-state
    information
  • X-ray relevant for metals, Se, I

9
Solution scattering
  • Proteins in solution scatter X-rays in
    characteristic, spherically-averaged ways
  • Low-resolution structural information available
  • Does not require crystals
  • Until 2000 you needed high protein
  • Thanks to BioCAT, SAXS on dilute proteins is
    becoming more feasible
  • Hypothesis-based analysis

10
Fiber Diffraction
  • Some proteins, like many DNA molecules, possess
    approximate fibrous order(2-D ordering)
  • Produce characteristic fiber diffraction patterns
  • Collagen, muscle proteins, filamentous viruses

11
Protein Structure Helps us Understand Protein
Function
  • If we do know what a protein does, its structure
    will tell us how it does it.
  • If we dont know what a protein does, its
    structure might give us what we need to know to
    figure out its function.

12
Levels of Protein Structure
  • We conventionally describe proteins at four
    levels of structure, from most local to most
    global
  • Primary linear sequence of peptide units and
    covalent disulfide bonds
  • Secondary main-chain H-bonds that define
    short-range order in structure
  • Tertiary three-dimensional fold of a polypeptide
  • Quaternary Folds of multiple polypeptide chains
    to form a complete oligomeric unit

13
What does the primary structure look like?
  • -ala-glu-val-thr-asp-pro-gly-
  • Can be determined by amino acid sequencing of the
    protein
  • Can also be determined by sequencing the gene and
    then using the codon information to define the
    protein sequence
  • Amino acid analysis means percentages thats
    less informative than the sequence

14
Components of secondary structure
  • ?, 310, ? helices
  • ??pleated sheets and the strands that comprise
    them
  • Beta turns
  • More specialized structures like collagen helices

15
An accounting for secondary structure
phospholipase A2
16
Alpha helix
17
Characteristics of ? helices
  • Hydrogen bonding from amino nitrogen to carbonyl
    oxygen in the residue 4 earlier in the chain
  • 3.6 residues per turn
  • Amino acid side chains face outward
  • 10 residues long in globular proteins

18
What would disrupt this?
  • Not much the side chains dont bump into one
    another
  • Proline residue will disrupt it
  • Main-chain N cant H-bond
  • The ring forces a kink
  • Glycines sometimes disrupt because they tend to
    be flexible

19
Other helices
  • NH to CO four residues earlier is not the only
    pattern found in proteins
  • 310 helix is NH to CO three residues earlier
  • More kinked 3 residues per turn
  • Often one H-bond of this kind at N-terminal end
    of an otherwise ?-helix
  • ? helix even rarer NH to CO five residues
    earlier

20
Beta strands
  • Structures containing roughly extended
    polypeptide strands
  • Extended conformation stabilized by inter-strand
    main-chain hydrogen bonds
  • No defined interval in sequence number between
    amino acids involved in H-bond

21
Sheets roughly planar
  • Folds straighten H-bonds
  • Side-chains roughly perpendicular from sheet
    plane
  • Consecutive side chains up, then down
  • Minimizes intra-chain collisions between bulky
    side chains

22
Anti-parallel beta sheet
  • Neighboring strands extend in opposite directions
  • Complementary CON bonds from top to bottom and
    bottom to top strand
  • Slightly pleated for optimal H-bond strength

23
Parallel Beta Sheet
  • N-to-C directions are the same for both strands
  • You need to get from the C-end of one strand to
    the N-end of the other strand somehow
  • H-bonds at more of an angle relative to the
    approximate strand directions
  • Therefore more pleated than anti-parallel sheet

24
Beta turns
  • Abrupt change in direction
  • ?,? angles arecharacteristic of beta
  • Main-chain H-bonds maintained almost all the way
    through the turn
  • Jane Richardson and others have characterized
    several types

25
Collagen triple helix
  • Three left-handed helical strands interwoven with
    a specific hydrogen-bonding interaction
  • Every 3rd residue approaches other strands
    closely so theyre glycines

26
Poll question
  • Remember that there are about 3.6 residues per
    turn in an alpha helix.
  • Suppose you had a helical protein that was
    sitting on, not in, a phospholipid bilayer so
    that the side chains point inward and outward
    along the surface.
  • Which of the following sequences would be the
    most stable in this environment?

27
Options
  • Assume side chain of residue 2 points DOWN into
    the bilayer
  • (a) GADHKYEKLRG
  • (b) GLDGIVESVGG
  • (c) AKRTTVWKDKD
  • (d) YRNNADRRKLG

28
Note about disulfides
  • Cysteine residues brought into proximity under
    oxidizing conditions can form a disulfide
  • Forms a cystine residue
  • Oxygen isnt always the oxidizing agent
  • Can bring sequence-distant residues close
    together and stabilize the protein

29
Hydrogen bonds, revisited
  • Biological settings, H-bonds are almost always
  • Between carbonyl oxygen and hydroxyl(CO
    H-O-)
  • between carbonyl oxygen and amine(CO H-N-)
  • These are stabilizing structures
  • Any stabilization is (on its own) entropically
    disfavored
  • Sufficient enthalpic optimization overcomes that!
  • In general the optimization is 1- 4 kcal/mol

30
Secondary structures in structural proteins
  • Structural proteins often have uniform secondary
    structures
  • Seeing instances of secondary structure provides
    a path toward understanding them in globular
    proteins
  • Examples
  • Alpha-keratin (hair, wool, nails, ) ?-helical
  • Silk fibroin (guess) is ?-sheet

31
Alpha-keratin
  • Actual ?-keratins sometimes contain helical
    globular domains surrounding a fibrous domain
  • Fibrous domain long segments of regular
    ?-helical bonding patterns
  • Side chains stick out from the axis of the helix

32
Silk fibroin
  • Antiparallel beta sheets running parallel to the
    silk fiber axis
  • Multiple repeats of (Gly-Ser-Gly-Ala-Gly-Ala)n

33
Secondary structure in globular proteins
  • Segments with secondary structure are usually
    short 2-30 residues
  • Some globular proteins are almost all helical,
    but even then there are bends between short
    helices
  • Other proteins mostly beta
  • Others regular alternation of ?, ?
  • Still others irregular ?, ?, coil

34
Tertiary Structure
  • The overall 3-D arrangement of atoms in a single
    polypeptide chain
  • Made up of secondary-structure elements locally
    unstructured strands
  • Described in terms of sequence, topology, overall
    fold, domains
  • Stabilized by van der Waals interactions,
    hydrogen bonds, disulfides, . . .

35
Quaternary structure
  • Arrangement of individual polypeptide chains to
    form a complete oligomeric, functional protein
  • Individual chains can be identical or different
  • If theyre the same, they can be coded for by the
    same gene
  • If theyre different, you need more than one gene

36
Not all proteins have all four levels of structure
  • Monomeric proteins dont have quaternary
    structure
  • Tertiary structure subsumed into 2ndry structure
    for many structural proteins (keratin, silk
    fibroin, )
  • Some proteins (usually small ones) have no
    definite secondary or tertiary structure they
    flop around!

37
Protein Topology
  • Description of the connectivity of segments of
    secondary structure and how they do or dont
    cross over

38
TIM barrel
  • Alternating ?, ? creates parallel ?-pleated sheet
  • Bends around as it goes to create barrel
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