Transcript Protein Structure - Research Centers

Protein Methods;
Fundamentals of Protein
Structure
Andy Howard
Introductory Biochemistry, Fall 2008
4 September 2008
Biochemistry: Methods & Structure
09/04/08
Plans for Today
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Protein methods
(Concluded)
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Electrophoresis
Spectroscopy
Scattering
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Levels of Structure
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Primary
Secondary
Tertiary
Quaternary
Why we care
about structure
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Electrophoresis
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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)
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SDS-PAGE
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Sodium dodecyl sulfate: strong
detergent, applied to protein
Charged species binds quantitatively
Denatures protein
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Good because initial shape irrelevant
Bad because it’s no longer folded
Larger proteins move slower because
they get tangled in the matrix
1/Velocity  √MW
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SDS PAGE illustrated
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Isoelectric focusing
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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 where
charge =0 (isoelectric point)
Sensitive to single changes in
charge (e.g. asp -> asn)
Readily used preparatively with
samples that are already semipure
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Ultraviolet spectroscopy
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Tyr, trp absorb and fluoresce:
abs ~ 280-274 nm; f = 348 (trp), 303nm
(tyr)
Reliable enough to use for estimating
protein concentration via Beer’s 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
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X-ray spectroscopy
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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
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Solution scattering
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Proteins in solution scatter X-rays
in characteristic, sphericallyaveraged 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
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Fiber
Diffraction
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Some proteins, like many
DNA molecules, possess
approximate fibrous order
(2-D ordering)
Produce characteristic fiber
diffraction patterns
Collagen, muscle proteins,
filamentous viruses
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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 don’t know what a protein
does, its structure might give us
what we need to know to figure out
its function.
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Levels of Protein Structure
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We conventionally describe proteins at
four levels of structure, from most local to
most global:
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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
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What does the primary
structure look like?
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-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; that’s
less informative than the sequence
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Components of secondary
structure
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, 310,  helices
pleated sheets and
the strands that
comprise them
Beta turns
More specialized
structures like
collagen helices
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An accounting for secondary
structure: phospholipase A2
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Alpha helix
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Characteristics of  helices
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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
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What would disrupt this?
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Not much: the side chains
don’t bump into one another
Proline residue will disrupt it:
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Main-chain N can’t H-bond
The ring forces a kink
Glycines sometimes disrupt
because they tend to be
flexible
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Other helices
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NH to C=O four residues earlier is
not the only pattern found in
proteins
310 helix is NH to C=O three
residues earlier
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More kinked; 3 residues per turn
Often one H-bond of this kind at Nterminal end of an otherwise -helix
  helix: even rarer: NH to C=O
five residues earlier
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Beta strands
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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
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Sheets: roughly
planar
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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
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Anti-parallel
beta sheet
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Neighboring strands extend in opposite
directions
Complementary C=O…N bonds from
top to bottom and bottom to top strand
Slightly pleated for optimal H-bond
strength
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Parallel
Beta Sheet
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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
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Beta turns
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Abrupt change in direction
, angles are
characteristic of beta
Main-chain H-bonds
maintained almost all the way
through the turn
Jane Richardson and others
have characterized several
types
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Collagen triple helix
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Three left-handed helical
strands interwoven with a
specific hydrogen-bonding
interaction
Every 3rd residue
approaches other strands
closely: so they’re glycines
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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?
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Options
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Assume side chain of
residue 2 points DOWN
into the bilayer:
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(a) GADHKYEKLRG
(b) GLDGIVESVGG
(c) AKRTTVWKDKD
(d) YRNNADRRKLG
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Note about disulfides
H
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Cysteine residues brought S
into proximity under
H
oxidizing conditions can C
form a disulfide
Forms a “cystine” residue
Oxygen isn’t always the
oxidizing agent
Can bring sequence-distant
residues close together and
stabilize the protein
Biochemistry: Methods & Structure
H
S
H
H
+
(1/2)O 2
H2O
H
H
C
C
S
H
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S
H
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H
C
Hydrogen bonds, revisited
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Biological settings, H-bonds are almost
always:
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Between carbonyl oxygen and hydroxyl:
(C=O ••• H-O-)
between carbonyl oxygen and amine:
(C=O ••• H-N-)
These are stabilizing structures
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Any stabilization is (on its own) entropically
disfavored;
Sufficient enthalpic optimization overcomes
that!
In general the optimization is ~ 1- 4 kcal/mol
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Secondary structures in
structural proteins
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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
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Alpha-keratin
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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
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Silk
fibroin
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Antiparallel beta
sheets running
parallel to the
silk fiber axis
Multiple repeats
of (Gly-Ser-GlyAla-Gly-Ala)n
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Secondary structure in
globular proteins
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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”
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Tertiary Structure
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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, . . .
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Quaternary
structure
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Arrangement of individual polypeptide
chains to form a complete oligomeric,
functional protein
Individual chains can be identical or
different
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If they’re the same, they can be coded for
by the same gene
If they’re different, you need more than
one gene
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Not all proteins have all four
levels of structure
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Monomeric proteins don’t 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!
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Protein Topology
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Description of the
connectivity of
segments of
secondary structure
and how they do or
don’t cross over
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TIM barrel
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Alternating ,  creates parallel pleated sheet
Bends around as it goes to create
barrel
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