Transcript Biochemical Thermodynamics - Illinois Institute of Technology

Protein Methods II
Andy Howard
Introductory Biochemistry
Fall 2009, IIT
Proteins are worth studying
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We’ll finish our quick overview of
methods of studying proteins
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Plans
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Purification methods
Analytical methods
Structural methods
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Ion-exchange
chromatography
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Charged species affixed to
column
 Phosphonates (-) retard
(+)charged proteins:
Cation exchange
 Quaternary ammonium salts
(+) retard (-)charged
proteins: Anion exchange
 Separations facilitated by
adjusting pH
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Affinity chromatography
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Stationary phase contains a species that
has specific favorable interaction with
the protein we want
DNA-binding protein specific to
AGCATGCT: bind AGCATGCT to a
column, and the protein we want will
stick; every other protein falls through
Often used to purify antibodies by
binding the antigen to the column
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Metal-ion affinity
chromatography
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Immobilize a metal ion, e.g. Ni, to
the column material
 Proteins with affinity to that metal
will stick
 Wash them off afterward with a
ligand with even higher affinity
 We can engineer proteins to
contain the affinity tag:
poly-histidine at N- or C-terminus
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High-performance liquid
chromatography
Many LC separations can happen faster
and more effectively under high
pressure
 Works for small molecules
 Protein application is routine too, both
for analysis and purification
 FPLC is a trademark, but it’s used
generically
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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)
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SDS-PAGE
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Sodium dodecyl sulfate: strong detergent,
applied to protein
Charged species binds quantitatively
Denatures protein
– Good because initial shape irrelevant
– Bad because it’s no longer folded
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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 I
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)
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Isoelectric focusing II
Sensitive to single changes in
charge (e.g. asp -> asn)
 Can be readily used preparatively
with samples that are already
semi-pure
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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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Warning: Specialty Content!
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I determine protein structures (and develop
methods for determining protein structures)
as my own research focus
 So it’s hard for me to avoid putting a lot of
emphasis on this material
 But today I’m allowed to do that, because it’s
one of the stated topics of the day.
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How do we determine structure?
We can distinguish between methods
that require little prior knowledge
(crystallography, NMR, ?CryoEM?)
and methods that answer specific
questions (XAFS, fiber, …)
 This distinction isn’t entirely clear-cut
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Crystallography: overview
Crystals are translationally ordered 3-D
arrays of molecules
 Conventional solids are usually crystals
 Proteins have to be coerced into
crystallizing
 … but once they’re crystals, they
behave like other crystals, mostly
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How are protein crystals
unusual?
Aqueous interactions required for
crystal integrity: they disintegrate if dried
 Bigger unit cells (~10nm, not 1nm)
 Small # of unit cells and static disorder
means they don’t scatter terribly well
 So using them to determine 3D
structures is feasible but difficult
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Crystal structures: Fourier
transforms of diffraction results
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Experiment:
– Grow crystal, expose it to X-ray
– Record diffraction spots
– Rotate through small angle and repeat ~180 times
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Position of spots tells you size, shape of unit
cell
Intensity tells you what the contents are
We’re using electromagnetic radiation, which
behaves like a wave, exp(2ik•x)
Therefore intensity Ihkl = C*|Fhkl|2
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What are these Fhkl values?
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Fhkl is a complex coefficient in the Fourier
transform of the electron density in the unit
cell:
(r) = (1/V) hkl Fhkl exp(-2ih•r)
Critical point: any single diffraction spot
contains information derived from all the
atoms in the structure; and any atom
contributes to all the diffraction spots
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The phase problem
Note that we said Ihkl = C*|Fhkl
 That means we can figure out
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|2
Fhkl
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ahkl
bhkl
|Fhkl| = (1/C)√Ihkl
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We can’t figure out the direction of F:
Fhkl = ahkl + ibhkl = |Fhkl|exp(ihkl)
 This direction angle is called a phase angle
 Because we can’t get it from Ihkl, we have a
problem: it’s the phase problem!
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What can we learn?
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Electron density map + sequence  we can
determine the positions of all the non-H
atoms in the protein—maybe!
Best resolution possible: Dmin =  / 2
Often the crystal doesn’t diffract that well, so
Dmin is larger—1.5Å, 2.5Å, worse
Dmin ~ 2.5Å tells us where backbone and
most side-chain atoms are
Dmin ~ 1.2Å: all protein non-H atoms, most
solvent, some disordered atoms; some H’s
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What does this look like?
Takes some
experience to
interpret
 Automated
fitting
programs work
pretty well with
Dmin < 2.1Å
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ATP binding to a protein of
unknown function: S.H.Kim
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How’s the field changing?
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1990: all structures done by professionals
Now: many biochemists and molecular
biologists are launching their own
structure projects as part of broader
functional studies
Fearless prediction: by 2020:
– crystallographers will be either technicians or
methods developers
– Most structures will be determined by cell
biologists & molecular biologists
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Macromolecular NMR
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NMR is a mature field
Depends on resonant interaction between EM
fields and unpaired nucleons (1H, 15N, 31S)
Raw data yield interatomic distances
Conventional spectra of proteins are too
muddy to interpret
Multi-dimensional (2-4D) techniques:
initial resonances coupled with additional ones
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Typical protein 2-D spectrum
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Challenge:
identify which
H-H distance is
responsible for a
particular peak
 Enormous
amount of
hypothesis
testing required
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Prof. Mark Searle,
University of Nottingham
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Results of NMR studies
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Often there’s a family of structures that
satisfy the NMR data equally well
Can be portrayed as a series of threads
tied down at unambiguous assignments
They portray the protein’s structure in
solution
The ambiguities partly represent real
molecular diversity; but they also represent
atoms that area in truth well-defined, but
the NMR data don’t provide the
unambiguous assignment
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Comparing NMR to X-ray
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NMR family of structures often reflects real
conformational heterogeneity
Nonetheless, it’s hard to visualize what’s
happening at the active site at any instant
Hydrogens sometimes well-located in NMR;
they’re often the least defined atoms in an Xray structure
The NMR structure is obtained in solution!
Hard to make NMR work if MW > 35 kDa
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What does it mean when NMR
and X-ray structures differ?
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Lattice forces may have tied down or moved
surface amino acids in X-ray structure
NMR may have errors in it
X-ray may have errors in it (measurable)
X-ray structure often closer to true atomic
resolution
X-ray structure has built-in reliability checks
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Cryoelectron
microscopy
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Like X-ray crystallography,
EM damages the samples
 Samples analyzed < 100K
survive better
 2-D arrays of molecules
– Spatial averaging to improve
resolution
– Discerning details ~ 4Å resolution
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Can be used with crystallography
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Circular dichroism
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Proteins in solution can
rotate polarized light
 Amount of rotation varies
with 
 Effect depends on
interaction with secondary
structure elements, esp. 
 Presence of characteristic
 patterns in presence of
other stuff enables
estimate of helical content
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Poll question:
discuss!
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QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Which protein would yield
a more interpretable CD
spectrum?
– (a) myoglobin
– (b) Fab fragment of
immunoglobulin G
– (c) both would be fully
interpretable
– (d) CD wouldn’t tell us
anything about either
protein
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Sperm
whale
myoglobin
PDB 2jho
1.4Å
16.9 kDa
Antifluorescein
Fab
PDB 1flr
1.85 Å
52 KDa
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
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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
All atoms absorb UV or
X-rays at characteristic
wavelengths
 Higher Z means higher
energy, lower for a
particular edge
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X-ray spectroscopy II
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, spherically-averaged ways
Low-resolution structural information
available
Does not require crystals
Until ~ 2000: 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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