Transcript structuremethods

Protein Structure and Function
Andrew Howard
Fourth Lecture
Introductory Biochemistry
31 January 2008
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.

IIT Biochemistry: 31 Jan 2008
Slide 2 of 60
Let’s finish up peptides,
though!

We need to talk about Ramachandran
angles, peptide bonds, and oligopeptide
and polypeptide chemistry before we go
on to techniques for determining
structures.
IIT Biochemistry: 31 Jan 2008
Slide 3 of 60
Ramachandran
angles
G.N. Ramachandran
IIT Biochemistry: 31 Jan 2008
Slide 4 of 60
Preferred Values
of  and 
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Steric hindrance makes some
values unlikely
Specific values are characteristic of
particular types of secondary
structure
Most structures with forbidden
values of  and  turn out to be
errors
IIT Biochemistry: 31 Jan 2008
Slide 5 of 60
How far from 180º can w
vary?
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Remember what we said about the
partial double bond character of
the C-N main-chain bond
That imposes planarity
In practice it rarely varies by more
than a few degrees from 180º.
IIT Biochemistry: 31 Jan 2008
Slide 6 of 60
Ramachandran plot
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Cf. fig. 4.9 in
Horton
If you submit a
structure to the
PDB with
Ramachandran
angles far from
the yellow
regions, be
prepared to justify
them!
IIT Biochemistry: 31 Jan 2008
Slide 7 of 60
How are oligo- and
polypeptides synthesized?
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Formation of the peptide linkages occurs
in the ribosome under careful enzymatic
control
Polymerization is endergonic and
requires energy in the form of GTP (like
ATP, only with guanosine):
GTP + n-length-peptide + amino acid 
GDP + Pi + (n+1)-length peptide
IIT Biochemistry: 31 Jan 2008
Slide 8 of 60
What happens at the ends?
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Usually there’s a free amino end
and a free carboxyl end:
H3N+-CHR-CO-(peptide)n-NH-COOCyclic peptides do occur
Cyclization doesn’t happen at the
ribosome: it involves a separate,
enzymatic step.
IIT Biochemistry: 31 Jan 2008
Slide 9 of 60
Reactivity in peptides &
proteins
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Main-chain acid-base reactivity
unavailable except on the ends
Side-chain reactivity available but
with slightly modified pKas.
Terminal main-chain pKavalues
modified too
Environment of protein side chain is
often hydrophobic, unlike free amino
acid side chain’s neighborhood
IIT Biochemistry: 31 Jan 2008
Slide 10 of 60
Discussion question
What’s the net charge in ELVIS
at pH 7?
 (a) 0
 (b) +1
 (c) -1
 (d) +2
 (e) -2
IIT Biochemistry: 31 Jan 2008
Slide 11 of 60
Disulfides
In oxidizing
environments, two
neighboring cysteine
residues can react
with an oxidizing
agent to form a
covalent bond
between the side
chains
IIT Biochemistry: 31 Jan 2008
H
H
S
S
H
H
C
H
+
(1/2)O 2
H2O
H
H
C
C
S
H
S
H
Slide 12 of 60
H
C
What could this do?
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Can bring portions of a protein
that are distant in amino acid
sequence into close proximity with
one another
This can influence protein stability
IIT Biochemistry: 31 Jan 2008
Slide 13 of 60
… and now, onward to protein
structure methods and results

We’ll look at several techniques, and
then focus on what we know on the basis
of the application of those techniques.
IIT Biochemistry: 31 Jan 2008
Slide 14 of 60
Plans for Today

Methods of
Determining
Protein Structure

Crystallography
 NMR
 CryoEM
 Specialty
techniques
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IIT Biochemistry: 31 Jan 2008
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Levels of Protein
Structure
Hydrogen Bonds
Secondary
structure in
globular proteins
Tertiary Structure
Domains
Slide 15 of 60
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 the stated topic of the day.
IIT Biochemistry: 31 Jan 2008
Slide 16 of 60
How do we determine
structure?
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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
IIT Biochemistry: 31 Jan 2008
Slide 17 of 60
Crystallography: overview
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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
IIT Biochemistry: 31 Jan 2008
Slide 18 of 60
How are protein crystals
unusual?
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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
IIT Biochemistry: 31 Jan 2008
Slide 19 of 60
Crystal structures: Fourier
transforms of diffraction
results
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Position of spots tells you how big the unit cell is
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
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)
IIT Biochemistry: 31 Jan 2008
Slide 20 of 60
F
The phase problem
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
a
Note that we said Ihkl = C*|Fhkl|2
That means we can figure out
|Fhkl| = (1/C)√Ihkl
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But 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!
IIT Biochemistry: 31 Jan 2008
Slide 21 of 60
b
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 atoms, most solvent,
some disordered atoms
IIT Biochemistry: 31 Jan 2008
Slide 22 of 60
What does this look like?
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Takes some
experience to
interpret
Automated fitting
programs work
pretty well with
Dmin < 2.1Å
ATP binding to a protein of
unknown function: S.H.Kim
IIT Biochemistry: 31 Jan 2008
Slide 23 of 60
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
IIT Biochemistry: 31 Jan 2008
Slide 24 of 60
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
IIT Biochemistry: 31 Jan 2008
Slide 25 of 60
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
IIT Biochemistry: 31 Jan 2008
Prof. Mark Searle,
University of Nottingham
Slide 26 of 60
Results
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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
IIT Biochemistry: 31 Jan 2008
Slide 27 of 60
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;
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 > 25 kDa
IIT Biochemistry: 31 Jan 2008
Slide 28 of 60
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
IIT Biochemistry: 31 Jan 2008
Slide 29 of 60
Cryoelectron
microscopy
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Like X-ray crystallography,
EM damages the samples
Samples analyzed < 100K
survive better
2-D arrays of molecules
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Spatial averaging to improve
resolution
Discerning details ~ 4Å resolution
Can be used with crystallography
IIT Biochemistry: 31 Jan 2008
Slide 30 of 60
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
IIT Biochemistry: 31 Jan 2008
Slide 31 of 60
Poll question:
discuss!
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Which protein would yield
a more interpretable CD
spectrum?
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(a) myoglobin
(b) Fab fragment of
immunoglobulin G
(c) both would be fully
interpretable
(d) CD wouldn’t tell us
anything about either
protein
IIT Biochemistry: 31 Jan 2008
Slide 32 of 60
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
IIT Biochemistry: 31 Jan 2008
Slide 33 of 60