Determining Protein Structure - 1 NMR Spectroscopy: Saturday, October 8, 2011

Determining protein structure - 1
NMR spectroscopy
1
Saturday, October 8, 2011
NMR spectroscopy
Require nucleus with “spin” (I ≥ ½):

Nuclei of biological interest with:
I = 0: 12C, 16O NMR active &
“useful” in solution
I = ½: 1H, 13C, 15N, 19F, 31P
NMR active but
I > ½: 2H, 14N, 33S, 23Na, 17O quadrupolar (or,
difficult to study)
Basis of the NMR signal [to the board...]
2
Now, ΔE - but same for all nuclei?
What if our B0 field differs locally?
Differences in local
electron density
modulate the local
magnetic field...
Figure: Levitt. Spin Dynamics. (2002) Wiley. 3

Protein NMR
1D 1H-NMR of 199
amino acid protein:
δ 1H (ppm) 4
Expected protein chemical shifts
To TopSpin to look at some of the data behind:
where peptides have sequence GGXAGG
5
Meaning (at least crudely):
N-H, O-H Side chain

& aromatics Hα aliphatics
δ 1H (ppm) 6
Move to 2-dimensions:
2D 1H-15N
correlation NMR
data of same 199
amino acid protein:
δ 15N (ppm)
δ 1H (ppm) 7
Chemical shift assignment strategies?
Using (bio)chemical intuition - expect variation

in δ patterns for different residue types:
Figure: Branden & Tooze (1999) Introduction to Protein Structure (2nd ed.) Garland. 8
Chemical shift assignment strategies?
Use experiments that correlate nuclei within vs.

between amino acid residues:
Sequential
assignment:
rely on
proximity-based
experiments
From: Wüthrich. “NMR of Proteins & Nucleic Acids” (1986) Wiley. 9

Alternatively, with 13C & 15N enrichment
Use 3-dimensional NMR experiments to carry

out “backbone walk”
To the board
for an
example
Figure: http://www.protein-nmr.org.uk/assignment_theory.html 10
Once we have chemical shifts?
Now, assign 1H-1H distances using nuclear

Overhauser enhancement (NOE) spectroscopy
(NOESY)
Just like the FRET process, the strength of the
NOE depends upon the inverse 6th power of the
distance between two nuclei
11
NOESY example from Voet & Voet
NOESY gives the 3

distance constraints
shown as i, j & k
Each provides
connectivity in different
regions of polypeptide
Fold final structure such
that all distances are
satisfied
12
More realistic 2D NMR data
800 MHz NMR data for isolated 31 residue transmembrane segment in DPC micelles
NOESY (blue) - through space

vs. TOCSY (white) through bond
13
Data used in Reddy et al. (2008) J Biol Chem
Effects of 2° & 3° structure?
α-Helices have specific

expected patterns of
distance restraints:
And, 3° structuring gives
rise to “longer-range”
(with respect to 1°
structure) restraints
Example of α-helicity
NOESY at 800 MHz - 27 residue peptide in detergent micelles
H! (ppm)
HN (ppm)
Data used in Rainey/Ding et al. (2006) J Biol Chem 15
Example of α-helicity
NOESY at 800 MHz - 27 residue peptide in detergent micelles
H! (ppm)
HN (ppm)
Data used in Rainey/Ding et al. (2006) J Biol Chem 15
Dissecting those contacts
See: contacts between Hα of residue i and:

HN of “self” (v. strong)
HN of i+1 & i+3 (strong)
HN of i+4 (medium)
HN of i+2 (weak)
16
β-sheets also have characteristic patterns
Note alternating pattern of

Hα and HN contacts
between green sheet &
each orange sheet
Question: would parallel β-

sheet be different?
Obtain a structural ensemble
Use distance constraints (& other experimental

data) to fold our structure. Get set (or ensemble)
of structures that obey restraints:
β-sandwich backbone
superposes very well (as do
illustrated side chains)
Note: loops less converged
18
Example of structural variability
19
Value of NMR for structural biology?
High-resolution structures in [arguably]

physiological conditions: buffers or “crowded”
environments; membrane mimetic environments:
detergent micelles, phospholipid bicelles; living
cells(!)
Since δ is highly sensitive to environment, we
can easily monitor structural perturbation on an
atomic level!
20
Beyond structure?
As movie and structural ensembles (vs.

snapshots) imply, molecular dynamics is
inherently seen by NMR
Can go beyond this and monitor
variation of dynamics at atomic level
21
Example: troponin regulatory domain
apo-troponin
Spyracopoulos et al. (2001) Biochemistry 40: 12541
troponin saturated with Ca2+
Spyracopoulos et al. (1997) Biochemistry 37: 18032
Structures: 1SPY (apo)

1AP4 (Ca2+-bound) 22

Determining Protein Structure - 1 NMR Spectroscopy: Saturday, October 8, 2011

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Determining protein structure - 1

Require nucleus with “spin” (I ≥ ½):

What if our B0 field differs locally?

Figure: Levitt. Spin Dynamics. (2002) Wiley. 3

To TopSpin to look at some of the data behind:

where peptides have sequence GGXAGG

N-H, O-H Side chain

Using (bio)chemical intuition - expect variation

Use experiments that correlate nuclei within vs.

From: Wüthrich. “NMR of Proteins & Nucleic Acids” (1986) Wiley. 9

Use 3-dimensional NMR experiments to carry

Now, assign 1H-1H distances using nuclear

NOESY gives the 3

NOESY (blue) - through space

α-Helices have specific

See: contacts between Hα of residue i and:

Note alternating pattern of

Question: would parallel β-

Use distance constraints (& other experimental

Note: loops less converged

High-resolution structures in [arguably]

As movie and structural ensembles (vs.

Spyracopoulos et al. (2001) Biochemistry 40: 12541

troponin saturated with Ca2+

Spyracopoulos et al. (1997) Biochemistry 37: 18032

Structures: 1SPY (apo)

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