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Solving NMR structures Part I: Experimentally derived restraints. 1. Distance restraints from crosspeak intensities in NOESY spectra; measuring and calibrating NOEs 2. Dihedral angle restraints from three-bond J couplings; measuring J couplings; chemical shifts and dihedral angles
1. Distance restraints
from crosspeak intensities in NOESY spectra; measuring and calibrating NOEs
2. Dihedral angle restraints
from three-bond J couplings; measuring J couplings;
chemical shifts and dihedral angles
3. Hydrogen bond restraints
from amide hydrogen exchange protection data
4. Orientational restraints from residual dipolar couplings (covered in a separate lecture)
basic NOESY pulse sequence
classrestraintdescription *for protein w/ Mr<20 kDa
strong 1.8-2.7 Å strong intensity in short tm (~50 ms*) NOESY
medium 1.8-3.3 Å weak intensity in short tm (~50 ms*) NOESY
weak 1.8-5.0 Å only visible in longer mixing time NOESY
always the same!
There exist relationships between three-bond scalar coupling constants3Jand the corresponding dihedral anglesq, called Karplus relations. These have the general form
3J = Acos2q + Bcosq + C
in solution vs. f angles from crystal
structure for BPTI
differ by 60°
because they are defined differently
from p. 167 Wuthrich textbook
3JHa,HN(f)= 6.4 cos2(f - 60°) -1.4 cos(f - 60°) + 1.9
3JHa,Hb2(c1)= 9.5 cos2(c1- 120°) -1.6 cos(c1- 120°) + 1.8
3JHa,Hb3(c1)= 9.5 cos2(c1) -1.6 cos(c1) + 1.8
3JN,Hb3(c1)= -4.5 cos2(c1+ 120°) +1.2 cos(c1+ 120°) + 0.1
3JN,Hb2(c1)= 4.5 cos2(c1- 120°) +1.2 cos(c1- 120°) + 0.1
ratio of crosspeak
and diagonal peak intensities
can be related to 3JHN-Ha
HN to Ha
this is one plane of a 3D spectrum of ubiquitin. The plane corresponds to this 15N chemical shift
Archer et al. J. Magn. Reson.
95, 636 (1991).
& Wuthrich Biopolymers
17, p. 2727 (1978).
for c1 =180 both 3JNb ~1 Hz for c1 =+60,-60 one is ~5, other is ~1
can’t tell the difference unless b’s are stereospecifically assigned
for a particular b proton,
if q=180, 3JC,Hb= ~8 Hz
if q=+60 or -60, 3JC,Hb= ~1 Hz
Grzesiek et al. J. Magn. Reson. 95,
from Bax et al. Meth. Enzym. 239, 79.
3JHa,HN(f) < 6 Hz f= -65° ± 25°
3JHa,HN(f) > 8 Hz f= -120 ± 40°
This accounts for errors in measurement as well as the fact that the Karplus relation itself is not exact.
Wishart, Sykes & Richards
J Mol Biol (1991) 222, 311.
exp’tl Ha shift rel. to referenceassignedCSI
within ± 0.1 ppm 0
>0.1 ppm lower -1
>0.1 ppm higher +1
*Wishart, Sykes & Richards Biochemistry (1992) 31, 1647-51.
from random coil values when in either a-helix or b-sheet conformation
for a-helices, Ca shifts are higher than normal, whereas
Cb shifts are lower than normal.
amide protons undergo acid- and base-catalyzed exchange with solvent protons at a rate which ranges from the second to minute time scale, depending upon solvent conditions (mostly pH)
if a protein is placed in D2O, the amide signals due to 1H nuclei will disappear over time due to this exchange
exchange rate in D2O
at 20 °C. Minimum with respect to pH is due to the fact that exchange is both acid and base catalyzed
pH but in D2O!
Englander & Mayne, Ann. Rev. Biophys. Biomol. Struct. (1992) 21, 243.
N-H --> N-D
N-H....O=C --> N-D....O=C
where U is the unfolded state and N is the native state
picture at left:
15N-1H HSQC of Arc repressor 5 min after resuspension in D2O (pH 4.7)--note that unprotected amides such as the unstructured N-terminus (1-7) have already exchanged. This is because intrinsic half-lives at this pH are in the vicinity of 10 seconds to a minute.
Burgering et al. Biopolymers (1995) 35, 217.
The plot above shows measured exchange rates from a series of15N-1H HSQC spectra for Arc repressor. Protected amides are usually within secondary structure elements, but the presence of secondary structure doesn’t guarantee that protection will be observed (note the lack of protection for the beta-sheet from residue 9-14). Lack of protection could indicate that the secondary structure element undergoes fairly rapid local unfolding. Note the strength of NMR and HSQC in particular here--you get a separate exchange rate for every amide proton--great residue-specific dynamic information!
surface is less well-protected
ends of helices less well-protected
blue spheres: P > 4650
green spheres: 370 < P < 4650
red spheres: P < 370
note that while all protected amides are hydrogen-bonded,
not all hydrogen-bonded
amides are equally well
protected. Rates differ both within secondary structure elements (buried positions near the center usually most protected) and between different secondary structure elements
Liu et al.Biochemistry (1999)