Protein Structure and Dynamics by NMR in Solution
101
3.3.3
Cross-Correlated Relaxation Rate Constants
There has been recently much excitement about the prospects for using relaxation not only to characterize the dynamical behavior of proteins but also to measure dihedral angles. We have mentioned earlier that the ultimate cause of relaxation is molecular motion: when two relaxation mechanisms are modulated by the
same motion (they are said to be cross-correlated), spurious effects appear: for
instance, the two components of a ISN resonance (split by the HN coupling)
exhibit different line-widths, under slow tumbling conditions. The theoretical
background for these effects is outside the scope of this review, but the feature of
most interest for a conformational use is their angular dependence in (3
cos 2 8-1), where 8 defines the relative orientation of the two relaxation mechanisms. This method has been proposed to quantify the dihedral angle underlying
two dipole-dipole interactions (NH-CUH) or a CSA tensor and a dipole-dipole
interaction (C' CSA-CUH) (Reif et al 1997, Yang et al 1997). It is expected that
these new probes will be widely used in the near future to complement Jcouplings, despite their experimental and theoretical complexities (passive coupling, internal motion, etc .... ).
The cross-correlation effect can be directly exploited simply to enhance the
spectral resolution: Pervushin et al (1997) have proposed an experiment called
TROSY (transverse relaxation optimized spectroscopy) for IH_lSN correlation.
For one of the lines of the correlation peak, a partial cancellation of the transverse relaxation (T2) arises due to the interference of the CSA and the dipolar
relaxation. The authors claim a several-fold increase of the molecular size accessible by NMR with these methods, despite the fact that the predicted narrowing
varies substantially from one nucleus to another and reaches its maximum at
very high fields (corresponding to IH frequencies larger than 1 GHz, actually, the
most performant NMR spectrometer operate at 800 MHz).
3.4
Exchange Rate Constants (H - D)
Some hydrogens (such as NH, NH2, OH) in macromolecules are in contino us
exchange with hydrogen atoms of the water. Measurements of hydrogenexchange rates can provide - in a non-perturbing way - information on protein
dynamics and stability at an atomic level. The labile hydrogens in a protein are
covalently bound and thus a chemical reaction is actually required for the
exchange with solvent. In aqueous solution, exchange of peptidic HN can be catalyzed either by H+ or OH- ions, showing a minimal rate between pH 2 and 3.
Structural effects can slow down protein hydrogen exchange by large protection
factors (> 10 9 ), that always involve hydrogen bonding. Therefore, an exchange
reaction requires two steps: a transient opening of the structure and the intrinsic
exchange. Two limiting cases can be distinguished, (EX2, EXl) depending upon
which step is rate-limiting. Hydrogen exchange is thus related to transient
unfolding of the protein, involving either the unfolding of the whole molecule or
of secondary structure elements. As compared to other spectroscopic techniques
101
3.3.3
Cross-Correlated Relaxation Rate Constants
There has been recently much excitement about the prospects for using relaxation not only to characterize the dynamical behavior of proteins but also to measure dihedral angles. We have mentioned earlier that the ultimate cause of relaxation is molecular motion: when two relaxation mechanisms are modulated by the
same motion (they are said to be cross-correlated), spurious effects appear: for
instance, the two components of a ISN resonance (split by the HN coupling)
exhibit different line-widths, under slow tumbling conditions. The theoretical
background for these effects is outside the scope of this review, but the feature of
most interest for a conformational use is their angular dependence in (3
cos 2 8-1), where 8 defines the relative orientation of the two relaxation mechanisms. This method has been proposed to quantify the dihedral angle underlying
two dipole-dipole interactions (NH-CUH) or a CSA tensor and a dipole-dipole
interaction (C' CSA-CUH) (Reif et al 1997, Yang et al 1997). It is expected that
these new probes will be widely used in the near future to complement Jcouplings, despite their experimental and theoretical complexities (passive coupling, internal motion, etc .... ).
The cross-correlation effect can be directly exploited simply to enhance the
spectral resolution: Pervushin et al (1997) have proposed an experiment called
TROSY (transverse relaxation optimized spectroscopy) for IH_lSN correlation.
For one of the lines of the correlation peak, a partial cancellation of the transverse relaxation (T2) arises due to the interference of the CSA and the dipolar
relaxation. The authors claim a several-fold increase of the molecular size accessible by NMR with these methods, despite the fact that the predicted narrowing
varies substantially from one nucleus to another and reaches its maximum at
very high fields (corresponding to IH frequencies larger than 1 GHz, actually, the
most performant NMR spectrometer operate at 800 MHz).
3.4
Exchange Rate Constants (H - D)
Some hydrogens (such as NH, NH2, OH) in macromolecules are in contino us
exchange with hydrogen atoms of the water. Measurements of hydrogenexchange rates can provide - in a non-perturbing way - information on protein
dynamics and stability at an atomic level. The labile hydrogens in a protein are
covalently bound and thus a chemical reaction is actually required for the
exchange with solvent. In aqueous solution, exchange of peptidic HN can be catalyzed either by H+ or OH- ions, showing a minimal rate between pH 2 and 3.
Structural effects can slow down protein hydrogen exchange by large protection
factors (> 10 9 ), that always involve hydrogen bonding. Therefore, an exchange
reaction requires two steps: a transient opening of the structure and the intrinsic
exchange. Two limiting cases can be distinguished, (EX2, EXl) depending upon
which step is rate-limiting. Hydrogen exchange is thus related to transient
unfolding of the protein, involving either the unfolding of the whole molecule or
of secondary structure elements. As compared to other spectroscopic techniques