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Study of the similar spectral patterns originating from different models and ways of telling them apart

Analysis of all three-site models (U_R, U_L, U_RL, U_R2, U_L2, U_R2L2) revealed that there is a limited number of spectral appearances generated by the model. In general case every titration spectral series may be proposed to be due to at least two alternative mechanisms. However, despite the similar visual appearance of the spectra the quantitative features of the line shapes are, in many cases, different enough to tell the models apart. In other cases, measurements of concentration dependence of peak positions combined with the forward and a reverse titration are capable of unambiguously identify the undelying mechanisms. In completely ambiguous cases, the additional experiments such as ITC, relaxation dispersion or stopped-flow measurements may resolve the ambiguity.

Contents

 

 

Summary of models included in analysis
   

 

 

Common spectral patterns and the models giving rise to them

In this section I summarize my observations from systematic study of the model behaviors. I will make a list of spectral patterns and list all the models in all conditions I saw this pattern occuring. The list is not considered to be exhaustive, however, I hope to pinpoint most of the major patterns which may be seen in NMR spectra of the 3-site systems.

I do NOT consider possible models such as U-R if R* has low population so the coupled transition is NOT significantly operative. In this case, this will be a U model.

Another scenario is if the chemical shift difference of the coupled or binding transition is close to zero. I will only consider here cases where both transitions have substantial dw. The variation of dw may then be envisioned and modelled.

1. Slow exchange between two peaks
Model Transition A Transition B How to separate?
 U slow    
       
U-R slow fast  
U-R slow slow, shifted to one side  
U-R2 slow slow, shifted to one side  
       
U-L slow any  
U-L2 slow any  
       
U-RL slow fast  
U-RL slow slow, shifted to one side  
U-R2L2 slow slow, shifted to one side  
       

 

2. Fast exchange between the two peaks

 
Model Transition A regime Transition B regime

How to separate?

 
U fast    
       
U-R fast fast 2-site fit pattern
U-R2 fast fast

2-site fit pattern, concentration dependence of the position of the initial peak
       
U-L fast any  
U-L2 fast any  
       
U-RL fast fast 2-site fit pattern
U-R2L2 fast fast 2-site fit pattern, concentration dependence of the position of the final peak

Additional models: if the other peak is NOT identified (overlapped/unassigned) and equilibrium in isomerization/dimerization is NOT strongly shifted to one side.

Model Transition A regime Transition B regime How to separate?
U-R fast slow, R* peak unassigned 2-site fit pattern
       
U-R2 fast slow, R2 peak unassigned 2-site fit pattern, concentration dependence of the intensity of the initial peak
U-R2 slow fast, titration incomplete 2-site fit pattern, concentration dependence of the position of the initial peak, further titration points
       
U-RL fast slow, R*L peak unassigned 2-site fit pattern
       
U-R2L2 fast slow, R2L2 peak unassigned 2-site fit pattern, concentration dependence of the intensity of the final peak

More peaks disappear than appear
Model Transition A regime Transition B regime How to separate?
U-R slow slow  
U-R2 slow slow concentration dependence of the intensity of the initial peaks
       
       

In the crowded spectrum a small peak of another residue may be mistaken for a minor conformer: if the final peak of that another residue is exchange-broadened and NOT detected/assigned. One needs a proof of the minor peak being a minor conformer for a residue in question via triple-resonance experiments (say, HNCO/HN(CA)CO).

 

 

More peaks appear than disappear
Model Transition A regime Transition B regime How to separate?
U-RL slow slow  
U-R2L2 slow slow concentration dependence of the intensity of the final peaks
       
       

In the crowded spectrum a small peak of another residue may be mistaken for a minor conformer: if the initial peak of that another residue is exchange-broadened and NOT detected/assigned. One needs a proof of the minor peak being a minor conformer for a residue in question via triple-resonance experiments (say, HNCO/HN(CA)CO).

 

The disappearing peak is stationary while the appearing peak is moving
Model Transition A regime Transition B regime How to separate?
U-R fast slow  
U-R2 fast slow concentration dependence of the intensity of the initial peaks
U-R2L2 slow fast concentration dependence of the position of the final peak
       

NOTE: when modeling Af regimes make sure to include both orientations of chemical shifts (crossing and non-crossing the chemical shift of end species)

The disappearing peak is moving while the appearing peak is stationary
Model Transition A regime Transition B regime How to separate?
U-RL fast slow  
U-R2 slow fast concentration dependence of the position of the initial peak
U-R2L2 fast slow concentration dependence of the intensity of the final peaks
       

NOTE: when modeling Af regimes make sure to include both orientations of chemical shifts (crossing and non-crossing the chemical shift of end species)

Transient narrowing of the peak before the full saturation is achieved

 

Model Transition A regime Transition B regime How to separate?
U-RL fast intermediate  
U-R2L2 fast intermediate concentration dependence of the position of the final peak

Conditions to observe this behavior:

1. The chemical shift of a dimer or isomer must be in between the free and bound species.

2. The equilbirium in isomerization/dimerization must be significantly shifted towards the isomer/dimer.

NOTE: when modeling Af regimes make sure to include both orientations of chemical shifts (crossing and non-crossing the chemical shift of end species)

Transient narrowing of the peak at the low receptor saturation
Model Transition A regime Transition B regime How to separate?
U-R fast intermediate  
U-R2 fast intermediate concentration dependence of the position of the initial peak

Conditions to observe this behavior:

1. The chemical shift of a dimer or isomer must be in between the free and bound species.

2. The equilbirium in isomerization/dimerization must be significantly shifted towards the isomer/dimer.

NOTE: when modeling Af regimes make sure to include both orientations of chemical shifts (crossing and non-crossing the chemical shift of end species)

 

General protocol for establishing the mechanism of the interactions:

Perform three experiments:

  1. HSQC of the free receptor at multiple concentrations
  2. HSQC of the free ligand at multiple concentrations
  3. HSQC of the saturated complex at multiple concentrations
  4. A forward titration (L=>R)
  5. A reverse titration (R=>L)
  6. Optional: A forward titration (L=>R)  at a different receptor concentration.
  7. Optional:  A reverse titration (R=>L)  at a different ligand concentrations.

This data will provide enough evidence for selecting the appropriate mechanism. All data together may be fit by a LineShapeKin Analysis 4 NMR line shape analysis package.

 

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Conclusions

Analysis is unfinished

 

 

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