Statistical strategy for stereospecific hydrogen NMR assignments: validation procedures for the floating prochirality...

Area of Science:

• Biochemistry
• Structural Biology
• Computational Chemistry

Background:

• Stereospecific hydrogen NMR assignments are crucial for determining molecular structures.
• The floating prochirality method offers a computational approach to these assignments.
• Statistical validation is essential for the reliability of NMR-derived structural data.

Purpose of the Study:

• To evaluate the statistical validity and reproducibility of stereospecific hydrogen NMR assignments.
• To identify limitations of current floating prochirality methods.
• To present validated protocols for assignment and statistical validation.

Main Methods:

• Utilized approximately 1300 distance constraints from NOESY spectra of oxidized horse cytochrome c.
• Performed repeated calculations using various computational strategies on the whole molecule and a fragment.
• Applied statistical analysis to the distribution of prochiral orientations during structure refinement.

Main Results:

• Reproducible assignments were achieved for up to one-third of centers using only NOE constraints with appropriate precautions.
• Adjustments for multiple statistical comparisons and inter-prochiral center interactions are critical.
• Inadequately constrained systems, like molecular fragments, can yield misleading results.

Conclusions:

• The floating prochirality method, when statistically validated, provides reliable stereospecific hydrogen NMR assignments.
• Careful consideration of statistical interactions and constraints is necessary for accurate structure determination.
• Protocols for assignment and validation enhance the robustness of NMR-based structural studies.