Increasing the accuracy of exchange parameters reporting on slow dynamics by performing CEST experiments with 'high'...

Area of Science:

• Biophysics
• Nuclear Magnetic Resonance (NMR) Spectroscopy
• Chemical Exchange Saturation Transfer (CEST)

Background:

• Chemical Exchange Saturation Transfer (CEST) NMR is vital for studying biomolecular conformational dynamics between visible and invisible states.
• Accurate characterization of these dynamics relies on selecting appropriate B1 field strengths during CEST experiments.
• Current methods typically select B1 fields around the exchange rate (kex), but the role of minor state relaxation (R2,B) is often overlooked.

Purpose of the Study:

• To investigate the influence of the minor state transverse relaxation rate (R2,B) on B1 field selection for CEST NMR.
• To demonstrate that using a higher B1 field criterion ([Formula: see text] ≥ kex) improves the accuracy of derived exchange parameters.
• To provide guidance for optimizing CEST experiments for studying slow conformational dynamics.

Main Methods:

• Utilized Chemical Exchange Saturation Transfer (CEST) NMR spectroscopy.
• Studied conformational exchange in two mutants of the 71-residue FF domain with distinct exchange rates (kex ≈ 11 s⁻¹ and 72 s⁻¹).
• Compared CEST datasets acquired using B1 field strengths guided by kex versus those guided by K (where K is related to kex and R2,B).

Main Results:

• CEST datasets analyzed with B1 fields guided by kex yielded imprecise exchange parameters for both FF domain mutants.
• Employing B1 field strengths guided by K resulted in precise, site-specific exchange parameters.
• The study highlights the critical role of R2,B in determining optimal B1 fields for accurate CEST analysis.

Conclusions:

• Selecting B1 field strengths guided by K, rather than solely kex, is crucial for obtaining accurate conformational dynamics parameters via CEST NMR.
• This approach is particularly valuable for studying slow processes in systems with significant intrinsic relaxation rates.
• The findings are applicable to diverse biological systems, including carbonyl and amide sites in proteins of various sizes.