Conformational flexibility of a microcrystalline globular protein: order parameters by solid-state NMR spectroscopy

Area of Science:

• Biophysics
• Structural Biology
• Nuclear Magnetic Resonance (NMR) Spectroscopy

Background:

• Protein structures are often determined in crystalline states, but characterizing dynamics in these states is challenging.
• Solid-state NMR (SSNMR) offers a powerful approach to probe dynamic information in crystalline biomolecules.
• Advancements in high-resolution SSNMR allow site-specific assignment of carbon-13 ((13)C) and nitrogen-15 ((15)N) nuclei in proteins.

Purpose of the Study:

• To investigate the backbone and side chain conformational dynamics of ubiquitin in a microcrystalline state.
• To obtain a global perspective on sub-microsecond dynamics using site-specific order parameters.
• To compare solid-state NMR findings with existing solution NMR data for ubiquitin.

Main Methods:

• Utilized multidimensional separated-local-field experiments in solid-state NMR.
• Measured molecular conformational order parameters based on heteronuclear dipolar couplings.
• Correlated order parameters with assigned chemical shifts for detailed dynamic analysis.

Main Results:

• Collected 38 Calpha, 35 Cbeta, and multiple side chain order parameters for microcrystalline ubiquitin.
• Revealed significant mobility in microcrystalline ubiquitin, with side chains generally exhibiting higher motion than backbone sites.
• Observed that SSNMR is sensitive to a broader timescale (low microseconds and faster) than solution NMR (low nanoseconds and faster), yielding generally lower order parameters.

Conclusions:

• Solid-state NMR provides extensive, site-specific dynamic information for crystalline biomolecules.
• The study highlights the potential of SSNMR for detailed dynamic studies of biopolymers in their crystalline state.
• SSNMR order parameters for (13)C(1)H(2) spin systems are readily measurable from powder line shape data, offering an alternative to solution NMR relaxation methods.