High-resolution solid-state NMR structure of a 17.6 kDa protein.

Area of Science:

• Biochemistry
• Structural Biology
• Biophysics

Background:

• Solid-state NMR is crucial for determining protein structures, but signal assignment can be challenging.
• Paramagnetic metal ions can provide long-range structural information through pseudocontact shifts.

Purpose of the Study:

• To develop and validate a strategy using pseudocontact shifts for unambiguous signal assignment in solid-state NMR.
• To enable accurate distance restraint extraction for protein structure calculation.

Main Methods:

• Utilized paramagnetic metal ion (cobalt(II)) substitution in a microcrystalline protein sample.
• Employed various solid-state NMR experiments including PDSD, DARR, CHHC, PAR, and PAIN-CP.
• Obtained pseudocontact shifts to derive distance restraints.

Main Results:

• Generated 777 unambiguous distance restraints (sequential, medium-range, long-range) for the catalytic domain of matrix metalloproteinase 12.
• Determined a high-resolution protein structure with a backbone RMSD of 1.0 ± 0.2 Å.
• The calculated structure showed good agreement with the existing X-ray structure (RMSD of 1.3 Å).

Conclusions:

• Pseudocontact shifts from paramagnetic ions are effective for unambiguous signal assignment in solid-state NMR.
• This strategy significantly advances protein structure determination using solid-state NMR.
• The approach is potentially generalizable to non-metalloproteins using paramagnetic tags.