Protein conformational changes studied by diffusion NMR spectroscopy: Application to helix‐loop‐helix calcium binding proteins

• Published in: Protein science Vol. 12; no. 2; pp. 228 - 236
• Main Authors: Weljie, Aalim M., Yamniuk, Aaron P., Yoshino, Hidenori, Izumi, Yoshinobu, Vogel, Hans J.
• Format: Journal Article
• Language: English
• Published: Bristol Cold Spring Harbor Laboratory Press 01-02-2003
• Subjects: Amino Acid Sequence; Apoproteins - chemistry; Apoproteins - metabolism; CaB, calbrain; calcium and integrin binding protein (CIB); Calcium-Binding Proteins - chemistry; Calcium-Binding Proteins - metabolism; calcium‐dependent protein kinase (CDPK); Calmodulin; Calmodulin - chemistry; Calmodulin - metabolism; CaM, calmodulin; CaMKI, calmodulin‐dependent protein kinase I; CDPK, calmodulin‐like domain protein kinase; Cf, compaction factor; CIB, calcium and integrin binding protein; CLD, calmodulin‐like domain; Diffusion; DLS, dynamic light scattering; GPC, gel permeation chromatography; Helix-Loop-Helix Motifs; HSQC, heteronuclear single quantum coherence; JD, junction domain; Magnetic Resonance Spectroscopy; Molecular Sequence Data; NMR, nuclear magnetic resonance; PDE, cyclic nucleotide phosphodiesterase; PFG diffusion; PFG, pulse field gradient; Protein Conformation; protein conformational change; protein–ligand interactions; protein–protein interactions; Rg, radius of gyration; Rh, hydrodynamic radius; SAXS, small‐angle X‐ray scattering; SIV, simian immunodeficiency virus; skMLCK, skeletal muscle myosin light‐chain kinase
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1110/ps.0226203

Pulsed‐field gradient (PFG) diffusion NMR spectroscopy studies were conducted with several helix‐loop‐helix regulatory Ca2+‐binding proteins to characterize the conformational changes associated with Ca2+‐saturation and/or binding targets. The calmodulin (CaM) system was used as a basis for evaluation, with similar hydrodynamic radii (Rh) obtained for apo‐ and Ca2+‐CaM, consistent with previously reported Rh data. In addition, conformational changes associated with CaM binding to target peptides from myosin light chain kinase (MLCK), phosphodiesterase (PDE), and simian immunodeficiency virus (SIV) were accurately determined compared with small‐angle X‐ray scattering results. Both sets of data demonstrate the well‐established collapse of the extended Ca2+‐CaM molecule into a globular complex upon peptide binding. The Rh of CaM complexes with target peptides from CaM‐dependent protein kinase I (CaMKI) and an N‐terminal portion of the SIV peptide (SIV‐N), as well as the anticancer drug cisplatin were also determined. The CaMKI complex demonstrates a collapse analogous to that observed for MLCK, PDE, and SIV, while the SIV‐N shows only a partial collapse. Interestingly, the covalent CaM–cisplatin complex shows a near complete collapse, not expected from previous studies. The method was extended to related calcium binding proteins to show that the Rh of calcium and integrin binding protein (CIB), calbrain, and the calcium‐binding region from soybean calcium‐dependent protein kinase (CDPK) decrease on Ca2+‐binding to various extents. Heteronuclear NMR spectroscopy suggests that for CIB and calbrain this is likely because of shifting the equilibrium from unfolded to folded conformations, with calbrain forming a dimer structure. These results demonstrate the utility of PFG‐diffusion NMR to rapidly and accurately screen for molecular size changes on protein–ligand and protein–protein interactions for this class of proteins.