HA-detected experiments for the backbone assignment of intrinsically disordered proteins.
We propose a new alpha proton detection based approach for the sequential assignment of natively unfolded proteins. The proposed protocol superimposes on following features: HA-detection (1) enables assignment of natively unfolded proteins at any pH, i.e., it is not sensitive to rapid chemical exchange undergoing in natively unfolded proteins even at moderately high pH. (2) It allows straightforward assignment of proline-rich polypeptides without additional proline-customized experiments. (3) It offers more streamlined and less ambiguous assignment based on solely intraresidual (15)N(i)-(13)C'(i)-H(alpha)(i) (or (15)N(i)-(13)C(alpha)(i)-H(alpha)(i)) and sequential (15)N(i + 1)-(13)C'(i)-H(alpha)(i) (or (15)N(i + 1)-(13)C(alpha)(i)-H(alpha)(i)) correlation experiments together with efficient use of chemical shifts of (15)N and (13)C' nuclei, which show smaller dependence on residue type. We have tested the proposed protocol on two proteins, small globular 56-residue GB1, and highly disordered, proline-rich 47-residue fifth repeat of EspF(U). Using the proposed approach, we were able to assign 90% of (1)H(alpha), (13)C(alpha), (13)C', (15)N chemical shifts in EspF(U). We reckon that the HA-detection based strategy will be very useful in the assignment of natively unfolded proline-rich proteins or polypeptide chains.
Mäntylahti
Sampo
S
Program in Structural Biology and Biophysics, Institute of Biotechnology/NMR Laboratory, University of Helsinki, P.O. Box 65, 00014, Helsinki, Finland.
Aitio
Olli
O
Hellman
Maarit
M
Permi
Perttu
P
D001426:Bacterial Proteins / Q000737:chemistry; D002247:Carbon Isotopes / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry; D003903:Deuterium / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry; D047908:Intracellular Signaling Peptides and Proteins; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry* / Q000145:classification
D001426:Bacterial Proteins; D002247:Carbon Isotopes; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C489109:EspFU protein, E coli; C034284:IgG Fc-binding protein, Streptococcus; D047908:Intracellular Signaling Peptides and Proteins; D009587:Nitrogen Isotopes; D011506:Proteins; D003903:Deuterium