Preprints
https://doi.org/10.5194/mr-2021-15
https://doi.org/10.5194/mr-2021-15

  19 Feb 2021

19 Feb 2021

Review status: this preprint is currently under review for the journal MR.

Conformational features and ionization states of Lys side chains in a protein revealed by the stereo-array isotope labeling (SAIL) method

Mitsuhiro Takeda1,2, Yohei Miyanoiri1,3, Tsutomu Terauchi4,5, and Masatsune Kainosho1,5 Mitsuhiro Takeda et al.
  • 1Structural Biology Research Center, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602 Japan
  • 2Department of Structural BioImaging, Faculty of Life Sciences, Kumamoto University, 5-1, Oe-honmachi, Chuo-ku, Kumamoto, 862-0973 Japan
  • 3Research Center for State-of-the-Art Functional Protein Analysis, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, 565-0871 Japan
  • 4SAIL Technologies Co., Inc., 2008-2 Wada, Tama-city, Tokyo, 206-0001 Japan
  • 5Graduate School of Science, Tokyo Metropolitan University, 1-1 Minami-ohsawa, Hachioji, Tokyo, 192-0397 Japan

Abstract. Although both the hydrophobic aliphatic chain and hydrophilic ζ-amino group of the Lys side chain presumably contribute to the structures and functions of proteins, the dual nature of the Lys residue has not been fully investigated by NMR spectroscopy, due to the lack of appropriate methods to acquire comprehensive information on its long consecutive methylene chain. We describe herein a robust strategy to address the current situation, using various isotope-aided NMR technologies. The feasibility of our approach is demonstrated for the Δ+PHS/V66K variant of Staphylococcal nuclease (SNase), which contains as many as 21 Lys residues, including the engineered Lys-66 with an unusually low pKa of ~5.6. All of the NMR signals for the 21 Lys residues were sequentially and stereo-specifically assigned by using the stereo-array isotope labeled Lys (SAIL-Lys), [U-13C,15N; β2223-D4]-Lys. The unambiguously assigned NMR signals for the β-, γ-, δ- and ε-methylene moieties afforded a variety of crucial structural information, which could not be obtained by other methods. For example, the 13Cε signals in the SNase variant, selectively labeled with [ε-13C; ε,ε-D2]-Lys, were ~0.3 ppm up-field shifted in D2O, as compared to those in H2O, except for Lys-66, which showed a ~0.2 ppm up-field shift in D2O. This result indicates that the deuterium-induced up-field shifts of the 13Cε signals depend on the ionization states of the ζ-amino group; i.e., ~ −0.3 ppm for Δδ13Cε [NζD3+-NζH3+] and ~ −0.2 ppm for Δδ13Cε [NζD2-NζH2]. Since the highly sensitive 1D-13C NMR spectrum of a protein selectively labeled with [ε-13C; ε,ε-D2]-Lys shows extremely narrow, well-dispersed 13C signals, the deuterium-induced isotope shifts will be a powerful alternative tool to characterize the ionization states of the Lys ζ-amino groups in larger proteins.

Mitsuhiro Takeda et al.

Status: open (until 19 Mar 2021)

Comment types: AC – author | RC – referee | CC – community | EC – editor | CEC – chief editor | : Report abuse
  • RC1: 'Comment on mr-2021-15', Anonymous Referee #1, 25 Feb 2021 reply
  • RC2: 'Comment on mr-2021-15', Anonymous Referee #2, 25 Feb 2021 reply

Mitsuhiro Takeda et al.

Mitsuhiro Takeda et al.

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Short summary
Although both the hydrophobic aliphatic chain and hydrophilic ζ-amino group of the lysine side chain presumably contribute to the structures and functions of proteins, the dual nature of the lysinyl residue has not been fully understood yet, due to the lack of appropriate methods to acquire comprehensive information on its long consecutive methylene chain at the atomic scale. We describe herein a novel strategy to address the current situation using nuclear magnetic resonance spectroscopy.