In:
Magnetic Resonance, Copernicus GmbH, Vol. 2, No. 1 ( 2021-04-26), p. 223-237
Abstract:
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 using 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 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 stereospecifically assigned
using the stereo-array isotope-labeled Lys (SAIL-Lys), [U-13C,15N;
β2,γ2,δ2,ε3-D4]-Lys. The complete set of assigned 1H, 13C, and
15N NMR signals for the Lys side-chain moieties affords useful
structural information. For example, the set includes the characteristic
chemical shifts for the 13Cδ, 13Cε, and
15Nζ signals for Lys-66, which has the deprotonated ζ-amino group, and the large upfield shifts for the 1H and 13C
signals for the Lys-9, Lys-28, Lys-84, Lys-110, and Lys-133 side chains, which are
indicative of nearby aromatic rings. The 13Cε and
15Nζ chemical shifts of the SNase variant selectively labeled
with either [ε-13C;ε,ε-D2]-Lys or SAIL-Lys, dissolved in H2O and D2O, showed that
the deuterium-induced shifts for Lys-66 were substantially different from
those of the other 20 Lys residues. Namely, the deuterium-induced shifts of
the 13Cε and 15Nζ signals depend on the
ionization states of the ζ-amino group, i.e., −0.32 ppm for Δδ13Cε [NζD3+-NζH3+] vs. −0.21 ppm for Δδ13Cε [NζD2-NζH2] and −1.1 ppm for Δδ15Nζ[NζD3+-NζH3+] vs. −1.8 ppm for Δδ15Nζ[NζD2-NζH2]. Since the 1D 13C NMR spectrum of a
protein selectively labeled with [ε-13C;ε,ε-D2]-Lys shows narrow (〉 2 Hz) and
well-dispersed 13C signals, the deuterium-induced shift difference of
0.11 ppm for the protonated and deprotonated ζ-amino groups, which
corresponds to 16.5 Hz at a field strength of 14 T (150 MHz for
13C), could be accurately measured. Although the isotope shift
difference itself may not be absolutely decisive to distinguish the
ionization state of the ζ-amino group, the 13Cδ,
13Cε, and 15Nζ signals for a Lys residue
with a deprotonated ζ-amino group are likely to exhibit distinctive
chemical shifts as compared to the normal residues with protonated ζ-amino groups. Therefore, the isotope shifts would provide a useful
auxiliary index for identifying Lys residues with deprotonated ζ-amino groups at physiological pH levels.
Type of Medium:
Online Resource
ISSN:
2699-0016
DOI:
10.5194/mr-2-223-2021
Language:
English
Publisher:
Copernicus GmbH
Publication Date:
2021
detail.hit.zdb_id:
2998533-X
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