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

Abstrak

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 [NζD<math xmlns="http://www.w3.org/1998/Math/MathML" id="M44" display="inline" overflow="scroll" dspmath="mathml"><mrow><msubsup><mi/><mn mathvariant="normal">3</mn><mo>+</mo></msubsup></mrow></math><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="8pt" height="15pt" class="svg-formula" dspmath="mathimg" md5hash="60d71c0ce434fa7b8a23fcab0d760b7e"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="mr-2-223-2021-ie00001.svg" width="8pt" height="15pt" src="mr-2-223-2021-ie00001.png"/></svg:svg>-NζH<math xmlns="http://www.w3.org/1998/Math/MathML" id="M46" display="inline" overflow="scroll" dspmath="mathml"><mrow><msubsup><mi/><mn mathvariant="normal">3</mn><mo>+</mo></msubsup></mrow></math><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="8pt" height="15pt" class="svg-formula" dspmath="mathimg" md5hash="be960de55958729edd7e309793a8788f"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="mr-2-223-2021-ie00002.svg" width="8pt" height="15pt" src="mr-2-223-2021-ie00002.png"/></svg:svg>] vs. −0.21 ppm for Δδ13Cε [NζD2-NζH2] and −1.1 ppm for Δδ15Nζ[NζD<math xmlns="http://www.w3.org/1998/Math/MathML" id="M58" display="inline" overflow="scroll" dspmath="mathml"><mrow><msubsup><mi/><mn mathvariant="normal">3</mn><mo>+</mo></msubsup></mrow></math><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="8pt" height="15pt" class="svg-formula" dspmath="mathimg" md5hash="a254698401948ff320b31e0593de61e3"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="mr-2-223-2021-ie00003.svg" width="8pt" height="15pt" src="mr-2-223-2021-ie00003.png"/></svg:svg>-NζH<math xmlns="http://www.w3.org/1998/Math/MathML" id="M60" display="inline" overflow="scroll" dspmath="mathml"><mrow><msubsup><mi/><mn mathvariant="normal">3</mn><mo>+</mo></msubsup></mrow></math><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="8pt" height="15pt" class="svg-formula" dspmath="mathimg" md5hash="cca525997fc0a5961a53aac790614a13"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="mr-2-223-2021-ie00004.svg" width="8pt" height="15pt" src="mr-2-223-2021-ie00004.png"/></svg:svg>] 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.

Topik & Kata Kunci

Electricity and magnetism

Penulis (8)

M

M. Takeda

M

M. Takeda

Y

Y. Miyanoiri

Y

Y. Miyanoiri

T

T. Terauchi

T

T. Terauchi

M

M. Kainosho

M

M. Kainosho

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Takeda, M., Takeda, M., Miyanoiri, Y., Miyanoiri, Y., Terauchi, T., Terauchi, T. et al. (2021). Conformational features and ionization states of Lys side chains in a protein studied using the stereo-array isotope labeling (SAIL) method. https://doi.org/10.5194/mr-2-223-2021

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

Abstrak

Topik & Kata Kunci

Penulis (8)

Format Sitasi

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