α-Ketoacids as precursors for phenylalanine and tyrosine labelling in cell-based protein overexpression

Lichtenecker, Roman; Weinhäupl, Katharina; Schmid, Walther; Konrat, Robert

doi:10.1007/s10858-013-9796-9

Cited by 38 publications

(82 citation statements)

References 26 publications

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“…[5] More recently, a number of isotopic labeling strategies have been introduced to enable relaxation studies to probe the ps-ns and μs-ms time scales. [9] These labeling strategies are designed to ameliorate confounding spin-spin interactions that have previously limited measurement of 13 C-relaxation in aromatic ring systems to natural abundance. [10] …”

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confidence: 99%

“…To isolate a 1 H- 13 C pair in the aromatic ring in an otherwise perdeuterated background we used a biosynthetic scheme based on 13 C 4 -eyrthrose. [9b] The protein was also uniformly labeled with 15 N. [12] …”

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confidence: 99%

“…[12] The 15 N- and 13 C-relaxation data was analyzed using the model-free treatment of Lipari & Szabo [13] as described previously. [9b] 15 N relaxation data was obtained to determine macromolecular tumbling and analyzed as described in detail elsewhere [12] (Tables S1 and S2 in the Supporting Information). Based on 15 N relaxation, the determined tumbling models at 283 and 303 K at all pressures (1 bar, 1200 bar, and 2500 bar) were fully anisotropic whereas those at 313 and 323 K at all pressures were axially symmetric (Table S1).…”

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confidence: 99%

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A Sharp Thermal Transition of Fast Aromatic‐Ring Dynamics in Ubiquitin

Kasinath

Sharp

et al. 2014

Angew Chem Int Ed

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Aromatic amino acid side chains have a rich role within proteins and are often central to their structure and function. Suitable isotopic labelling strategies enable studies of sub-nanosecond aromatic ring dynamics using solution NMR relaxation methods. Surprisingly, we find that the three aromatic side chains in human ubiquitin show a sharp thermal dynamical transition at ~312 K. Hydrostatic pressure has little effect on the low temperature behaviour but decreases somewhat the amplitude of motion in the high temperature regime. Thus below the transition temperature ring motion is largely librational. Above it complete ring rotation that is most consistent with a continuous rotational diffusion not requiring transient creation of a large activated free volume occurs. Molecular dynamics simulations qualitatively corroborate this view and reinforce the notion that the dynamical character of the protein interior has a much more liquid alkane-like properties than previously appreciated.

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A Sharp Thermal Transition of Fast Aromatic‐Ring Dynamics in Ubiquitin

Kasinath

Sharp

et al. 2014

Angew Chem Int Ed

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“…Another important step was the development of a novel approach for independent leucine labelling using α-ketoisocaproate as a direct metabolic precursor without interfering with the valine metabolic pathway (Lichtenecker et al 2013a(Lichtenecker et al , 2013b. Most recently, this approach was extended to the aromatic amino acids phenylalanine and tyrosine (further extensions to tryptophan are underway) (Lichtenecker et al 2013c). Although originally designed for the generation of unique isotope-labeling patterns of methyl groups and aromatic ring systems the approach also allows for specific backbone labelling (Cα and/or C′ positions).…”

Section: Specific Isotope-labelling Strategiesmentioning

confidence: 99%

“…Although originally designed for the generation of unique isotope-labeling patterns of methyl groups and aromatic ring systems the approach also allows for specific backbone labelling (Cα and/or C′ positions). As has been shown already, specific backbone labelling can be efficiently used to edit, for example, 2D 15 N-1 H HSQC (by employing HNCO or HNCA-type pulse schemes and only recording the 2D HN plane) (Lichtenecker et al 2013a(Lichtenecker et al , 2013b(Lichtenecker et al , 2013c. The availability of specifically labeled amino acids offers exciting possibilities for spectral simplification of large and complex IDPs.…”

Section: Specific Isotope-labelling Strategiesmentioning

confidence: 99%

NMR Spectroscopic Studies of the Conformational Ensembles of Intrinsically Disordered Proteins

Kurzbach

Kontaxis

Coudevylle

et al. 2015

Advances in Experimental Medicine and Biology

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Intrinsically disordered proteins (IDPs) are characterized by substantial conformational flexibility and thus not amenable to conventional structural biology techniques. Given their inherent structural flexibility NMR spectroscopy offers unique opportunities for structural and dynamic studies of IDPs. The past two decades have witnessed significant development of NMR spectroscopy that couples advances in spin physics and chemistry with a broad range of applications. This chapter will summarize key advances in NMR methodology. Despite the availability of efficient (multi-dimensional) NMR experiments for signal assignment of IDPs it is discussed that NMR of larger and more complex IDPs demands spectral simplification strategies capitalizing on specific isotope-labeling strategies. Prototypical applications of isotope labeling-strategies are described. Since IDP-ligand association and dissociation processes frequently occur on time scales that are amenable to NMR spectroscopy we describe in detail the application of CPMG relaxation dispersion techniques to studies of IDP protein binding. Finally, we demonstrate that the complementary usage of NMR and EPR data provide a more comprehensive picture about the conformational states of IDPs and can be employed to analyze the conformational ensembles of IDPs.

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A Sharp Thermal Transition of Fast Aromatic‐Ring Dynamics in Ubiquitin

Kasinath

Sharp

et al. 2014

Angewandte Chemie

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Aromatic amino acid side chains have a rich role within proteins and are often central to their structure and function. Suitable isotopic‐labelling strategies enable studies of sub‐nanosecond aromatic‐ring dynamics using solution NMR relaxation methods. Surprisingly, it was found that the three aromatic side chains in human ubiquitin show a sharp thermal dynamical transition at approximately 312 K. Hydrostatic pressure has little effect on the low‐temperature behavior, but somewhat decreases the amplitude of motion in the high‐temperature regime. Therefore, below the transition temperature, ring motion is largely librational. Above this temperature, a complete ring‐rotation process that is fully consistent with a continuous diffusion not requiring the transient creation of a large activated free volume occurs. Molecular dynamics simulations qualitatively corroborate this view and reinforce the notion that the dynamical character of the protein interior has much more liquid‐alkane‐like properties than previously appreciated.

show abstract

α-Ketoacids as precursors for phenylalanine and tyrosine labelling in cell-based protein overexpression

Cited by 38 publications

References 26 publications

A Sharp Thermal Transition of Fast Aromatic‐Ring Dynamics in Ubiquitin

A Sharp Thermal Transition of Fast Aromatic‐Ring Dynamics in Ubiquitin

NMR Spectroscopic Studies of the Conformational Ensembles of Intrinsically Disordered Proteins

A Sharp Thermal Transition of Fast Aromatic‐Ring Dynamics in Ubiquitin

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