Time-saving methods for heteronuclear multidimensional NMR of (13C, 15N) doubly labeled proteins

Abstract

Heteronuclear 2D ((13)C, (1)H) and ((15)N, (1)H) correlation spectra of ((13)C, (15)N) fully enriched proteins can be acquired simultaneously with virtually no sensitivity loss or increase in artefact levels. Three pulse sequences are described, for 2D 'time-shared' or TS-HSQC, 2D TS-HMQC and 2D TS-HSMQC spectra, respectively. Independent spectral widths can be sampled for both heteronuclei. The sequences can be greatly improved by combining them with field-gradient methods. By applying the sequences to 3D and 4D NMR spectroscopy, considerable time savings can be obtained. The method is demonstrated for the 18 kDa HU protein.

Time‐shared NMR experiments

“…Figure 2 shows the basic conversion between two separate 13 C and 15 N HMQC pulse trains to a single TS version. The resulting TS-HMQC experiment would result to overlap the 13 C and the 15 N pulse sequence.…”

“…Because 1 H bonded to 13 C is not bonded to 15 N, two independent coherence pathways are generated in TS experiments and they can be analyzed separately. In addition, the independence of these two evolutions during fixed or variable periods comes because at natural isotopic abundance is quiet improbably to find protons coupled both 13 C and 15 N (1.07 and 0.364% natural abundance).…”

“…The J(CH) (145 Hz), respectively. Gradients G2 and G3 are optimized to select 13 C magnetization, whereas all four G1-G4 gradients select the equivalent 15 N coherence pathway (see Eqs. [1] and [2]) G1 ¼ 60, G2 ¼ 50, G3 ¼ 30, and G4 ¼ 60.…”

“…1). For instance, it can be advisable to record 1 H/ 13 C and 1 H/ 15 N HMBC spectra in many nitrogen-containing compounds, such as natural products, peptides and proteins, synthetic organic derivatives, organometallic and inorganic molecules, nucleotides, or nucleic acids. Both pulse sequences present strong similarities, and the possibility to simultaneously acquire both spectra by redesigning the original HMBC pulse Conversion of two independent but complementary HMQC pulse sequences in a single one by using the concept of time-sharing (TS) multiple-frequency evolution during a variable t 1 evolution period.…”

Biomolekulare NMR‐Spektroskopie auf der Überholspur

“…Um ausreichende spektrale Auflösung in den indirekten Dimensionen zu erzielen, sind längere Messzeiten dennoch erforderlich, daher werden diese Experimente als auflösungslimitiert bezeichnet. Neben verschiedenen Strategien zur optimierten Aufnahme der Messpunkte in indirekten Dimensionen, wie nichtlinearer Datenpunktsammlung [13,14] oder simultaner Aufnahme, [15][16][17] wurde auch die Reduzierung der Dimensionalität schon vor zehn Jahren vorgeschlagen. [18,19] In Experimenten mit reduzierter Dimensionalität (RD) werden nach dem Prinzip der Akkordeon-Spektroskopie [6] zwei oder mehr [2,20,21] indirekte Zeitdimensionen gemeinsam aufgenommen, indem die entsprechenden Zeitintervalle synchron inkrementiert werden (Abbildung 1 b).…”