Application of DTD to DGK Spectra

An example of the application of DTD to a homo-nuclear correlation spectrum of an integral membrane protein is shown in chapter 8, Fig. 8.23.

Solid state spectra of integral membrane proteins intrinsically carry the disadvan-tage of limited sensitivity. This is caused by several different factors. Firstly, the expression yield is generally low for membrane proteins, and the proteins have to be reconstituted into an membrane mimetic environment, if their natural surroundings are to be emulated. Although for most samples polarization enhancement of hetero-nuclei from ¹H can be used, proton detection is normally not possible in the solid state, as the homo-nuclear dipolar couplings lead to a severe broadening of the spec-tra. Most of the time, the interplay of several such factors makes measuring times unfeasibly long.

This was also the case for a ²H−¹³C−¹⁵N labeled preparation of the integral membrane protein diacylglycerol kinase. For this sample, the amount of protein was limited and no polarization enhancement schemes could be used, due to the perdeuter-ation of the protein.

This resulted in a very limited sensitivity and the spectrum could only be acquired with 140 increments in the indirect dimension, as 2048 scans were needed per increment to obtain a workable signal to noise ratio. This number of increments is normally insufficient to obtain¹³C−¹³C spectra of good quality, as the digital resolution in the indirect dimension is limited to about 200Hz, given the necessary spectral width.

Still, the measuring time was approx. 6 days.

Dual Transformation Denoising presents an opportunity to increase the quality of spectra recorded for such difficult samples. It was therefore applied to the TOBSY

7.5. CONCLUSION AND OUTLOOK

spectrum of²H−¹³C−¹⁵N labeled DGK shown in chapter 8, Fig. 8.23.

For the given spectrum, no additional or missing resonances could be detected upon DTD processing and no significant change of the line shapes in the direct dimension was observed.

7.5 Conclusion and Outlook

DTD has been demonstrated to be an effective tool which increases the S/N of a homo-nuclear spectrum, while at the same time reducing the number of necessary increments in a given two dimensional spectrum. This can lead to a significant reduction of measuring time for homo-nuclear 2D experiments, as was shown for the example of PDSD spectra of bradykinin, recorded in one fourth of the normal measuring time.

Alternatively, the experimental time could be kept constant by increasing the number of scans per increment at the expense of the number of increments recorded. This would make use of the fact that the S/ N improves steeply with a growing number of scans for DTD processed spectra, while utilizing the suppression of truncation artifacts to compensate for the decreased number of increments. This should lead to spectra of high quality with an enhanced S/N ratio. Although DTD is demonstrated here using a combination of covariance and FFT on 2D data sets, spectra of higher dimensionality should show the same reduction of measuring time, if a homo-nuclear mixing period is included.

8.1 Introduction

Diacylglycerol Kinase (DGK) fromEscherichia coli[122–124] is a small integral mem-brane protein, with 121 amino acids and a molecular weight of 13.2 kDa. It functions as a kinase, catalyzing the phosphorylation of diacylglycerol (DAG) by the transfer theγ-phosphate from ATP to DAG, to form phosphatidic acid (PA) and ADP.

InE.coli, the functionality of DGK has been shown to influence on the rate of cell proliferation under conditions of low osmolarity [125]. This observation is based on the role of DAG in the membrane derived oligosaccharide (MDO) cycle.

During the synthesis of MDOs, the pre-MDOs need to be ferried to the site of synthesis. This involves phosphatidylglycerol (PG) as a carrier molecule for the pre-MDOs.

After the pre-MDO has dissociated, DAG remains, which has been shown to ac-cumulate in the membrane in the absence of DGK function [125].

Phosphorylation to PA by DGK recycles DAG, as PA can act as a precursor molecule for the biosynthesis of phosphatidylethanolamine (PE) and cardiolipin (CL).

In this biochemical pathway, CDP-Diacylglycerol is formed from PA by CDP - Dia-cylglycerol Synthetase. One of the intermediate products in the CL synthesis pathway is PG, which can again be used by the cell for MDO biosynthesis as outlined above, closing the PG-DAG-PA catalytic cycle.

DGK spans the membrane three times as elucidated by Smith et al. [126] via β-lactamase andβ-galactosidase fusion experiments and is mostlyα-helical [127]. The topology is proposed [128] to consist of two small N-terminal amphipathic helices, fol-lowed by two transmembrane helices connected by a small loop of four amino acids.

These domains are followed by a third amphipathic helix, containing most of the residues highly conserved in all procaryotic DGK sequences and proposed to be in-volved in the active site of the protein [128–130], which in turn is followed by a third transmembrane helix and the C-terminus.

The N-terminus of DGK is proposed to reside in the cytoplasm and the C-terminus is oriented towards the periplasm inE.coli [126]. This topology model was further refined by¹⁹F solution state NMR studies [128, 131].

DGK forms trimers [132] with transmembrane helix 2 acting as the center of