Structure and stability of the potato cysteine protease inhibitor group (cv. Elkana).

The conformational stability of potato cysteine protease inhibitor (PCPI), the second most abundant protease inhibitor group in potato tuber, was investigated at ambient temperature and upon heating using far- and near-UV circular dichroism spectroscopy, fluorescence spectroscopy, and differential scanning calorimetry (DSC). The PCPI isoforms investigated have a highly similar structure at both the secondary and the tertiary level. PCPI isoforms show structural properties similar to those of the potato serine protease inhibitor group and the Kunitz type soybean trypsin inhibitor, a known beta-II protein. Therefore, PCPI isoforms are also classified as members of the beta-II protein subclass. Results show that the thermal unfolding of PCPI isoforms does not follow a two-state mechanism and that at least one intermediate is present. The occurrence of this intermediate is most apparent in the thermal unfolding of PCPI 8.3 as indicated by the presence of two peaks in the DSC thermogram. Additionally, the formation of aggregates (>100 kDa), especially at low scan rates, increases the apparent cooperativity of the unfolding.

In vivo uniform (15)N-isotope labelling of plants: using the greenhouse for structural proteomics.

Isotope labelling of proteins is important for progress in the field of structural proteomics. It enables the utilisation of the power of nuclear magnetic resonance spectroscopy (NMR) for the characterisation of the three-dimensional structures and corresponding dynamical features of proteins. The usual approach to obtain isotopically labelled protein molecules is by expressing the corresponding gene in bacterial or yeast host organisms, which grow on isotope-enriched media. This method has several drawbacks. Here, we demonstrate that it is possible to fully label a plant with (15)N-isotopes. The advantage of in vivo labelling of higher organisms is that all constituting proteins are labelled and become available as functional, post-translationally modified, correctly folded proteins. A hydroponics set-up was used to create the first example of a uniformly (15)N-labelled (> 98%) plant species, the potato plant (Solanum tuberosum L., cv. Elkana). Two plants were grown at low costs using potassium-[(15)N]-nitrate as the sole nitrogen source. At harvest time, a total of 3.6 kg of potato tubers and 1.6 kg of foliage, stolons and roots were collected, all of which were fully (15)N-labelled. Gram quantities of soluble (15)N-labelled proteins (composed mainly of the glycoprotein patatin and Kunitz-type protease inhibitors) were isolated from the tubers. NMR results on the complete proteome of potato sap and on an isolated protease inhibitor illustrate the success of the labelling procedure. The presented method of isotope labelling is easily modified to label other plants. Its envisioned impact in the field of structural proteomics of plants is discussed.