THERMAL DENATURATION OF BARLEY LIPID TRANSFER PROTEIN 1 STUDIED BY NUCLEAR MAGNETIC RESONANCE AND DIFFERENTIAL SCANNING CALORIMETRY

Jitka Žídková¹, Michaela Matejková², Lukáš Žídek², Michaela Wimmerová^2,3, Vladimír Sklenář², Janette Bobáľová¹

¹Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, v.v.i., Veveří 97, 60200 Brno, Czech Republic

²National Centre for Biomolecular Research and ³Department of Biochemistry, Faculty of Science, Masaryk University, Kotlářská 2, 61137 Brno, Czech Republic

Lipid transfer protein 1 (LTP1) is a ubiquitous plant protein able to transfer lipids between membranes in vitro. Barley LTP1 consists of 91 amino acids and adopts a typical structure comprised of four α -helices and a C-terminal tail both stabilized by four disulfide bridges.

LTP1 isolated from barley grain contains a lipid-like molecule of 294 Da as a postranslational modification. Recently, this lipid-like adduct has been identified as a reactive oxylipin (α-ketol of 9-hydroxy-10-oxo-12-Z-octadecenoic acid). LTP1 is extremely heat and protease resistant, survives all procedure of making beer, and is therefore present in beer and has effect on foaming.

In spite of the number of thermal stability studies reported in the literature, we felt that two issues still remained to be addressed in order to understand the impact of the thermal denaturation of LTP1 on the quality of beer and other barley products. First, the LTP1 denaturation was so-far systematically studied up to 100 °C, which is not enough to observe unfolding of the native protein. The second and more important issue is the irreversibility of the thermal denaturation of LTP1. While reversible denaturation curves can be interpreted in terms of van't Hoff enthalpy and of melting temperature directly related to the entropy, such approach is not relevant for irreversible denaturation.

In our study, process of thermal denaturation of LTP1 covalently modified at Asp 7 was monitored by nuclear magnetic resonance (NMR) and differential scanning calorimetry (DSC) up to 120 °C. While DSC provided a complete picture of heat capacity changes, NMR selectively described the structural changes during protein unfolding. The denaturation was found to be irreversible and highly cooperative. A method of numerical quantitative analysis

allowing to fit the NMR data to a transition-state model without further simplification was developed. Based on the obtained values of transition state enthalpy and entropy, rate of denaturation was calculated as a simple measure of protein stability at various temperatures. Effects of reducing agents were studied and discussed in the context of quality control of barley products during storage and processing.

Acknowledgment:

This work was supported by the Grants 1M0570, MSM0021622413, and LC06030 of the Ministry of Education, Youth, and Physical Culture of the Czech Republic and by Grant AV0Z40310501 of the Academy of Sciences of the Czech Republic.