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The knowledge of the three-dimensional structure of proteins is the key to understanding their biological function. X-ray structure analysis of protein crystals is a well-established method to obtain this structural information. However, the method has the disadvantage that proteins must be crystallized, which is often a serious problem, and that the protein structure in the crystal does not necessarily reflect its structure under biological conditions.
The great advantage of small-angle X-ray scattering (SAXS) for protein research is that investigations can be performed in solution under biological conditions and that structural changes can be studied upon changing the external conditions.
Cyanogenesis, the release of hydrogen cyanide (HCN), has been observed in more than 3000 plant species including crop plants and is widely considered to be a defense mechanism against e.g. fungal attack. A second physiological role for cyanogenesis is the utilization of the liberated HCN for the synthesis of asparagine, an important component of many proteins.
One of the key enzymes of the cyanogenesis process is hydroxynitrile lyase (HNL). Besides its biological interest, HNL has attracted much attention as potent biocatalyst for the industrial synthesis of chiral cyanohydrins making use of the reverse cyano-genesis reaction.
Chiral cyanohydrins are important synthetic intermediates for a wide range of pharmaceuticals and agrochemicals. Therefore, understanding the function mechanism of hydroxynitrile lyase is of fundamental biological and industrial relevance. Three-dimensional structure information in the biological environment is indispensable towards this objective.
In this study the structure of (S)-hydroxynitrile lyase from rubber tree (Hb-HNL) is investigated with the new SAXSess small-angle X-ray scattering system. In the crystal Hb-HNL was observed to occur as dimers. It has been a long-standing question whether these dimers also persist in solution.
For the SAXS measurements (S)-Hb-HNL was made available in a 20 mM citrate phosphate buffer of pH 6.5 (101.8 U/mg and 46.1 mg/ml) by Roche Diagnostics.
The small-angle X-ray scattering set-up comprised the SAXSess small-angle X-ray scattering instrument from Anton Paar GmbH and the PW3830 laboratory X-ray generator (40 kV, 50mA) with a long-fine focus sealed X-ray tube (CuKa wavelength of λ = 0.1542 nm) from PANalytical. Detection was performed with the 2D imaging-plate reader Cyclone® by Perkin Elmer.
Measurements were performed in a 1 mm quartz capillary for 60 minutes.
The data were collected up to a q value of 5 nm-1, where q = (4 π/λ) sin(θ/2) is the length of the scattering vector and θ is the scattering angle, i.e. the angle of observation.
The measured scattering function of Hb-HNL (scattered intensity vs. scattering angle) was calibrated to an absolute scale using water as a secondary standard.
The experimental data were evaluated with the program GIFT and were transformed into the pair-distance distribution function p(r).
The p(r) function of a particle in solution gives the distribution of all possible distances between any two points within that particle. The location on the abscissa, where p(r) goes to zero, indicates the largest possible distance between two points within the particle, i.e. the largest particle diameter. The result for the Hb-HNL sample was compared to the theoretical p(r) functions of the monomer and the dimer calculated from the crystal structure data with the program MULTIBODY.
The comparison between the experimental and the simulated data for the dimer (Fig. 1) leave no doubt that under the experimental conditions Hb-HNL persists as a dimer in solution. This is in excellent agreement with the dimeric Hb-HNL observed in the crystal. Fivefold and tenfold dilutions of the sample in the same buffer yielded the same result within experimental error.
Figure 1 p(r) functions of Hb-HNL sample (circles with error bars), monomer (dash dotted) and dimer (full line) as calculated from crystal structure data; ø = diameter
Hydroxynitrile lyase is an enzyme of great biological and industrial relevance as a biocatalyst.
Protein crystallography revealed that Hb-HNL occurs as dimers in the crystalline state.
However, for the understanding of biocatalytic processes the structure information under biological conditions in solution is also important. Small-angle X-ray scattering is the method of choice in this respect.
The new SAXSess was used for structural investigations into Hb-HNL that confirmed unequivocally that this protein persists as a dimer in solution.
Due to its high X-ray flux in the sample, the measuring times with the SAXSess are low. This is especially important for the investigation of biological/protein samples, where long radiation exposure time may lead to radiation damage.
Source: Anton Paar GmbH.
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