Arne Meyer

Efficient UV detection of protein crystals enabled by fluorescence excitation at wavelengths longer than 300 nm

It is well known that most proteins and many other biomolecules fluoresce when illuminated with UV radiation, but it is also commonly accepted that utilizing this property to detect protein crystals in crystallization setups is limited by the opacity of the materials used to contain and seal them. For proteins, this fluorescence property arises primarily from the presence of tryptophan residues in the sequence. Studies of protein crystallization results in a variety of setup configurations show that the opacity of the containment hardware can be overcome at longer excitation wavelengths, where typical hardware materials are more transparent in the UV, by the use of a powerful UV-light source that is effective in excitation even though not at the maximum of the excitation response. The results show that under these circumstances UV evaluation of crystallization trials and detection of biomolecular crystals in them is not limited by the hardware used. It is similarly true that a deficiency in tryptophan or another fluorescent component that limits the use of UV light for these purposes can be effectively overcome by the addition of fluorescent prostheses that bind to the biomolecule under study. The measurements for these studies were made with a device consisting of a potent UV-light source and a detection system specially adapted (i) to be tunable via a motorized and software-controlled absorption-filter system and (ii) to convey the excitation light to the droplet or capillary hosting the crystallization experiment by quartz-fibre light guides.

Structure of mistletoe lectin I from Viscum album in complex with the phytohormone zeatin.

The crystal structure of mistletoe lectin I (ML-I) isolated from the European mistletoe Viscum album in complex with the most active phytohormone zeatin has been analyzed and refined to 2.54 A resolution. X-ray suitable crystals of ML-I were obtained by the counter-diffusion method using the Gel-Tube R crystallization kit (GT-R) onboard the Russian Service Module on the international space station ISS. High quality hexagonal bipyramidal crystals were grown during 3 months under microgravity conditions. Selected crystals were soaked in a saturated solution of zeatin and subsequently diffraction data were collected applying synchrotron radiation. A distinct F(o)-F(c) electron density has been found inside a binding pocket located in subunit B of ML-I and has been interpreted as a single zeatin molecule. The structure was refined to investigate the zeatin-ML-I interactions in detail. The results demonstrate the ability of mistletoe to protect itself from the host transpiration regulation by absorbing the most active host plant hormones as part of a defense mechanism.

Design and application of a microfluidic device for protein crystallization using an evaporation-based crystallization technique

A new crystallization system is described, which makes it possible to use an evaporation-based microfluidic crystallization technique for protein crystallization. The gas and water permeability of the used polydimethylsiloxane (PDMS) material enables evaporation of the protein solution in the microfluidic device. The rates of evaporation are controlled by the relative humidity conditions, which are adjusted in a precise and stable way by using saturated solutions of different reagents. The protein crystals could nucleate and grow under different relative humidity conditions. Using this method, crystal growth could be improved so that approximately 1 mm-sized lysozyme crystals were obtained more successfully than using standard methods. The largest lysozyme crystal obtained reached 1.57 mm in size. The disadvantage of the good gas permeability in PDMS microfluidic devices becomes an advantage for protein crystallization. The radius distributions of aggregrates in the solutions inside the described microfluidic devices were derived from in situ dynamic light scattering measurements. The experiments showed that the environment inside of the microfluidic device is more stable than that of conventional crystallization techniques. However, the morphological results showed that the protein crystals grown in the microfluidic device could lose their morphological stability. Air bubbles in microfluidic devices play an important role in the evaporation progress. A model was constructed to analyze the relationship of the rates of evaporation and the growth of air bubbles to the relative humidity.

Monitoring and scoring counter-diffusion protein crystallization experiments in capillaries by in situ dynamic light scattering.

In this paper, we demonstrate the feasibility of using in situ Dynamic Light Scattering (DLS) to monitor counter-diffusion crystallization experiments in capillaries. Firstly, we have validated the quality of the DLS signal in thin capillaries, which is comparable to that obtained in standard quartz cuvettes. Then, we have carried out DLS measurements of a counter-diffusion crystallization experiment of glucose isomerase in capillaries of different diameters (0.1, 0.2 and 0.3 mm) in order to follow the temporal evolution of protein supersaturation. Finally, we have compared DLS data with optical recordings of the progression of the crystallization front and with a simulation model of counter-diffusion in 1D.

Latest methods of fluorescence‐based protein crystal identification

Fluorescence, whether intrinsic or by using trace fluorescent labeling, can be a powerful aid in macromolecule crystallization. Its use in screening for crystals is discussed here.

Systematic analysis of protein-detergent complexes applying dynamic light scattering to optimize solutions for crystallization trials.

Application of in situ dynamic light scattering to solutions of protein–detergent complexes permits characterization of these complexes in samples as small as 2 µl in volume.

Structure of the recombinant BPTI/Kunitz-type inhibitor rShPI-1A from the marine invertebrate Stichodactyla helianthus

The BPTI/Kunitz-type inhibitor family includes several extremely potent serine protease inhibitors. To date, the inhibitory mechanisms have only been studied for mammalian inhibitors. Here, the first crystal structure of a BPTI/Kunitz-type inhibitor from a marine invertebrate (rShPI-1A) is reported to 2.5 Å resolution. Crystallization of recombinant rShPI-1A required the salt-induced dissociation of a trypsin complex that was previously formed to avoid intrinsic inhibitor aggregates in solution. The rShPI-1A structure is similar to the NMR structure of the molecule purified from the natural source, but allowed the assignment of disulfide-bridge chiralities and the detection of an internal stabilizing water network. A structural comparison with other BPTI/Kunitz-type canonical inhibitors revealed unusual ϕ angles at positions 17 and 30 to be a particular characteristic of the family. A significant clustering of ϕ and ψ angle values in the glycine-rich remote fragment near the secondary binding loop was additionally identified, but its impact on the specificity of rShPI-1A and similar molecules requires further study.

Single-drop optimization of protein crystallization.

A completely new crystal-growth device has been developed that permits charting a course across the phase diagram to produce crystalline samples optimized for diffraction experiments. The utility of the device is demonstrated for the production of crystals for the traditional X-ray diffraction data-collection experiment, of microcrystals optimal for data-collection experiments at a modern microbeam insertion-device synchrotron beamline and of nanocrystals required for data collection on an X-ray laser beamline.

Comparative crystallization and preliminary X-ray diffraction studies of locked nucleic acid and RNA stems of a tenascin C-binding aptamer

The pharmacokinetic properties of an aptamer against the tumour-marker protein tenascin-C have recently been successfully improved by the introduction of locked nucleic acids (LNAs) into the terminal stem of the aptamer. Since it is believed that this post-SELEX optimization is likely to provide a more general route to enhance the in vitro and in vivo stability of aptamers, elucidation of the structural basis of this improvement was embarked upon. Here, the crystallographic and X-ray diffraction data of the isolated aptamer stem encompassed in a six-base-pair duplex both with and without the LNA modification are presented. The obtained all-LNA crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 52.80, c = 62.83 angstroms; the all-RNA crystals belong to space group R32, with unit-cell parameters a = b = 45.21, c = 186.97 angstroms, gamma = 120.00 degrees.

Reliably distinguishing protein nanocrystals from amorphous precipitate by means of depolarized dynamic light scattering

Crystallization of biological macromolecules such as proteins implies several prerequisites, for example, the presence of one or more initial nuclei, sufficient amounts of the crystallizing substance and the chemical potential to provide the free energy needed to force the process. The initiation of a crystallization process itself is a stochastic event, forming symmetrically assembled nuclei over kinetically preferred protein-dense liquid clusters. The presence of a spatial repetitive orientation of macromolecules in the early stages of the crystallization process has so far proved undetectable. However, early identification of the occurrences of unit cells is the key to nanocrystal detection. The optical properties of a crystal lattice offer a potential signal with which to detect whether a transition from disordered to ordered particles occurs, one that has so far not been tested in nanocrystalline applications. The ability of a lattice to depolarize laser light depends on the different refractive indices along different crystal axes. In this study a unique experimental setup is used to detect nanocrystal formation by application of depolarized scattered light. The results demonstrate the successful detection of nano-sized protein crystals at early stages of crystal growth, allowing an effective differentiation between protein-dense liquid cluster formation and ordered nanocrystals. The results are further verified by complementary methods like X-ray powder diffraction, second harmonic generation, ultraviolet two-photon excited fluorescence and scanning electron microscopy.