Protein Single Crystals Research Articles

Growing a single suspended perfect protein crystal in a fully noncontact manner

Nucleation is a fundamental process that determines the structure, morphology, and properties of crystalline materials, and is difficult to control because it is unpredictable. Here, we demonstrate a new method to control the protein crystal nucleation using a magnetic force, where we manipulate the movement and coalescence of nucleation precursors by adding paramagnetic salt into the crystallization solution to constrain the number and position of nucleation. We found that protein nucleation could be significantly affected by the magnetic force that the gradient magnetic fields generate. When the magnetization force is sufficiently enough, nucleation can be confined to the crystallization solution with no interface contact; therefore, only one crystal nucleus appears, which results in noncontact suspension growth of a single crystal in the crystallization solution system. Under these situations, the nucleation rate significantly decreases due to the coalescence of the dense liquid phase, and the crystal growth rate also decreases due to the suppression of convection, which increases the crystal quality. Our findings provide a new method for the noncontact control of crystal nucleation and demonstrate that externally applied physical environments can be used to affect the liquid-liquid phase separation process.

A Tutorial Review on the Methodologies and Theories Utilized to Handle Proteins toward Obtaining Single Protein Crystals

A Tutorial Review on the Methodologies and Theories Utilized to Handle Proteins toward Obtaining Single Protein Crystals

Obtaining anomalous and ensemble information from protein crystals from 220 K up to physiological temperatures.

X-ray crystallography has been invaluable in delivering structural information about proteins. Previously, an approach has been developed that allows high-quality X-ray diffraction data to be obtained from protein crystals at and above room temperature. Here, this previous work is built on and extended by showing that high-quality anomalous signal can be obtained from single protein crystals using diffraction data collected at 220 K up to physiological temperatures. The anomalous signal can be used to directly determine the structure of a protein, i.e. to phase the data, as is routinely performed under cryoconditions. This ability is demonstrated by obtaining diffraction data from model lysozyme, thaumatin and proteinase K crystals, the anomalous signal from which allowed their structures to be solved experimentally at 7.1 keV X-ray energy and at room temperature with relatively low data redundancy. It is also demonstrated that the anomalous signal from diffraction data obtained at 310 K (37°C) can be used to solve the structure of proteinase K and to identify ordered ions. The method provides useful anomalous signal at temperatures down to 220 K, resulting in an extended crystal lifetime and increased data redundancy. Finally, we show that useful anomalous signal can be obtained at room temperature using X-rays of 12 keV energy as typically used for routine data collection, allowing this type of experiment to be carried out at widely accessible synchrotron beamline energies and enabling the simultaneous extraction of high-resolution data and anomalous signal. With the recent emphasis on obtaining conformational ensemble information for proteins, the high resolution of the data allows such ensembles to be built, while the anomalous signal allows the structure to be experimentally solved, ions to be identified, and water molecules and ions to be differentiated. Because bound metal-, phosphorus- and sulfur-containing ions all have anomalous signal, obtaining anomalous signal across temperatures and up to physiological temperatures will provide a more complete description of protein conformational ensembles, function and energetics.

X-ray Emission Spectroscopy of Single Protein Crystals Yields Insights into Heme Enzyme Intermediates.

Enzyme reactivity is often enhanced by changes in oxidation state, spin state, and metal-ligand covalency of associated metallocofactors. The development of spectroscopic methods for studying these processes coincidentally with structural rearrangements is essential for elucidating metalloenzyme mechanisms. Herein, we demonstrate the feasibility of collecting X-ray emission spectra of metalloenzyme crystals at a third-generation synchrotron source. In particular, we report the development of a von Hamos spectrometer for the collection of Fe Kβ emission optimized for analysis of dilute biological samples. We further showcase its application in crystals of the immunosuppressive heme-dependent enzyme indoleamine 2,3-dioxygenase. Spectra from protein crystals in different states were compared with relevant reference compounds. Complementary density functional calculations assessing covalency support our spectroscopic analysis and identify active site conformations that correlate to high- and low-spin states. These experiments validate the suitability of an X-ray emission approach for determining spin states of previously uncharacterized metalloenzyme reaction intermediates.

Growth of gold-aryl nanoparticles in lysozyme crystals

Hen egg-white lysozyme crystals are an exemplary system for the growth of gold-aryl nanoparticles within proteins. We investigated the growth of AuNPs functionalized with carboxyl groups in a single protein crystal of lysozyme (Lys@AuNPs) by the protein reduction of aryldiazonium tetrachloroaurate(III) salt [COOH-4-C6H4NN]AuCl4 over 30, 60, and 90 days. We studied the time-dependent, in situ, and gradual growth of AuNPs within single crystals of lysozyme in precipitant solution of 6.5% NaCl in 0.1 M sodium acetate at pH 4.5. The growth and incorporation of AuNPs inside the lysozyme crystals were confirmed by HR-TEM measurements and microscopy imaging. Investigation of TEM images indicates that AuNPs matured progressively beginning with an average size of 48.5 ± 5.7 nm after 30 days. More numbers of AuNPs of 20.8 ± 4.4 nm and 19.2 ± 5.4 nm after 60 and 90 days, respectively, were formed within the lysozyme crystals. Furthermore, we observed that the addition of Hg2+ can expedite the formation of Lys@AuNPs crystals. The color of the gold nanoparticles was developed after 8 days in the presence of Hg2+. The formed AuNPs were mostly spherical with an average size of 13.9 ± 5.2 nm.

Nucleation and growth of a single suspended protein crystal by merging phase separated droplets

Nucleation and growth of a single suspended protein crystal by merging phase separated droplets

Laboratory diffracted x-ray blinking to monitor picometer motions of protein molecules and application to crystalline materials.

In recent years, real-time observations of molecules have been required to understand their behavior and function. To date, we have reported two different time-resolved observation methods: diffracted x-ray tracking and diffracted x-ray blinking (DXB). The former monitors the motion of diffracted spots derived from nanocrystals labeled onto target molecules, and the latter measures the fluctuation of the diffraction intensity that is highly correlated with the target molecular motion. However, these reports use a synchrotron x-ray source because of its high average flux, resulting in a high time resolution. Here, we used a laboratory x-ray source and DXB to measure the internal molecular dynamics of three different systems. The samples studied were bovine serum albumin (BSA) pinned onto a substrate, antifreeze protein (AFP) crystallized as a single crystal, and poly{2-(perfluorooctyl)ethyl acrylate} (PC8FA) polymer between polyimide sheets. It was found that not only BSA but also AFP and PC8FA molecules move in the systems. In addition, the molecular motion of AFP molecules was observed to increase with decreasing temperature. The rotational diffusion coefficients (DR) of BSA, AFP, and PC8FA were estimated to be 0.73 pm2/s, 0.65 pm2/s, and 3.29 pm2/s, respectively. Surprisingly, the DR of the PC8FA polymer was found to be the highest among the three samples. This is the first report that measures the molecular motion of a single protein crystal and polymer by using DXB with a laboratory x-ray source. This technique can be applied to any kind of crystal and crystalline polymer and provides atomic-order molecular information.

Structural resolution of switchable states of a de novo peptide assembly

De novo protein design is advancing rapidly. However, most designs are for single states. Here we report a de novo designed peptide that forms multiple α-helical-bundle states that are accessible and interconvertible under the same conditions. Usually in such designs amphipathic α helices associate to form compact structures with consolidated hydrophobic cores. However, recent rational and computational designs have delivered open α-helical barrels with functionalisable cavities. By placing glycine judiciously in the helical interfaces of an α-helical barrel, we obtain both open and compact states in a single protein crystal. Molecular dynamics simulations indicate a free-energy landscape with multiple and interconverting states. Together, these findings suggest a frustrated system in which steric interactions that maintain the open barrel and the hydrophobic effect that drives complete collapse are traded-off. Indeed, addition of a hydrophobic co-solvent that can bind within the barrel affects the switch between the states both in silico and experimentally.

Purification and Preliminary X-Ray Crystallographic Analysis of the Peptidyl-Prolyl cis–trans Isomerase Alr5059 from Anabaena sp. PCC 7120

Peptidyl-prolyl cis–trans isomerases (PPIases) have a wide range of functions in all cells. They are typically classified as cyclophilin, the FK506-binding protein-like or parvulins. Most PPIases are two-domain enzymes. While the peptidyl-prolyl cis–trans isomerase domain is common to all PPIlases, different proteins differ in the function of the second domain. Plant PPIases of the cyclophilin family (Cyp38 in A. thaliana) contain a second domain at the N-terminus, but its function is still not known. They are localized in the thylakoid lumen and are involved in the assembly of photosystem II. The protein is conserved among plants and cyanobacteria with high similarity in the PPIase domain, but with some divergence in the N-terminal region of the protein. This prompted protein crystallization to analyze whether a unique feature of plant proteins originates from a cyanobacterial ancestor. We expressed the Alr5059 protein from Anabaena sp. PCC 7120 in E. coli and crystallized protein by the sitting drop method. Single crystals of the Alr5059 protein appeared after 5–7 days. The best crystal gave a diffraction pattern to a resolution of 1.1 A. The crystal belongs to the monoclinic space group P1211 with unit cell parameters a = 41.2 A, b = 103.2 A, c = 44.2 A and β = 114.7° and contains one molecule per asymmetric unit.

Instrumentation and experimental procedures for robust collection of X-ray diffraction data from protein crystals across physiological temperatures.

Traditional X-ray diffraction data collected at cryo-temperatures have delivered invaluable insights into the three-dimensional structures of proteins, providing the backbone of structure-function studies. While cryo-cooling mitigates radiation damage, cryo-temperatures can alter protein conformational ensembles and solvent structure. Furthermore, conformational ensembles underlie protein function and energetics, and recent advances in room-temperature X-ray crystallography have delivered conformational heterogeneity information that can be directly related to biological function. Given this capability, the next challenge is to develop a robust and broadly applicable method to collect single-crystal X-ray diffraction data at and above room temperature. This challenge is addressed herein. The approach described provides complete diffraction data sets with total collection times as short as ∼5 s from single protein crystals, dramatically increasing the quantity of data that can be collected within allocated synchrotron beam time. Its applicability was demonstrated by collecting 1.09-1.54 Å resolution data over a temperature range of 293-363 K for proteinase K, thaumatin and lysozyme crystals at BL14-1 at the Stanford Synchrotron Radiation Lightsource. The analyses presented here indicate that the diffraction data are of high quality and do not suffer from excessive dehydration or radiation damage.

Microprocessing on Single Protein Crystals Using Femtosecond Pulse Laser.

Proteins with different micropatterns have various applications in biosensing, structural analysis, and other biomedical fields. However, processing of micropatterns on single protein crystals remains a challenge due to the fragility of protein molecules. In this work, we studied femtosecond laser processing on single hen egg white lysozyme protein crystals. Optimized laser parameters were found to achieve micropatterning without cracking of protein crystals. The ablation morphology dependence on the laser fluence and the pulse number was discussed to control the processing results. Under a laser fluence higher than 1 J/cm2, the ablation hole was formed. While multipulses with fluence lower than the ablation threshold were applied, the foaming area was observed due to the denaturation of protein. The numerical simulation shows that the ablation results were influenced by the ionization and energy deposition process. Micropatterns including lines, areas, and microarrays can be processed with a minimum size of 2 μm. Processed patterns on the crystal surface can be used for biosensing microarrays and the enhancement of crystal growth. The microprocessing method proposed in this study has potential applications in different fields including biodevices and biomedicine.

Mechanical‐Bending‐Induced Fluorescence Enhancement in Plastically Flexible Crystals of a GFP Chromophore Analogue

AbstractSingle crystals of optoelectronic materials that respond to external stimuli, such as mechanical, light, or heat, are immensely attractive for next generation smart materials. Here we report single crystals of a green fluorescent protein (GFP) chromophore analogue with irreversible mechanical bending and associated unusual enhancement of the fluorescence, which is attributed to the strained molecular packing in the perturbed region. Soft crystalline materials with such fluorescence intensity modulations occurring in response to mechanical stimuli under ambient pressure conditions will have potential implications for the design of technologically relevant tunable fluorescent materials.

Mechanical-Bending-Induced Fluorescence Enhancement in Plastically Flexible Crystals of a GFP Chromophore Analogue.

Single crystals of optoelectronic materials that respond to external stimuli, such as mechanical, light, or heat, are immensely attractive for next generation smart materials. Here we report single crystals of a green fluorescent protein (GFP) chromophore analogue with irreversible mechanical bending and associated unusual enhancement of the fluorescence, which is attributed to the strained molecular packing in the perturbed region. Soft crystalline materials with such fluorescence intensity modulations occurring in response to mechanical stimuli under ambient pressure conditions will have potential implications for the design of technologically relevant tunable fluorescent materials.

DNA-Directed Protein Packing within Single Crystals

Designed DNA-DNA interactions are investigated for their ability to modulate protein packing within single crystals of mutant green fluorescent proteins (mGFPs) functionalized with a single DNA strand (mGFP-DNA). We probe the effects of DNA sequence, length, and protein-attachment position on the formation and protein packing of mGFP-DNA crystals. Notably, when complementary mGFP-DNA conjugates are introduced to one another, crystals form with nearly identical packing parameters, regardless of sequence if the number of bases is equivalent. DNA complementarity is essential, because experiments with non-complementary sequences produce crystals with different protein arrangements. Importantly, the DNA length and its position of attachment on the protein markedly influence the formation of and protein packing within single crystals. This work shows how designed DNA interactions can be used to influence the growth and packing in X-ray diffraction quality protein single crystals and is thus an important step forward in protein crystal engineering.

Structure Determination of the Transactivation Domain of P53 in Complex with S100A4 Using Annexin A2 as a Crystallization Chaperone

In spite of the fact that structure solving methods are constantly improving, the biggest challenge of protein crystallography remains the production of well diffracting single protein crystals. Full understanding the environmental factors that influence crystal packing would be an enormous task, therefore crystallographers are still forced to work “blindly” trying as many crystallizing conditions as possible. Numerous times the random attempts simply fail even when using crystallization robots. As an alternative option in these cases, crystallization chaperones can be used. These proteins have a unique property, namely they easily form protein crystals, which can be exploited by using them as a heterologous fusion partner to promote crystal contact formation. Today, the most frequently used crystallization chaperone is the maltose-binding protein (MBP) and crystallographers are in need of other options. Our previous results showed the outstanding crystallization properties of a non-EF- hand calcium-binding protein annexin A2 (ANXA2). Here, we compared ANXA2 with the wild type MBP and found that ANXA2 is just as good, if not a better crystallization chaperone. Using ANXA2 for this purpose, we were able to solve the atomic resolution structure of a challenging crystallization target, the transactivation domain (TAD) of p53 in complex with S100A4, an EF-hand calcium-binding protein associated with metastatic tumors. The full-length TAD forms an asymmetric fuzzy complex with S100A4 and could interfere with its function.

A prototype system for dynamically polarized neutron protein crystallography

Abstract The sensitivity of Neutron Macromolecular Crystallography to the presence of hydrogen makes it a powerful tool to complement X-ray crystallographic studies using protein crystals. The power of this technique is currently limited by the relative low neutron flux provided by even the most powerful neutron sources . The strong polarization dependence of the neutron scattering cross section of hydrogen will allow us to use Dynamic Nuclear Polarization to dramatically improve the signal to noise ratio of neutron diffraction data, delivering order of magnitude gains in performance, and enabling measurements of radically smaller crystals of larger protein systems than are possible today. We present a prototype frozen spin system, built at Oak Ridge National Laboratory to polarize single protein crystals on the IMAGINE beamline at the High Flux Isotope Reactor (HFIR). Details of the design and construction will be described, as will the performance of the system offline and during preliminary tests at HFIR.

On-demand droplet loading of ultrasonic acoustic levitator and its application for protein crystallography experiments

Higher throughput has been ever demanded in the state-of-the-art protein crystallography beamlines for applications such as the screening of drug targets in protein-ligand complex structures at room temperature. As a potential method to achieve an order of magnitude higher throughput, we explore capturing of ejected droplets by an acoustic levitator to remotely load single protein crystal samples to an acoustic levitation diffractometer at the Swiss Light Source synchrotron facility. The results from X-ray diffraction experiments support the feasibility of this method as a fully automated sample delivery for high-throughput serial crystallography experiments using the acoustic levitation.

A novel handling-free method of mounting single protein crystals for synchrotron structure analyses at room temperature.

We have developed a handling-free mounting method for X-ray crystallography of protein crystals at room temperatures-the glass capillary method. In this method, crystals were nucleated and grown on the capillary walls, and then, growth solutions were gently removed. The procedures for collecting data on the crystals were conducted by simply setting the capillary on the goniometer of a synchrotron beamline without touching the crystals. Crystal quality was characterized using mosaicity, resolution at I/σ(I) = 2, I/σ(I) at resolution = 2.0 Å, Rmerge, and completeness. Wilson plots were also used to characterize the quality of crystals. In particular, all samples showed very low mosaicity; the handling-free method successfully retained their low mosaicity and effectively maintained the crystal quality.

Serial crystallography captures enzyme catalysis in copper nitrite reductase at atomic resolution from one crystal.

Relating individual protein crystal structures to an enzyme mechanism remains a major and challenging goal for structural biology. Serial crystallography using multiple crystals has recently been reported in both synchrotron-radiation and X-ray free-electron laser experiments. In this work, serial crystallography was used to obtain multiple structures serially from one crystal (MSOX) to study in crystallo enzyme catalysis. Rapid, shutterless X-ray detector technology on a synchrotron MX beamline was exploited to perform low-dose serial crystallography on a single copper nitrite reductase crystal, which survived long enough for 45 consecutive 100 K X-ray structures to be collected at 1.07-1.62 Å resolution, all sampled from the same crystal volume. This serial crystallography approach revealed the gradual conversion of the substrate bound at the catalytic type 2 Cu centre from nitrite to nitric oxide, following reduction of the type 1 Cu electron-transfer centre by X-ray-generated solvated electrons. Significant, well defined structural rearrangements in the active site are evident in the series as the enzyme moves through its catalytic cycle, namely nitrite reduction, which is a vital step in the global denitrification process. It is proposed that such a serial crystallography approach is widely applicable for studying any redox or electron-driven enzyme reactions from a single protein crystal. It can provide a 'catalytic reaction movie' highlighting the structural changes that occur during enzyme catalysis. The anticipated developments in the automation of data analysis and modelling are likely to allow seamless and near-real-time analysis of such data on-site at some of the powerful synchrotron crystallographic beamlines.

A microfluidic-based protein crystallization method in 10 micrometer-sized crystallization space

Protein crystallization and subsequent X-ray diffraction analysis of the three-dimensional structure are necessary for elucidation of the biological functions of proteins and effective rational drug design. Therefore, controlling protein crystallization is important to obtain high resolution X-ray diffraction data. Here, a simple microfluidic method using chips with 10 and 50 μm high crystallization chambers to selectively form suitable single protein crystals for X-ray analysis is demonstrated. As proof of concept, three different types of proteins: lysozyme, glucokinase from Pseudoalteromonas sp. AS-131 (PsGK), and NADPH-cytochrome P450 oxidoreductase–heme oxygenase complex were crystallized. We demonstrate that the crystal growth orientation depends on the height of the crystallization chamber regardless of the protein type. Our results suggest that the confined micro space induces the protein molecules to adhere to a specific crystal face and affects the growth kinetics of each crystal face. The present microfluidic-based protein crystallization method can reform a suitable single protein crystal for X-ray analysis from aggregates of needle-shaped protein crystals.