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NASA NTRS1986

Protein crystal growth; Proceedings of the First International Conference, Stanford University, CA, August 14-16, 1985

Papers are presented on mechanisms of nucleation and growth of protein crystals, the role of purification in the crystallization of proteins and nucleic acids, and the effect of chemical impurities in polyethylene glycol on macromolecular crystallization. Also considered are growth kinetics of tetragonal lysozyme crystals, thermodynamic and kinetic considerations for crystal growth of complex molecules from solution, protein single-crystal growth under microgravity, and growth of organic crystals in a microgravity environment. Papers are also presented on preliminary investigations of protein crystal growth using the Space Shuttle, convective diffusion in protein crystal growth, and the growth and characterization of membrane protein crystals.

Feigelson, R. S.↗

NASA NTRS2001

Lab-on-a-Chip Based Protein Crystallization

We are developing a novel technique with which we will grow protein crystals in very small volumes, utilizing chip-based, microfluidic ("LabChip") technology. This development, which is a collaborative effort between NASA's Marshall Space Flight Center and Caliper Technologies Corporation, promises a breakthrough in the field of protein crystal growth. Our initial results obtained from two model proteins, Lysozyme and Thaumatin, show that it is feasible to dispense and adequately mix protein and precipitant solutions on a nano-liter scale. The mixtures have shown crystal growth in volumes in the range of 10 nanoliters to 5 microliters. In addition, large diffraction quality crystals were obtained by this method. X-ray data from these crystals were shown to be of excellent quality. Our future efforts will include the further development of protein crystal growth with LabChip(trademark) technology for more complex systems. We will initially address the batch growth method, followed by the vapor diffusion method and the liquid-liquid diffusion method. The culmination of these chip developments is to lead to an on orbit protein crystallization facility on the International Space Station. Structural biologists will be invited to utilize the on orbit Iterative Biological Crystallization facility to grow high quality macromolecular crystals in microgravity.

vanderWoerd, Mark J.↗

NASA NTRS1991

Containerless protein crystal growth method

A method of growing protein crystals from levitated drops is introduced and unique features of containerless approach in 1-g and micro-G laboratories are discussed. Electrostatic multidrop levitation system which is capable of simultaneous four drop levitation is described. A method of controlling protein saturation level in a programmed way is introduced and discussed. Finally, some of the unique features of containerless approach of protein crystal growth in space are discussed and summarized.

Rhim, Won-Kyu↗

NASA NTRS1997

Protein Crystal Growth Apparatus for Microgravity

Apparatus for growing protein crystals under microgravity environment includes a plurality of protein growth assemblies stacked one above the other within a canister. Each of the protein growth assemblies includes a tray having a number of spaced apart growth chambers recessed below an upper surface. the growth chambers each having an upstanding pedestal and an annular reservoir about the pedestal for receiving a wick and precipitating agents. A well is recessed below the top of each pedestal to define a protein crystal growth receptacle. A flexible membrane is positioned on the upper surface of each tray and a sealing plate is positioned above each membrane, each sealing plate having a number of bumpers corresponding in number and alignment to the pedestals for forcing the membrane selectively against the upper end of the respective pedestal to seal the reservoir and the receptacle when the sealing plate is forced down.

Carter, Daniel C.↗

NASA NTRS1988

Protein crystal growth in low gravity

The solubility and growth of the protein canavalin, and the application of the schlieren technique to study fluid flow in protein crystal growth systems were investigated. These studies have resulted in the proposal of a model to describe protein crystal growth and the preliminary plans for a long-term space flight experiment. Canavalin, which may be crystallized from a basic solution by the addition of hydrogen (H+) ions, was shown to have normal solubility characteristics over the range of temperatures (5 to 25 C) and pH (5 to 7.5) studies. The solubility data combined with growth rate data gathered from the seeded growth of canavalin crystals indicated that the growth rate limiting step is a screw dislocation mechanism. A schlieren apparatus was constructed and flow patterns were observed in Rochelle salt (sodium potassium tartrate), lysozyme, and canavalin. The critical parameters were identified as the change in density with concentration (dp/dc) and the change in index of refraction with concentration (dn/dc). Some of these values were measured for the materials listed. The data for lyrozyme showed non-linearities in plots of optical properties and density vs. concentration. In conjunction with with W. A. Tiller, a model based on colloid stability theory was proposed to describe protein crystallization. The model was used to explain observations made by ourselves and others. The results of this research has lead to the development for a preliminary design for a long-term, low-g experiment. The proposed apparatus is univeral and capable of operation under microprocessor control.

Feigelson, Robert S.↗

NASA NTRS2003

Phase Sensitive X-Ray Diffraction Imaging Study of Protein Crystals

The study of defects and growth of protein crystals is of importance in providing a fundamental understanding of this important category of systems and the rationale for crystallization of better ordered crystals for structural determination and drug design. Yet, as a result of the extremely weak scattering power of x-rays in protein and other biological macromolecular crystals, the extinction lengths for those crystals are extremely large and, roughly speaking, of the order of millimeters on average compared to the scale of micrometers for most small molecular crystals. This has significant implication for x-ray diffraction and imaging study of protein crystals, and presents an interesting challenge to currently available x-ray analytical techniques. We proposed that coherence-based phase sensitive x-ray diffraction imaging could provide a way to augment defect contrast in x-ray diffraction images of weakly diffracting biological macromolecular crystals. I shall examine the principles and ideas behind this approach and compare it to other available x-ray topography and diffraction methods. I shall then present some recent experimental results in two model protein systems-cubic apofemtin and tetragonal lysozyme crystals to demonstrate the capability of the coherence-based imaging method in mapping point defects, dislocations, and the degree of perfection of biological macromolecular crystals with extreme sensitivity. While further work is under way, it is intended to show that the observed new features have yielded important information on protein crystal perfection and nucleation and growth mechanism otherwise unobtainable.

Hu, Z. W.↗

NASA NTRS1994

Automated protein crystal growth facility

A customer for the protein crystal growth facility fills the specially designed chamber with the correct solutions, fills the syringes with their quenching solutions, and submits the data needed for the proper growth of their crystal. To make sure that the chambers and syringes are filled correctly, a NASA representative may assist the customer. The data needed is the approximate growth time, the growth temperature, and the desired crystal size, but this data can be changed anytime from the ground, if needed. The chambers are gathered and placed into numbered slots in special drawers. Then, data is entered into a computer for each of the chambers. Technicians map out when each chamber's growth should be activated so that all of the chambers have enough time to grow. All of this data is up-linked to the space station when the previous growth session is over. Anti-vibrational containers need to be constructed for the high forces encountered during the lift off and the landing of the space shuttle, and though our team has not designed these containers, we do not feel that there is any reason why a suitable one could not be made. When the shuttle reaches the space station, an astronaut removes a drawer of quenched chambers from the growth facility and inserts a drawer of new chambers. All twelve of the drawers can be replaced in this fashion. The optical disks can also be removed this way. The old drawers are stored for the trip back to earth. Once inside the growth facility, a chamber is removed by the robot and placed in one of 144 active sites at a time previously picked by a technician. Growth begins when the chamber is inserted into an active site. Then, the sensing system starts to determine the size of the protein crystal. All during the crystal's growth, the customer can view the crystal and read all of the crystal's data, such as growth rate and crystal size. When the sensing system determines that the crystal has reached the predetermined size, the robot is told to pick up a syringe filled with the correct quenchant solution and inject it into the chamber to stop the crystal growth. The chamber is then removed from the active site and placed into its original storage slot. Another chamber is then placed into the active site and the process is repeated in all of the active sites until all of the chambers have complted their growth. After ninety days (the scheduled time between shuttle visits), the crystal growth is completed, and the old drawers are replaced with new ones. Once the customer extracts the crystals, the chambers are retrieved for future customers.

Donald, Stacey↗

NASA NTRS2010

Screening and Crystallization Plates for Manual and High-throughput Protein Crystal Growth

In one embodiment, a crystallization and screening plate comprises a plurality of cells open at a top and a bottom, a frame that defines the cells in the plate, and at least two films. The first film seals a top of the plate and the second film seals a bottom of the plate. At least one of the films is patterned to strongly pin the contact lines of drops dispensed onto it, fixing their position and shape. The present invention also includes methods and other devices for manual and high-throughput protein crystal growth.

Thorne, Robert E.↗

NASA NTRS1992

Protein crystal growth (5-IML-1)

Proteins (enzymes, hormones, immunoglobulins) account for 50 pct. or more of the dry weight of most living systems. A detailed understanding of the structural makeup of a protein is essential to any systematic research pertaining to it. Most macromolecules are extremely difficult to crystallize, and many otherwise exciting projects have terminated at the crystal growth stage. In principle, there are several aspects of microgravity that might be exploited to enhance protein crystal growth. The major factor is the elimination of density driven convective flow. Other factors that can be controlled in the absence of gravity is the sedimentation of growing crystals in a gravitational field, and the potential advantage of doing containerless crystal growth. As a result of these theories and facts, one can readily understand why the microgravity environment of an Earth orbiting vehicle seems to offer unique opportunities for the protein crystallographer. This perception has led to the establishment of the Protein Crystal Growth in a Microgravity Environment (PCG/ME) project. The results of experiments already performed during STS missions have in many cases resulted in large protein crystals which are structurally correct. Thus, the near term objective of the PCG/ME project is to continue to improve the techniques, procedures, and hardware systems used to grow protein crystals in Earth orbit.

Bugg, Charles E.↗

NASA NTRS2000

Molecular View of Protein Crystal Growth: Molecular Interactions, Surface Reconstruction and Growth Mechanism

Studies of the growth and molecular packing of tetragonal lysozyme crystals suggest that there is an underlying molecular growth mechanism, in addition to the classical one involving screw dislocation/2D) nucleation growth. These crystals are constructed by strongly bonded molecular chains forming helices about the 43 axes. The helices are connected to each other by weaker bonds. Crystal growth proceeds by the formation of these 4(sub 3) helices, which would explain some unexpected observations by earlier investigators, such as bimolecular growth steps on the (110) face. Another consequence of these molecular considerations is that only one of two possible packing arrangements could occur on the crystal faces and that their growth unit was at least a tetramer corresponding to the 4(sub 3) helix. Two new high resolution atomic force microscopy (AFM) techniques were developed to directly confirm these predictions on tetragonal lysozyme crystals. Most earlier investigations of protein crystal growth with AFM were in the low resolution mode which is adequate to investigate the classical growth mechanisms, but cannot resolve molecular features and mechanisms. Employing the first of the newly developed techniques, high resolution AFM images of the (110) face were compared with the theoretically constructed images for the two possible packing arrangements on this face. The prediction that the molecular packing arrangement of these faces corresponded to that for complete 4(sub 3) helices was confirmed in this manner. This investigation also showed the occurrence of surface reconstruction on protein crystals. The molecules on the surface of the (110) face were found to pack closer along the 4(sub 3) axes than those in the interior. The second new AFM technique was used to follow the growth process by measuring the dimensions of individual growth units on the (110) face. Linescans across a growth step, performed near the saturation limit of the crystals, allowed the growth unit dimensions to be measured as they were being incorporated into the crystal. These measurements showed that growth on the (110) face proceeded by the formation of new 4(sub 3) helices from the addition of many growth units of at least tetramer size. This is the first time that a time-resolved AFM technique has been used to observe individual molecular events during crystal growth.

Nadarajah, Arunan↗

NASA NTRS1998

Nucleation and Convection Effects in Protein Crystal Growth

Our work under this grant has significantly contributed to the goals of the NASA supported protein crystallization program. We have achieved the main objectives of the proposed work, as outlined in the original proposal: (1) We have provided important insight into protein nucleation and crystal growth mechanisms to facilitate a rational approach to protein crystallization; (2) We have delineated the factors that currently limit the x-ray diffraction resolution of protein crystals, and their correlation to crystallization conditions; (3) We have developed novel technologies to study and monitor protein crystal nucleation and growth processes, in order to increase the reproducibility and yield of protein crystallization. We have published 17 papers in peer-reviewed scientific journals and books and made more than 15 invited and 9 contributed presentations of our results at international and national scientific meetings.

Vekilow, Peter G.↗

NASA NTRS1986

An apparatus for protein crystal growth studies

An apparatus is described for observing protein crystals growing under varying growth conditions, and for changing or manipulating crystal orientation for viewing selected faces or growth modes. This flexibility permits a wide variety of protein crystal growth experiments to be performed using the same crystal(s).

Pusey, Marc L.↗

NASA NTRS1998

Effects of Convective Solute and Impurity Transport in Protein Crystal Growth

High-resolution optical interferometry was used to investigate the effects of forced solution convection on the crystal growth kinetics of the model protein lysozyme. Most experiments were conducted with 99.99% pure protein solutions. To study impurity effects, approx. 1% of lysozyme dimer (covalently bound) was added in some cases. We show that the unsteady kinetics, corresponding to bunching of growth steps, can be characterized by the Fourier components of time traces of the growth rate. Specific Fourier spectra are uniquely determined by the solution conditions (composition, temperature, and flow rate) and the growth layer source activity. We found that the average step velocity and growth rate increase by approx. I0% with increasing flow rate, as a result of the enhanced solute supply to the interface. More importantly, faster convective transport results in lower fluctuation amplitudes. This observation supports our rationale for system-dependent effects of transport on the structural perfection of protein crystals. We also found that solution flow rates greater than 500 microns/s result in stronger fluctuations while the average growth rate is decreased. This can lead to growth cessation at low supersaturations. With the intentionally contaminated solutions, these undesirable phenomena occurred at about half the flow rates required in pure solutions. Thus, we conclude that they are due to enhanced convective supply of impurities that are incorporated into the crystal during growth. Furthermore, we found that the impurity effects are reduced at higher crystal growth rates. Since the exposure time of terraces is inversely proportional to the growth rate, this observation suggests that the increased kinetics instability results from impurity adsorption on the interface. Finally, we provide evidence relating earlier observations of "slow protein crystal growth kinetics" to step bunch formation in response to nonsteady step generation.

Vekilov, Peter G.↗

NASA NTRS1989

Protein crystal growth in microgravity

The crystals of most proteins or other biological macromolecules are poorly ordered and diffract to lower resolutions than those observed for most crystals of simple organic and inorganic compounds. Crystallization in the microgravity environment of space may improve crystal quality by eliminating convection effects near growing crystal surfaces. A series of 11 different protein crystal growth experiments was performed on U.S. Space Shuttle flight STS-26 in September 1988. The microgravity-grown crystals of gamma-interferon D1, porcine elastase, and isocitrate lyase are larger, display more uniform morphologies, and yield diffraction data to significantly higher resolutions than the best crystals of these proteins grown on earth.

Delucas, Lawrence J.↗

NASA NTRS2001

Laboratory multiple-crystal X-ray topography and reciprocal-space mapping of protein crystals: influence of impurities on crystal perfection

Double-axis multiple-crystal X-ray topography, rocking-curve measurements and triple-axis reciprocal-space mapping have been combined to characterize protein crystals using a laboratory source. Crystals of lysozyme and lysozyme crystals doped with acetylated lysozyme impurities were examined. It was shown that the incorporation of acetylated lysozyme into crystals of lysozyme induces mosaic domains that are responsible for the broadening and/or splitting of rocking curves and diffraction-space maps along the direction normal to the reciprocal-lattice vector, while the overall elastic lattice strain of the impurity-doped crystals does not appear to be appreciable in high angular resolution reciprocal-space maps. Multiple-crystal monochromatic X-ray topography, which is highly sensitive to lattice distortions, was used to reveal the spatial distribution of mosaic domains in crystals which correlates with the diffraction features in reciprocal space. Discussions of the influence of acetylated lysozyme on crystal perfection are given in terms of our observations.

Muramidase/chemistry↗

NASA NTRS2004

Using Magnetic Fields to Control Convection during Protein Crystallization: Analysis and Validation Studies

The effect of convection during the crystallization of proteins is not very well understood. In a gravitational field, convection is caused by crystal sedimentation and by solutal buoyancy induced flow and these can lead to crystal imperfections. While crystallization in microgravity can approach diffusion limited growth conditions (no convection), terrestrially strong magnetic fields can be used to control fluid flow and sedimentation effects. In this work, we develop the analysis for magnetic flow control and test the predictions using analog experiments. Specifically, experiments on solutal convection in a paramagnetic fluid were conducted in a strong magnetic field gradient using a dilute solution of Manganese Chloride. The observed flows indicate that the magnetic field can completely counter the settling effects of gravity locally and are consistent with the theoretical predictions presented. This phenomenon suggests that magnetic fields may be useful in mimicking the microgravity environment of space for some crystal growth ana biological applications where fluid convection is undesirable.

Ramachandran, N.↗

NASA NTRS1992

Advanced protein crystal growth programmatic sensitivity study

The purpose of this study is to define the costs of various APCG (Advanced Protein Crystal Growth) program options and to determine the parameters which, if changed, impact the costs and goals of the programs and to what extent. This was accomplished by developing and evaluating several alternate programmatic scenarios for the microgravity Advanced Protein Crystal Growth program transitioning from the present shuttle activity to the man tended Space Station to the permanently manned Space Station. These scenarios include selected variations in such sensitivity parameters as development and operational costs, schedules, technology issues, and crystal growth methods. This final report provides information that will aid in planning the Advanced Protein Crystal Growth Program.

Source record↗

NASA NTRS1991

Protein crystal growth in microgravity

A series of protein-crystal growth experiments were performed on Space Shuttle flight STS-26 in September 1988 and STS-29 in March 1989. These proteins had been studied extensively in crystal-growth experiments on earth prior to the microgravity experiments. For those proteins which produced crystals of adequate size, 3D intensity data sets with electronic area detector systems were collected. Comparisons show that the microgravity-grown crystals of these proteins are larger, display more uniform morphologies, and yield diffraction data to significantly higher resolutions. Analyses of the 3D data sets indicate that the space-grown crystals are more highly ordered at the molecular level than their earth-grown counterparts.

Snyder, Robert↗