Dynamically controlled crystal growth system

Crystal growth can be initiated and controlled by dynamically controlled vapor diffusion or temperature change. In one aspect, the present invention uses a precisely controlled vapor diffusion approach to monitor and control protein crystal growth. The system utilizes a humidity sensor and various interfaces under computer control to effect virtually any evaporation rate from a number of different growth solutions simultaneously by means of an evaporative gas flow. A static laser light scattering sensor can be used to detect aggregation events and trigger a change in the evaporation rate for a growth solution. A control/follower configuration can be used to actively monitor one chamber and accurately control replicate chambers relative to the control chamber. In a second aspect, the invention exploits the varying solubility of proteins versus temperature to control the growth of protein crystals. This system contains miniature thermoelectric devices under microcomputer control that change temperature as needed to grow crystals of a given protein. Complex temperature ramps are possible using this approach. A static laser light scattering probe also can be used in this system as a non-invasive probe for detection of aggregation events. The automated dynamic control system provides systematic and predictable responses with regard to crystal size. These systems can be used for microgravity crystallization projects, for example in a space shuttle, and for crystallization work under terrestial conditions. The present invention is particularly useful for macromolecular crystallization, e.g. for proteins, polypeptides, nucleic acids, viruses and virus particles

Use of dye to distinguish salt and protein crystals under microcrystallization conditions

An improved method of screening crystal growth conditions is provided wherein molecules are crystallized from solutions containing dyes. These dyes are selectively incorporated or associated with crystals of particular character thereby rendering crystals of particular character colored and improving detection of the dyed crystals. A preferred method involves use of dyes in protein solutions overlayed by oil. Use of oil allows the use of small volumes of solution and facilitates the screening of large numbers of crystallization conditions in arrays using automated devices that dispense appropriate solutions to generate crystallization trials, overlay crystallization trials with an oil, provide appropriate conditions conducive to crystallization and enhance detection of dyed (colored) or undyed (uncolored) crystals that result

Method for acquiring, storing and analyzing crystal images

A system utilizing a digital computer for acquiring, storing and evaluating crystal images. The system includes a video camera (12) which produces a digital output signal representative of a crystal specimen positioned within its focal window (16). The digitized output from the camera (12) is then stored on data storage media (32) together with other parameters inputted by a technician and relevant to the crystal specimen. Preferably, the digitized images are stored on removable media (32) while the parameters for different crystal specimens are maintained in a database (40) with indices to the digitized optical images on the other data storage media (32). Computer software is then utilized to identify not only the presence and number of crystals and the edges of the crystal specimens from the optical image, but to also rate the crystal specimens by various parameters, such as edge straightness, polygon formation, aspect ratio, surface clarity, crystal cracks and other defects or lack thereof, and other parameters relevant to the quality of the crystals

Crystal Structure of \u3cem\u3eYersinia pestis\u3c/em\u3e Virulence Factor YfeA Reveals Two Polyspecific Metal-Binding Sites

Gram-negative bacteria use siderophores, outer membrane receptors, inner membrane transporters and substrate-binding proteins (SBPs) to transport transition metals through the periplasm. The SBPs share a similar protein fold that has undergone significant structural evolution to communicate with a variety of differentially regulated transporters in the cell. In Yersinia pestis, the causative agent of plague, YfeA (YPO2439, y1897), an SBP, is important for full virulence during mammalian infection. To better understand the role of YfeA in infection, crystal structures were determined under several environmental conditions with respect to transition-metal levels. Energy-dispersive X-ray spectroscopy and anomalous X-ray scattering data show that YfeA is polyspecific and can alter its substrate specificity. In minimal-media experiments, YfeA crystals grown after iron supplementation showed a threefold increase in iron fluorescence emission over the iron fluorescence emission from YfeA crystals grown from nutrient-rich conditions, and YfeA crystals grown after manganese supplementation during overexpression showed a fivefold increase in manganese fluorescence emission over the manganese fluorescence emission from YfeA crystals grown from nutrient-rich conditions. In all experiments, the YfeA crystals produced the strongest fluorescence emission from zinc and could not be manipulated otherwise. Additionally, this report documents the discovery of a novel surface metal-binding site that prefers to chelate zinc but can also bind manganese. Flexibility across YfeA crystal forms in three loops and a helix near the buried metal-binding site suggest that a structural rearrangement is required for metal loading and unloading

Self-interaction chromatography as a tool for optimizing conditions for membrane protein crystallization

The second virial coefficient, or B value, is a measurement of how well a protein interacts with itself in solution. These interactions can lead to protein crystallization or precipitation, depending on their strength, with a narrow range of B values (the `crystallization slot') being known to promote crystallization. A convenient method of determining the B value is by self-interaction chromatography. This paper describes how the light-harvesting complex 1-reaction centre core complex from Allochromatium vinosum yielded single straight-edged crystals after iterative cycles of self-interaction chromatography and crystallization. This process allowed the rapid screening of small molecules and detergents as crystallization additives. Here, a description is given of how self-interaction chromatography has been utilized to improve the crystallization conditions of a membrane protein

Protein crystal growth results from the United States Microgravity Laboratory-1 mission

Protein crystal growth experiments have been performed by this laboratory on 18 Space Shuttle missions since April, 1985. In addition, a number of microgravity experiments also have been performed and reported by other investigators. These Space Shuttle missions have been used to grow crystals of a variety of proteins using vapor diffusion, liquid diffusion, and temperature-induced crystallization techniques. The United States Microgravity Laboratory - 1 mission (USML-1, June 25 - July 9, 1992) was a Spacelab mission dedicated to experiments involved in materials processing. New protein crystal growth hardware was developed to allow in orbit examination of initial crystal growth results, the knowledge from which was used on subsequent days to prepare new crystal growth experiments. In addition, new seeding hardware and techniques were tested as well as techniques that would prepare crystals for analysis by x-ray diffraction, a capability projected for the planned Space Station. Hardware that was specifically developed for the USML-1 mission will be discussed along with the experimental results from this mission

Membrane protein stability can be compromised by detergent interactions with the extramembranous soluble domains: Detergents Destabilize Extramembranous Soluble Domains

Detergent interaction with extramembranous soluble domains (ESDs) is not commonly considered an important determinant of integral membrane protein (IMP) behavior during purification and crystallization, even though ESDs contribute to the stability of many IMPs. Here we demonstrate that some generally nondenaturing detergents critically destabilize a model ESD, the first nucleotide-binding domain (NBD1) from the human cystic fibrosis transmembrane conductance regulator (CFTR), a model IMP. Notably, the detergents show equivalent trends in their influence on the stability of isolated NBD1 and full-length CFTR. We used differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy to monitor changes in NBD1 stability and secondary structure, respectively, during titration with a series of detergents. Their effective harshness in these assays mirrors that widely accepted for their interaction with IMPs, i.e., anionic > zwitterionic > nonionic. It is noteworthy that including lipids or nonionic detergents is shown to mitigate detergent harshness, as will limiting contact time. We infer three thermodynamic mechanisms from the observed thermal destabilization by monomer or micelle: (i) binding to the unfolded state with no change in the native structure (all detergent classes); (ii) native state binding that alters thermodynamic properties and perhaps conformation (nonionic detergents); and (iii) detergent binding that directly leads to denaturation of the native state (anionic and zwitterionic). These results demonstrate that the accepted model for the harshness of detergents applies to their interaction with an ESD. It is concluded that destabilization of extramembranous soluble domains by specific detergents will influence the stability of some IMPs during purification

A Stable Human-Cell System Overexpressing Cystic Fibrosis Transmembrane Conductance Regulator Recombinant Protein at the Cell Surface

Recent human clinical trials results demonstrated successful treatment for certain genetic forms of cystic fibrosis (CF). To extend treatment opportunities to those afflicted with other genetic forms of CF disease, structural and biophysical characterization of CF transmembrane conductance regulator (CFTR) is urgently needed. In this study, CFTR was modified with various tags, including a His10 purification tag, the SUMOstar (SUMO*) domain, an extracellular FLAG epitope, or an enhanced green fluorescent protein (EGFP), each alone or in various combinations. Expressed in HEK293 cells, recombinant CFTR proteins underwent complex glycosylation, compartmentalized with the plasma membrane, and exhibited regulated chloride-channel activity with only modest alterations in channel conductance and gating kinetics. Surface CFTR expression level was enhanced by the presence of SUMO* on the N-terminus. Quantitative mass-spectrometric analysis indicated approximately 10% of the total recombinant CFTR (SUMO*-CFTRFLAG-EGFP) localized to the plasma membrane. Trial purification using dodecylmaltoside for membrane protein extraction reproducibly recovered 178 ± 56 μg SUMO*-CFTRFLAG-EGFP per billion cells at 80% purity. Fluorescence size-exclusion chromatography indicated purified CFTR was monodisperse. These findings demonstrate a stable mammalian cell expression system capable of producing human CFTR of sufficient quality and quantity to augment futrure CF drug discovery efforts, including biophysical and structural studies