Lawrence J. DeLucas

Crystal structure of calcium bound domain VI of calpain at 1.9 Å resolution and its role in enzyme assembly, regulation, and inhibitor binding

The three dimensional structure of calcium-bound domain VI of porcine calpain has been determined to 1.9 Å resolution. The crystal structure reveals five EF-hands, one more than previously suggested. There are two EF-hand pairs, one pair (EF1-EF2) displays an ‘open’ conformation and the other (EF3-EF4) a ‘closed’ conformation. Unusually, a calcium atom is found at the C-terminal end of the calcium binding loop of EF4. With two additional residues in the calcium binding loop, the fifth EF-hand (EF5) is in a ‘closed’ conformation. EF5 pairs up with the corresponding fifth EF-hand of a non-crystallographically related molecule. Considering the EFSs role in a homodimer formation of domain VI, we suggest a model for the assembly of heterodimeric calpain. The crystal structure of a Ca2+ bound domain VI–inhibitor (PD150606) complex has been refined to 2.1 Å resolution. A possible mode for calpain inhibition is discussed.

His-tag impact on structure.

Crystallographers are increasingly determining structures of protein constructs that include His tags. Many have taken for granted that these tags have little effect on the native structure. This paper surveys and compares crystal structures with and without His tags. It is observed that actual refined tag residues fitted into density occur in less that 10% of the tagged sequences. However, higher resolution crystals are observed when this occurs. It is shown that these purification tags generally have no significant effect on the structure of the native protein. Resolution and R factors are not affected, but the overall B factors are slightly higher. Additional annotation in the PDB format to make tag definition explicit is suggested.

Preliminary investigations of protein crystal growth using the Space Shuttle

Abstract Protein crystal growth in space is of interest because of the potential applications for unique studies of crystallization processes. Theoretical and experimental research indicates that gravitational fields produce density-driven convective flow patterns which can influence crystal growth, and these convective effects can be controlled under microgravity conditions. Microgravity can also be used to control sedimentation effects. As part of a program to investigate the influence of gravity on protein crystal growth, ground and shuttle-based experiments are in progress, and suitable techniques and equipment for protein crystal growth in space are being developed. The research program includes several phases of hardware development, beginning with a simple prototype system, and evolving to an automated protein crystal growth unit that will permit the major variables in protein crystallization to be monitored and controlled during the crystal growth processes. As part of the first step in hardware development, protein crystal growth experiments have been performed on four different shuttle flight missions.

Crystal Structure of the Cytoskeleton-associated Protein Glycine-rich (CAP-Gly) Domain*

Cytoskeleton-associated proteins (CAPs) are involved in the organization of microtubules and transportation of vesicles and organelles along the cytoskeletal network. A conserved motif, CAP-Gly, has been identified in a number of CAPs, including CLIP-170 and dynactins. The crystal structure of the CAP-Gly domain ofCaenorhabditis elegans F53F4.3 protein, solved by single wavelength sulfur-anomalous phasing, revealed a novel protein fold containing three β-sheets. The most conserved sequence, GKNDG, is located in two consecutive sharp turns on the surface, forming the entrance to a groove. Residues in the groove are highly conserved as measured from the information content of the aligned sequences. The C-terminal tail of another molecule in the crystal is bound in this groove.

INTERNATIONAL SPACE STATION

Abstract The International Space Station represents the largest scientific and technological cooperative program in history, drawing on the resources of thirteen nations. The early stages of construction will involve significant participation from the Russian Space Agency (RSA), numerous nations of the European Space Agency (ESA), and the space agencies of Canada (CSA), Japan (NASDA) and the United States Space Agency (NASA). Its purpose is to place a unique, highly capable laboratory in tower orbit, where high value scientific research can be performed in microgravity. In addition to providing facilities where an international crew of six astronaut-scientists can live and work in space, it will provide important laboratory research facilities for performing basic research in life science, biomedical and material sciences, as well as space and engineering technology development which cannot be accomplished on Earth. The Space Station will be comprised of numerous interlocking components which are currently being constructed on Earth. Space Station will be assembled in orbit over a period of time and will provide several experimentation modules as well as habitation modules and interfaces for logistic modules. Including the four extensive solar rays from which it will draw electrical power, the Station will measure more than 300 feet wide by 200 feet long. This paper will present an overview of the various phases of construction of the Space Station and the planned science thought will be performed during the construction phase and after completion.

Purification of octyl β-d-glucopyranoside and re-estimation of its micellar size

Abstract The commercial non-ionic detergent octyl β- d -glucopyranoside is often contaminated by significant amounts of UV absorbing and/or ionic compounds that can associate with membrane proteins. Such impurities can be monitored by several techniques (i.e., spectrophotometry, size exclusion chromatography, and pH, conductivity, and surface tension measurements) and can be removed using mixed-bed ion exchange chromatography. High performance of size exclusion chromatography, dynamic light scattering, and ultracentrifugation have been used to re-estimate the size of micelles of octyl β- d _glucopyranoside since previously published data varied over a wide range. Aggregation numbers were 27 to 100 for micellar molecular weights 8000 to 29 000. Direct physical methods that do not perturbate the sample indicated a large size for the micelles (hydrodynamic radius 23 ± 3 A ; Mr 22 000 ± 3000; aggregation number 75 ± 10 for a 34 mM aqueous solution). In contrast the chromatographic micellar size appeared to be smaller (hydrodynamic radius 15 ± 1 A ; Mr 8000 ± 1000; aggregation number 27). This underestimation may be the result of adsorption and/or alteration of the micelles.

Structural basis of profactor D activation: from a highly flexible zymogen to a novel self-inhibited serine protease, complement factor D

The crystal structure of profactor D, determined at 2.1 Å resolution with an Rfree and an R‐factor of 25.1 and 20.4%, respectively, displays highly flexible or disordered conformation for five regions: N‐22, 71–76, 143–152, 187–193 and 215–223. A comparison with the structure of its mature serine protease, complement factor D, revealed major conformational changes in the similar regions. Comparisons with the zymogen–active enzyme pairs of chymotrypsinogen, trypsinogen and prethrombin‐2 showed a similar distribution of the flexible regions. However, profactor D is the most flexible of the four, and its mature enzyme displays inactive, self‐inhibited active site conformation. Examination of the surface properties of the N‐terminus‐binding pocket indicates that Ile16 may play the initial positioning role for the N‐terminus, and Leu17 probably also helps in inducing the required conformational changes. This process, perhaps shared by most chymotrypsinogen‐like zymogens, is followed by a factor D‐unique step, the re‐orientation of an external Arg218 to an internal position for salt‐bridging with Asp189, leading to the generation of the self‐inhibited factor D.

Recent results and new hardware developments for protein crystal growth in microgravity

Abstract Protein crystal growth experiments have been performed on 16 space shuttle missions since April 1985. The initial experiments used vapor diffusion crystallization techniques similar to those used in laboratories for earth-based experiments. More recent experiments have assessed temperature-induced crystallization as an alternative method for growing high quality protein crystals in microgravity. Results from both vapor-diffusion and temperature-induced crystallization experiments indicate that protein crystals grown in microgravity may be larger, display more uniform morphologies, and yield diffraction data to significantly higher resolutions than the best crystals of these proteins grown on earth.

Experimental and theoretical analysis of the rate of solvent equilibration in the hanging drop method of protein crystal growth

Abstract In the hanging drop method of protein crystal growth, a water droplet containing protein, buffer and a precipitating agent, such as ammonium sulfate, is suspended from a glass coverslip above a well containing an aqueous solution of the precipitating agent at a concentration double that in the drop. We present a comprehensive theoretical study of the rate of water evaporation in the hanging drop method. We find that in earths gravity the rate controlling step in the evaporation is the rate of diffusion of water vapor across the air space separating the drop from the well. Using ammonium sulfate as the precipitating agent, we have made careful measurements at both 4°C and 25°C of the evaporation times for some 25 μL droplets at various concentrations. These results are in good agreement with our theory. As determined by the theory, the parameters affecting the rate of evaporation include the temperature, the vapor pressure of water, the ionization constant of the salt, the volume of the droplet, the contact angle between the droplet and the coverslip, the number of moles of salt in the droplet, the number of moles of water and salt in the well, the molar volumes of water and salt, the distance from the droplet to the well, and the coefficient of diffusion of water vapor through air. These parameters do not act independently; rather, they combine to form three dimensionless groups upon which the rate of evaporation depends. We evaluate numerically 18 different drop and well arrangements commonly encountered in the laboratory. In all cases considered at 25°C, the number of moles of water in the droplet achieves 95% of its final value in 3—30 h, after which further evaporation is quite slow. Our experiments confirm this. We consider qualitatively the effect of weightlessness (spaceflight) on the rate of evaporation and find it to be most likely controlled by the rate of the interdiffusion of salt and water in the droplet.

Protein crystal growth in microgravity review of large scale temperature induction method : bovine insulin, human insulin and human alpha interferon

Abstract The protein crystal growth facility (PCF) is space-flight hardware that accommodates large scale protein crystal growth experiments using temperature change as the inductive step. Recent modifications include specialized instrumentation for monitoring crystal nucleation with laser light scattering. This paper reviews results from the PCFs first seven flights on the Space Shuttle, the last with laser light scattering instrumentation. The PCFs objective is twofold: (1) production of high quality protein crystals for X-ray analysis and subsequent structure based drug design and (2) preparation of a large quantity of relatively contaminant free crystals for use as time-release protein pharmaceuticals. The first three Shuttle flights with bovine insulin constituted the PCFs proof of concept, demonstrating that the space-grown crystals were larger and diffracted to higher resolution than their earth-grown counterparts. The later four PCF missions were used to grow recombinant human insulin crystals for X-ray analysis and to continue productions trials aimed at the development of a processing facility for crystalline recombinant alpha interferon.