Keith B. Ward

Automatic preparation of protein crystals using laboratory robotics and automated visual inspection

Abstract A significant concern in the field of protein crystallography remains the routine preparation of single crystals suitable for x-ray diffraction studies. Crystallization techniques developed within the last three decades typically require accurate manipulation of microliter quantities of protein, buffer, and precipitating solutions. The accuracy and reproducibility necessary for such processes, together with the repetitiveness of the tasks, make several of the crystallization methods particularly appropriate for automation techniques. We describe here our efforts to automate the preparation of protein crystals by the sandwiched drop method using commercially available laboratory robotics equipment. The system incorporates a video monitoring station which permits automated inspection of individual crystallization droplets. Results obtained from the automatic monitoring of crystallization experiments will be utilized to redesign and prepare new experiments which will then be carried out by the robotics system, thereby allowing a more fully automated system for crystallization.

Methods of analysis of protein crystal images

Abstract Several protein crystallization techniques, including the vapor diffusion method, lend themselves well to automation techniques. Up until the present time, automation techniques have been restricted to setting up crystallization experiments, and procedures to monitor and analyze the experiments have not been developed. These procedures require additional hardware for video monitoring of crystallization chambers and automatic recognition of protein crystals. An automated image acquisition and analysis system makes use of both image processing routines and pattern recognition procedures. In order to design and implement such a system, we are presently developing algorithms which can recognize and locate protein crystals in video images of crystallization droplets. Images of crystallization experiments are acquired and digitized, and analyses of the droplet images are conducted on the microcomputer which also acts as a host in our laboratory robotics system. We describe here our current progress in designing the image analysis system, including the development of appropriate pattern recognition methods. In addition, the usefulness of various pattern recognition schemes for monitoring the progress of crystallization is explored.

Atomic models for the polypeptide backbones of myohemerythrin and hemerythrin

Abstract A tentative atomic model, including the polypeptide backbone and side chains of residues in the active center, has been constructed for myohemerythrin. The model was built to fit a low resolution electron density map for this molecule while meeting several conditions for stereochemical reasonableness. An adaptation of this model serves also to explain an electron density map of octameric hemerythrin.

Crystal structure analysis of recombinant human uteroglobin and molecular modeling of ligand binding.

Abstract: Uteroglobin, a steroid‐inducible, cytokine‐like, secreted protein with immunomodulatory properties, has been reported to bind progesterone, polychlorinated biphenyls (PCB), and retinol. Structural studies may delineate whether binding of ligands is a likely physiological function of human uteroglobin (hUG). We report a refined crystal structure of uncomplexed recombinant hUG (rhUG) at 2.5‐Å resolution and the results of our molecular modeling studies of ligand binding to the central hydrophobic cavity of rhUG. The crystal structure of rhUG is very similar to that of reported crystal structures of uteroglobins. Using molecular modeling techniques, the three ligands‐PCB, progesterone, and retinol‐were docked into the hydrophobic cavity of the dimer structure of rhUG. We undocked the progesterone ligand by pulling the ligand from the cavity into the solvent. From our modeling and undocking studies of progesterone, it is clear that these types of hydrophobic ligands could slip into the cavity between helix‐3 and helix‐3′ of the dimer instead of between helix‐1 and helix‐4 of the monomer, as proposed earlier. Our results suggest that at least one of the physiological functions of UG is to bind to hydrophobic ligands, such as progesterone and retinol.

Dynamic telerobotic control of crystallization experiments

Abstract A dynamically controlled system has been used to prepare crystals of lysozyme. The temperature of the crystallization chamber was adjusted based upon a scintillation signal used to detect the degree of nucleation and incipient crystal growth. Experiments concluded in one country were controlled and monitored by researchers in another, providing the first demonstration of telerobotic control of a protein crystallization experiment.

A partial primary structure of squid hepatopancreas organophosphorus acid anhydrolase

Earlier studies of OPA anhydrolase from the squid, Loligo pealei, report that the enzyme has a molecular weight near 26 kDa, despite the common observation that SDS-PAGE experiments do not support this conclusion. Recent results from protein sequencing and cloning experiments now suggest that the enzyme found in squid hepatopancreas has a molecular weight of about 42 kDa. The enzyme easily degrades into two fragments of 16 kDa and approximately 26 kDa. N-terminal sequence analyses of the intact enzyme and the 16 kDa fragment blotted from an SDS gel and sequenced from the blot have shown conclusively that the intact 42 kDa protein has a blocked N-terminus. Sequence data obtained previously are from the N-terminal portion of the 16 kDa fragment. Additional support for this interpretation has been obtained from PCR analysis of L. pealei mRNA and cDNA. The partial (30 residue) sequence presented here reveals no indication of similarity to any other OPA anhydrolase or aryldialkylphosphatase (EC 3.1.8.1.).

Comparative Anatomy of Phospholipase A2 Structures

For a number of years our laboratory has been interested in phospholipases A2, especially those that are neurotoxic. Within the last year or two we have begun to direct our interest to nontoxic phospholipases that are of importance in human health. The goal of our presentation is to set the stage for you, and to introduce you to many of the terms and concepts that will be given in other presentations of this symposium. Many of these will deal with enzyme mechanisms, enzyme regulation, enzyme activation and so forth, and will be given by researchers actively involved in these areas. A full appreciation of their presentations will be facilitated by an understanding of the detailed structural anatomy of phospholipases A2.

Comparison of organophosphorous acid anhydrolases from different species using monoclonal antibodies

1. Monoclonal antibodies were raised against squid hepatopancreas organophosphorous acid (OPA) anhydrolase (EC 3.1.8.2) and were used to study structural similarities with OPA anhydrolases isolated from different sources. 2. Common epitopes were identified in OPA anhydrolases with diverse origins, and with different substrate specificities. 3. Epitopes unique to the squid hepatopancreas OPA anhydrolase were identified; optic ganglion and hepatopancreas contain different enzymes which can be distinguished by their epitopes.

A PC-based spreadsheet for tracking results of crystallization experiments

Abstract Crystallization of proteins often involves many individual experiments entailing a great deal of recordkeeping. A method is used in our laboratory to track crystallization experiments with a commercially available spreadsheet program and an 80286 microprocessor-based personal computer (PC). The system takes advantage of a windowing environment. Results are entered directly into the spreadsheet as experiments are examined. The data are stored and later analyzed. They are sorted by pertinent fields and analyzed for significant trends. New experiments are then designed based upon these results. We have prepared crystals of several proteins, including the photoprotein aequorin, from the jellyfish Aequorea , in part by using information obtained from this tracking system.

Video Monitoring And Analysis Subsystem: A CCD-Based Monitoring System For The Protein Crystal Growth Apparatus

Of significant importance to the field of protein crystallography is the preparation of single crystals suitable for x-ray diffraction studies. Recent experiments have been conducted on the Space Shuttle to study in detail the effects of microgravity on protein crystallization. The device used on these Shuttle missions, the Protein Crystal Growth Apparatus (PCGA), utilizes the vapor diffusion crystallization method and has yielded extremely favorable results. We describe here our efforts to modify the PCGA in order to incorporate an automated CCD-based video system, the Video Monitoring and Analysis Subsystem (VMAS) for direct monitoring of crystallization experiments. The video system discussed allows frequent monitoring and observation of crystal growth. In addition, images of crystallization experiments obtained with the VMAS will be processed and analyzed on-board with a dedicated microcomputer to automatically determine the presence and rate of crystal growth.