Malcolm Capel

Structural genomics: beyond the Human Genome Project

With access to whole genome sequences for various organisms and imminent completion of the Human Genome Project, the entire process of discovery in molecular and cellular biology is poised to change. Massively parallel measurement strategies promise to revolutionize how we study and ultimately understand the complex biochemical circuitry responsible for controlling normal development, physiologic homeostasis and disease processes. This information explosion is also providing the foundation for an important new initiative in structural biology. We are about to embark on a program of high-throughput X-ray crystallography aimed at developing a comprehensive mechanistic understanding of normal and abnormal human and microbial physiology at the molecular level. We present the rationale for creation of a structural genomics initiative, recount the efforts of ongoing structural genomics pilot studies, and detail the lofty goals, technical challenges and pitfalls facing structural biologists.

Structure of a bacterial 30S ribosomal subunit at 5.5 Å resolution

The 30S ribosomal subunit binds messenger RNA and the anticodon stem-loop of transfer RNA during protein synthesis. A crystallographic analysis of the structure of the subunit from the bacterium Thermus thermophilus is presented. At a resolution of 5.5 Å, the phosphate backbone of the ribosomal RNA is visible, as are the α-helices of the ribosomal proteins, enabling double-helical regions of RNA to be identified throughout the subunit, all seven of the small-subunit proteins of known crystal structure to be positioned in the electron density map, and the fold of the entire central domain of the small-subunit ribosomal RNA to be determined.

Thermal annealing of roll-cast triblock copolymer films

Abstract Polystyrene—polybutadiene—polystyrene triblock copolymers were roll-cast from toluene solutions to form globally oriented films. Microstructural changes following thermal annealing of films with cylindrical and lamellar morphology were monitored using two-dimensional small angle X-ray scattering, transmission electron microscopy and thermomechanical analysis. The microdomains in the unannealed films of cylindrical morphology were found to be assembled on a distorted hexagonal lattice, due to the roll-casting flow field. Thermal annealing significantly improved the alignment and packing of the cylinders, increased grain size, reduced the number of morphological defects and resulted in a 12% decrease in the area per junction. The microstructure of the unannealed films of lamellar morphology was observed to be composed of many small grains with low-angle helicoid surface twist boundaries. Annealing significantly reduced the number of grains and twist boundaries and resulted in a 7% decrease in the area per junction. Molecular models are presented for the relaxation of the chains during the annealing process in both cylindrical and lamellar morphologies based upon 2-D SAXS data and thermomechanical analysis.

Solvent swelling of roll-cast triblock copolymer films

Abstract Polystyrene-polybutadiene-polystyrene triblock copolymers were roll-cast from toluene solutions to form globally oriented films. As-processed films, containing process-related residual stresses, were exposed to solvent vapours. Three solvents were used in this study: toluene, which is a non-preferential solvent for polystyrene and polybutadiene; methyl-ethyl-ketone, which is a preferential solvent for the polystyrene blocks; and hexane, which is a preferential solvent for the polybutadiene block. Microstructural changes accompanying the solvent swelling of films with cylindrical and lamellar morphology were monitored using two-dimensional small angle X-ray scattering. Solvent swelling significantly improved the symmetry of the hexagonal packing of the cylindrical domains, which was initially distorted due to the roll-casting flow field. Solvent swelling was also found to improve the long range order in roll-cast film with lamellar morphology. Various phenomena were found to accompany the swelling and deswelling of films with both cylindrical and lamellar morphology with the three different solvents used. Especially intriguing results were observed for the case of swelling both morphologies in hexane. For films with a lamellar morphology, after 1 h of swelling and 2 h of subsequent deswelling the d-spacing decreased by 18%. For films with cylindrical morphology, a similar decrease of 9% was observed. Molecular models are presented to explain these microstructural changes, that are closely linked to the mobility of the glassy polystyrene blocks, the relaxation of process-related stress and the ability of the polybutadiene-polystyrene junctions at the interfaces to reposition and accommodate volume changes.

Thermal stability and thermal expansion studies of PEEK and related polyimides

Abstract Thermal stability and thermal expansion properties are reported for several high performance polymers which contain carbonyl and/or ether linking groups designed to increase molecular mobility while retaining high temperature properties. The polymers studied include poly(ether ether ketone), PEEK, and three related novel thermoplastic polyimides: NEW-TPI, LARC-CPI, and LARC-IA. Thermogravimetric analysis showed that LARC-CPI exhibited major weight loss by about 450°C, and both PEEK and LARC-CPI lost 50% of their initial weight by 750°C. Thermomechanical analysis (t.m.a.) was used to determine linear coefficients of thermal expansion (CTE). All materials showed a large change in CTE at the glass transition temperature (T g ). Bulk linear CTEs ranged from 54–70 × 10 −6 °C −1 below T g , to 105–200 × 10 −6 °C −1 above T g . These CTEs are larger than those typically observed for more rigid chain polyimides. Using a two-phase model, the amorphous phase CTE was deduced for the semicrystalline materials. From t.m.a. data, amorphous phase CTEs above T g ranged from 120–245 × 10 −6 °C −1 . Small angle X-ray scattering (SAXS) was used to examine changes in long period with temperature for PEEK and NEW-TPI. Below T g , the long period did not change much while above T g , larger increases in the long period were observed. These increases in the long period with increasing temperature may be the result either of large amorphous phase expansion, or possibly of melting of thin lamellae.

X-ray scattering and thermal analysis study of the effects of molecular weight on phase structure in blends of poly(butylene terephthalate) with polycarbonate

Blends of Poly(butylene terephthalate), PBT, with Polycarbonate, PC, were studied for a range of molecular weights and blend compositions. Blends were available in PBT/PC compositions 80/20 and 40/60, and with Mw designated by H (high) or L (low). Samples were prepared by melt crystallization, or by cold crystallization following a rapid quench from the melt. Addition of PC reduces the crystallization kinetics of PBT so that the resulting crystals are more perfect than those which form in the homopolymer. Degree of crystallinity of the blends followed the rank ordering: L/L > L/H > H/L = H/H. The glass transition behavior was investigated using dynamic mechanical analysis (DMA) and modulated differential scanning calorimetry (MDSC). All blends exhibited two glass transitions at intermediate temperatures between the Tgs of the homopolymers, indicating existence of a PBT-rich phase and a PC-rich phase. Blends L/L were most, and H/H the least, miscible. Small-angle X-ray scattering was performed at room temperature on cold crystallized blends, or at elevated temperature during melt crystallization. The long period was consistently larger, and the linear stack crystallinity was consistently smaller, in blends L/L or H/L. These results indicate that in blends containing low Mw PC, there is more PC located within the PBT-rich phase. The long period was consistently smaller in cold crystallized samples, while the linear stack crystallinity was nearly the same, regardless of melt or cold crystallization treatment. Reduction of the average long period in cold crystallized samples could result from crystallization of PBT within the PC-rich phase. This is consistent with thermal analysis results, which indicate that cold crystallized samples have greater overall crystallinity than melt crystallized samples. A hypothetical liquid phase diagram is presented to explain the differences between melt and cold crystallized blends.

Preparative ion-exchange high-performance liquid chromatography of bacterial ribosomal proteins

We have developed analytical and preparative ion-exchange HPLC methods for the separation of bacterial ribosomal proteins. Proteins separated by the TSK SP-5-PW column were identified with reverse-phase HPLC and gel electrophoresis. The 21 proteins of the small ribosomal subunit were resolved into 18 peaks, and the 32 large ribosomal subunit proteins produced 25 distinct peaks. All peaks containing more than one protein were resolved using reverse-phase HPLC. Peak volumes were typically a few milliliters. Separation times were 90 min for analytical and 5 h for preparative columns. Preparative-scale sample loads ranged from 100 to 400 mg. Overall recovery efficiency for 30S and 50S subunit proteins was approximately 100%. 30S ribosomal subunit proteins purified by this method were shown to be fully capable of participating in vitro reassembly to form intact, active ribosomal subunits.

Macular corneal dystrophy type II: Multiple studies on a cornea with low levels of sulphated keratan sulphate

We investigated an individual macular corneal dystrophy (MCD) type II cornea from a 42-year-old woman with markedly reduced antigenic keratan sulphate levels. A characteristic 4.6 Å X-ray reflection was evident, and the mid-stroma contained 30% less sulphur than normal. Close packing of collagen was restricted to the superficial stroma. Abnormally large proteoglycan filaments were noted throughout the extracellular matrix and Descemets membranes posterior non-banded zone, but not its anterior banded zone. Small, collagen-associated stromal proteoglycans were susceptible to digestion with chondroitinase ABC, but not keratanase I or N-glycanase. On occasion, collagen fibrils ranged in size from 20 nm to 58 nm, with preferential diameters of 34 nm and 42 nm. Corneal guttae were evident, as were numerous endothelial inclusions, most probably due to intracellular fibrillogranular vacuoles similar to those found in the stroma. The endothelium expressed reduced anti-keratan sulphate labelling.

Oriented double gyroid films via roll casting

Films of an isoprene-rich poly (styrene-b-isoprene-b-styrene) (SIS) triblock copolymer thermoplastic elastomer having a highly textured double gyroid (DG) morphology were produced via roll casting and annealing. The symmetries of the extensional and shear flows involved in the roll casting process are not commensurate with those of the cubic Ia 3d symmetry of the tricontinuous, triply periodic cubic DG phase. Upon roll casting the microdomain morphology produced is predominantly that of cylinders, oriented with the axes along the roll cast direction. Upon annealing, the DG phase nucleates and grows with the [111] direction oriented along the roll cast direction. The resultant textured films are suitable for investigating the directional dependence of physical properties. q 2000 Elsevier Science Ltd. All rights reserved.

A synchrotron x-ray diffraction study of developing chick corneas.

To study some ultrastructural aspects of developing chick corneas we performed a synchrotron x-ray diffraction analysis of 22 specimens obtained daily from developmental day 10 through day 19. Before day 12 of development in chicks we were unable to detect a meridional x-ray diffraction pattern from cornea. Neither were we able to record a first-order equatorial x-ray reflection at this time. Normally, these reflections are present in corneal x-ray patterns, arising from, respectively, the periodic axial electron density of fibrillar collagen and the lattice-like arrangement of the fibrils. By day 12 of development we could detect the third- and fifth-order meridional reflections (indicating increased amounts of collagen) and a first-order equatorial reflection (implying that more collagen was regularly arranged). The third- and fifth-order meridional reflections became more intense as the tissue matured, suggestive of a continued deposition of fibrillar collagen, and the scattering angle of the interfibrillar maximum increased, suggesting that regularly arranged collagen was becoming more closely packed with maturation. In embryonic chick corneas, the establishment of an orderly, fairly compacted matrix of collagen fibrils may be one of the main events underlying the acquisition of corneal transparency.