Rod E. Hubbard

Phytochemistry: Heat-stable antifreeze protein from grass

We have discovered an antifreeze protein in an overwintering perennial ryegrass, Lolium perenne. The protein is stable at 100 °C and although it is a less effective antifreeze than proteins found in antarctic fish and insects, it is better at preventing ice recrystallization. This property enables grasses to tolerate ice formation in their tissues without being damaged, suggesting that the control of ice-crystal growth rather than the prevention of freezing may have evolved to be the critical factor in their survival at very low temperatures.

High resolution crystal structures and comparisons of T-state deoxyhaemoglobin and two liganded T-state haemoglobins: T(alpha-oxy)haemoglobin and T(met)haemoglobin.

The origin of co-operativity in haemoglobin (Hb) resides in the reduced affinity of the T-state. T-state Hb crystals grown from polyethyleneglycol can be liganded without the molecule switching to the R high affinity state. X-ray analysis of T-state alpha-oxy Hb and T-state met Hb has identified the structural basis for reduced affinity. The nature of the chemical tension at the haem environment is different in the alpha and beta haems. There are small but definite structural changes associated with ligation in the T-state: these prove to be mostly in the same direction as the larger changes that occur in the T-->R transition.

The design, synthesis, and evaluation of novel conformationally rigid analogues of sialyl Lewisx

Abstract The design and synthesis of a series of analogues of sialyl Lewisx (1) which incorporate conformationally rigid tetralin and naphthalene ring systems (2–4) has led to novel compounds which have similar potency to 1 as inhibitors of cell adhesion.

THE SYNTHESIS OF NOVEL STRUCTURAL ANALOGUES OF SIALYL LEWISX

Abstract The synthesis of conformationally rigid tetralin-based mimics of the tetrasaccharide sialyl Lewisx are described. Palladium catalysed coupling reactions are employed in the key synthetic steps.

Solid-phase binding assay to study interaction of chemokines with glycosaminoglycans.

Publisher Summary This chapter develops a solid-phase binding assay for chemokine-glycosaminoglycan interactions. Optimization and validation studies were performed to recognize and avoid significant ligand depletion, retain a significant signal-to-noise ratio, explore the effects of buffer composition, and examine nonspecific binding. Results show that variations in immobilized heparin concentration and ionic strength will affect the IC values in competition experiments. The results from this type of assay are relative, rather than absolute measurements of the affinity between chemokines and immobilized heparin. Considering this limitation, the assay is suitable for making comparisons between the relative potencies of different glycosaminoglycans or other small molecule competitors, and it may be useful in identifying molecules that can selectively inhibit the interactions between chemokines and glycosaminoglycans. Molecules identified by this assay could be further tested in other systems to characterize their suitability as anti-inflammatory molecules.

The stability of the lattice structure in low salt T-state haemoglobin crystals

The reconstruction of the electron density of molecules in crystals from X-ray diffraction measurements depends on the exactness of the packing of the molecules in the unit cell and the crystal lattice. Crystals of T-state haemoglobin, the low affinity form of the molecule, grow from high salt solutions or from low salt solutions in the presence of polyethylene glycol. The low salt lattice has the special property that it allows the haemoglobin molecule to bind oxygen and other ligands without the crystal breaking up. The stability of the low salt T-state crystals appears to arise from a small number of well-defined salt bridges and hydrogen bonds that are concentrated in specific lattice directions. These together form a framework within which the molecule can make adjustments which are sufficient to accommodate ligand binding but in which the larger quaternary movements normally associated with oxygenation are prevented. In these crystals therefore interactions with ligands can be studied directly by X-ray diffraction and the structural basis of the T-state’s low affinity for oxygen can be analysed.

The Water Structure in 2Zn Insulin Crystals

Proteins have evolved in an aqueous medium and they have exploited with advantage the bonding and steric properties of the water molecule in achieving their 3 dimensional folding, stability, control of substrate bonding and catalytic reactions. The study of water protein interactions has relied on spectroscopic and calorific methods which generally confirm the essential role water plays in protein structure and folding. The detailed description of the water-protein contacts and the dynamical behaviour of the protein surface atoms and the surrounding water molecules however is a much more challenging undertaking. It must be emphasized that the water molecules on protein surfaces are extremely mobile. They exchange sites very rapidly, even when well ordered by contacts to the protein, and move extensively along the connected networks across the protein. Where there are few specific sites with suitable H bond and other contacts the water molecules exist in a practically continuous population of networks (J Finney, et al., 1982).

Design, Synthesis and Testing of Novel Inhibitors of Cell Adhesion

The Selectin family of proteins comprises three carbohydrate binding proteins (E, P and L) involved in cell adhesion events1. In response to inflammatory stimuli, these proteins play a crucial role in the recognition of sialyl Lewis X (sLeX) and related carbohydrates present on the surface of neutrophils. Following recognition and binding the white blood cells are free to migrate to the sites of injury and infection2. In pathogenic states this sequence of events can lead to pain and inflammation. Blocking the binding of sLeX could potentially be of benefit in the treatment of inflammatory and autoimmune disorders such as, rheumatoid arthritis, asthma, psoriasis, IBD etc.

Heat-stable antifreeze protein from grass: Phytochemistry

We have discovered an antifreeze protein in an overwintering perennial ryegrass, Lolium perenne. The protein is stable at 100 °C and although it is a less effective antifreeze than proteins found in antarctic fish and insects, it is better at preventing ice recrystallization. This property enables grasses to tolerate ice formation in their tissues without being damaged, suggesting that the control of ice-crystal growth rather than the prevention of freezing may have evolved to be the critical factor in their survival at very low temperatures.