P. S. Stayton

pH-sensitive hemolysis by random copolymers of alkyl acrylates and acrylic acid

We have been designing and synthesizing synthetic polymers that mimic viral fusogenic peptides, which contain peptide residues having alkyl groups and carboxyl groups. We have synthesized two different types of such polymers, and their abilities to hemolyse red blood cells at pH 7.4 and 5.5 are compared here. The polymers are poly(2-alkylacrylic acid)s such as poly(2-propylacrylic acid), and random copolymers of poly(alkyl acrylate-co-acrylic acid) where the alkyl group is propyl or butyl. We have found that the poly(2-alkylacrylic acid)s such as poly(2-propylacrylic acid) are significantly more hemolytic at acidic pH than the random copolymers of equivalent propyl and carboxyl contents.

Structural characterization and comparison of RGD cell-adhesion recognition sites engineered into streptavidin

The RGD (arginine-glycine-aspartic acid) sequence is found in several important extracellular matrix proteins and serves as an adhesion ligand for members of the integrin family of cell-surface receptors. This sequence and flanking residues from fibronectin or osteopontin have been engineered into an accessible surface loop of streptavidin to create two new streptavidin variants (FN-SA or OPN-SA, respectively) that bind cells through the alpha(v)beta(3) and/or alpha(5)beta(1) integrin receptors. Their crystal structures confirm the design and construction of the mutants and provide evidence about the conformational dynamics of the RGD loops. The loops in the isomorphous crystal structures are involved in crystal-packing interactions and this stabilizes their structures. Even so, the loop in OPN-SA is slightly disordered and two of the residues are not seen in difference electron-density maps. Comparison with other experimentally determined structures of RGD loops in cell-adhesion molecules shows that these loops occupy a large subset of conformational space. This is consistent with the view that RGD loops, at least those involved in cell adhesion, sample a number of structures dynamically, a few of which display high affinity for appropriate receptors.

Quantitative interrogation of micropatterned biomolecules by surface force microscopy

Synthetic biomaterials are widely used in medical implants with success in improving and extending quality of life. However, these materials were not originally designed to interact with cells through specific signaling pathways. As a result, the interaction with the body is mediated through passive adsorption of a disorganized protein monolayer. Next generation biomaterials have been proposed to be active in modifying the biological response of the host through the incorporation of specific biorecognition moieties. An important tool in the development of these novel active biomaterials is the scanning force microscope (SFM). The SFM allows for interrogation of bioactive biomaterials in mapping or spectroscopic modes. In this work, micropatterned protein surfaces were prepared using biomolecules implicated in wound healing. The surfaces were imaged via SFM and the specific binding forces between surface associated biomolecules and antibody functionalized tips were quantified.

Atomic resolution structure of biotin-free Tyr43Phe streptavidin: what is in the binding site?

The streptavidin-biotin system is an example of a high-affinity protein-ligand pair (Ka approximately 10(13) mol-1). The thermodynamic and structural properties have been extensively studied as a model system for protein-ligand interactions. Here, the X-ray crystal structure of a streptavidin mutant of a residue hydrogen bonding to biotin [Tyr43Phe (Y43F)] is reported at atomic resolution (1.14 A). The biotin-free structure was refined with anisotropic displacement parameters (SHELXL97 program package). The high-resolution data also allowed interpretation of side-chain and residue disorder in 41 residues where alternate conformations were refined. The Y43F mutation is unambiguously observed in difference maps, although only a single O atom per monomer is altered. The atomic resolution enabled the identification of 2-methyl-2, 4-pentanediol (MPD) molecules in the biotin-binding pocket for the first time. Electron density for MPD was observed in all four subunit binding sites of the tetrameric protein. This was not possible with data at lower resolution (1.8-2.3 A) for wild-type streptavidin or mutants in the same crystal form using MPD in the crystallization. The impact of MPD binding on these studies is discussed.

Some personal reflections on the career of Allan Hoffman in honor of his 70th birthday

These special issues were organized by the three of us in honor of Allan Hoffmans 70th birthday, as explained in the Foreword. In the Preface, Buddy Ratner has given a concise and broad overview of the public aspects of Allan Hoffmans career that help to explain the overall impact of his work. As Buddy has described, these contributions can be associated with his creativity and quality research, effectively communicated through his many publications and presentations around the world. However, as we learn in the progression through our careers, personal interactions with our more experienced colleagues often have profound impacts on us. These less formal, seldom described interactions are important contributions to the development of young investigators, and this was certainly true for the three of us under the tutelage of Allan Hoffman. So we have decided to further honor Allan for these contributions by describing a few of our personal highlights from our long association with him. Each of us has taken the opportunity to reflect a bit on what Allan has meant to us as a colleague and a scientist, and also about those notable characteristics we see in Allan that perhaps only those working closely with him for a long time really notice.

Effect of tone burst ultrasound in combination with PPAA (poly‐propyl acrylic acid) on hemolysis of erythrocytes

In this study, high intensity focused ultrasound (HIFU) and PPAA (poly‐propyl‐acrylic acid) were combined at sublethal doses to achieve hemolysis of human erythrocytes. In a series of experiments, a suspension of erythrocytes (1A passive cavitation detection system) was utilized to monitor the acoustic emissions from the cell suspension during exposure to ultrasound. In the presence of PPAA, there is a significant increase in cavitation and hemolysis during ultrasound exposure at a pH of 6.1, but not at a pH of 7.4. PPAA is a pH‐sensitive polymer, changing its conformation and its activity as the solution becomes more acidic, achieving its optimal active state at a pH of 6.1. The acoustic data and the activity of PPAA suggest that the polymer, in its active state, provides nucleation sites to the cell suspension and that cell/bubble interaction is the primary mechanism for cell damage.