Spencer J. Anthony-Cahill

Metastability of native proteins and the phenomenon of amyloid formation.

An experimental determination of the thermodynamic stabilities of a series of amyloid fibrils reveals that this structural form is likely to be the most stable one that protein molecules can adopt even under physiological conditions. This result challenges the conventional assumption that functional forms of proteins correspond to the global minima in their free energy surfaces and suggests that living systems are conformationally as well as chemically metastable.

Expanding the natural repertoire of protein structure and function.

This review considers chemical and genetic approaches to the modification of protein structure. The historical interest in chemical and site-directed modifications will be briefly covered. Current chemical modification strategies will be presented. Biosynthetic mutagenesis with unnatural aminoacyl-tRNAs and current synthetic peptide ligation technologies will be covered in greater detail. The application of combinatorial genetic methods (e.g. phage display, DNA shuffling) to protein engineering with unnatural amino acids will be briefly discussed, with emphasis on the in vitro evolution of new enzymatic function (i.e. aminoacyl-tRNA synthetases). Throughout the review, the powerful insights gained from the combined use of these technologies will be illustrated by examples that focus on the elucidation of protein-ligand interactions.

The morphology of decorated amyloid fibers is controlled by the conformation and position of the displayed protein.

Self-assembled structures capable of mediating electron transfer are an attractive scientific and technological goal. Therefore, systematic variants of SH3-Cytochrome b(562) fusion proteins were designed to make amyloid fibers displaying heme-b(562) electron transfer complexes. TEM and AFM data show that fiber morphology responds systematically to placement of b(562) within the fusion proteins. UV-vis spectroscopy shows that, for the fusion proteins under test, only half the fiber-borne b(562) binds heme with high affinity. Cofactor binding also improves the AFM imaging properties and changes the fiber morphology through changes in cytochrome conformation. Systematic observations and measurements of fiber geometry suggest that longitudinal registry of subfilaments within the fiber, mediated by the interaction and conformation of the displayed proteins and their interaction with surfaces, gives rise to the observed morphologies, including defects and kinks. Of most interest is the role of small molecule modulation of fiber structure and mechanical stability. A minimum complexity model is proposed to capture and explain the fiber morphology in the light of these results. Understanding the complex interplay between these factors will enable a fiber design that supports longitudinal electron transfer.

Using the protein folding literature to teach biophysical chemistry to undergraduates

An understanding of physical chemistry principles enhances student understanding of biochemical phenomena; however, the application of these principles to biological examples is frequently missing in the standard undergraduate physical chemistry curriculum. The topics of protein folding and stability are based in thermodynamics and can serve as a vehicle for presenting essential thermodynamics in a context that is highly relevant to undergraduate biochemistry majors. The outline of a course that replaces the standard thermodynamics offering in physical chemistry is described. The protein folding literature is used to illustrate thermodynamic concepts in this course and students are expected to read and comprehend the assigned literature. The course is offered as a separate biophysical chemistry course for B. S. Biochemistry majors; however, elements of this course may be useful in crafting a more standard thermodynamics course for B. S. Chemistry majors in chemistry departments seeking to fulfill ACS guidelines for approved B. S. Chemistry majors.

Monodisperse 130 kDa and 260 kDa Recombinant Human Hemoglobin Polymers as Scaffolds for Protein Engineering of Hemoglobin-Based Oxygen Carriers

A recombinant 130 kDa dihemoglobin which is made up of a single-chain tetra-α globin and four β globins has been expressed as a soluble protein in E. coli. The sequence of the single chain tetra-α is: αI-Gly-αII-(SerGlyGly)5Ser-αIII-Gly-αIV. This dihemoglobin has been purified and characterized in vitro by size exclusion chromatography, electrospray mass spectroscopy, equilibrium oxygen binding, and analytical ultracentrifugation. The observed values of P50 and nmax for the dihemoglobin are slightly lower than those observed for the recombinant hemoglobin rHb1.1 (a “monohemoglobin” comprised of two β globins and an αI-Gly-αII diα-globin chain). Titration of the deoxy form of dihemoglobin with CO shows that all eight heme centers bind ligand. In vivo, dihemoglobin showed increased circulating halflife and a reduced pressor response in conscious rats when compared to rHb1.1. These observations suggest that dihemoglobin is an oxygen carrying molecule with desirable in vivo properties and provides a platform for an isooncotic hemoglobin solution derived solely from a recombinant source. A 260 kDa tetrahemoglobin has also been produced by chemical crosslinking of a dihemoglobin that contains a Lys16Cys mutation in the C-terminal α-globin subunit. Tetrahemoglobin also shows reduced vasoactivity in conscious rats that is comparable to that observed for dihemoglobin.