Thomas L. Mason’s research while affiliated with University of Massachusetts Amherst and other places

The Var1 protein (Var1p) is an essential, stoichiometric component of the yeast mitochondrial small ribosomal subunit, and it is the only major protein product of the mitochondrial genetic system that is not part of an energy transducing complex of the inner membrane. Interestingly, no mutations have been reported that affect the function of Var1p, presumably because loss of a functional mitochondrial translation system leads to an instability of mtDNA. To study the structure, function and synthesis of Var1p, we have engineered yeast strains for the expression of this protein from a nuclear gene, VAR1U, in which 39 nonstandard mitochondrial codons were converted to the universal code. Immunoblot analysis using an epitope-tagged form of Var1Up showed that the nuclear-encoded protein was expressed and imported into the mitochondria. VAR1U was tested for its ability to complement a mutation in mtDNA, PZ206, which disrupts '3-end processing of the VARI mRNA, causing greatly reduced synthesis of Var1p and a respiratory-deficient phenotype. Respiratory growth was restored in PZ206 mutants by transformation with a centromere plasmid carrying VAR1U under ADH1 promoter control, thus proving that VAR1 function can be relocated from the mitochondrion to the nucleus. Moreover, epitope-tagged Var1Up co-sedimented specifically with small ribosomal subunits in high salt sucrose gradients. The relocation of VAR1 from the mitochondrion to the nucleus provides an excellent system for the molecular genetic analysis of structure-function relationships in the unusual Var1 protein.

The most fundamental goal of the synthetic chemist is control of molecular architecture. With respect to small molecules (i.e., those of molecular weight less than a few thousand), this means absolute control of chemical connectivity and stereochemistry – complete specification of molecular structure. But in macromolecular chemistry, controlled architecture has meant something quite different. Because polymerizations are in general statistical processes, conventional polymeric materials are characterized by substantial heterogeneity in chain length, sequence and stereochemistry. Control is exercised in a statistical sense only, and considerable skill is required to control even the average properties of the chain population and the dispersity in those properties.

We have expressed in E. coli a series of periodic polypeptides represented by sequence 1. Our objective has been an understanding of the role of chemical sequence in determining the chain folding behavior of periodic macromolecules. Molecular organization has been examined by infrared spectroscopy and ^1H and ^(13)C NMR methods and a preliminary model of the folded structure has been developed.

The second half of the 20th century has witnessed a productive interplay between chemistry and materials science—in fields as varied as polymers, catalysis, interface science, ceramics, and electronic materials. Society also has enjoyed the spectacular results-most notably in information and communications technologies—achieved by merging condensed matter physics and materials research. In contrast, connections between materials science and biology have been relatively weak. To be sure, materials research has made important contributions to medicine, but the discipline has yet to draw on biology in the same way that it has absorbed people, ideas, and skills from chemistry and physics. This is beginning to change. With increasing frequency, new materials or processing strategies are emerging, inspired by biological examples or developed directly from biological systems. Already, researchers are engineering bacteria or other organisms to synthesize monomers for polymer production. They are synthesizing and expressing artificial genes to produce proteinlike materials with the mechanical properties of silk, collagen, or other materials containing elastic fibers. They are beginning to use phospholipids as templates for electronic materials; make synthetic phospholipid vesicles that, like proteins, respond to signals; use stereoselective catalyses to produce optically active polymers from racernic starting materials; and develop protein- and lipid-based sensors. Chemists are playing a vital role in these developments, providing synthetic skills as well as insights into the behavior of complex molecular systems.

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This chapter summarizes work from the authors’ laboratories in the area of de novo design of proteins with novel solid state structures and properties. Interest in this problem arises from two sources: first, from the fact that conventional polymeric solids are necessarily composed of complex mixtures of chains, and second, from the remarkable structures and properties exhibited by proteins of natural origin. In considering protein engineering as a synthetic tool, the polymer chemist is presented with striking new opportunities, from making simple polymers of precisely defined architectures to designing complex macromolecules with highly specialized and efficient catalytic, transport, and biological functions.

The experiments reported herein constitute part of an investigation of the relationship between primary sequence and higher order structure in repetitive polypeptides. Three series of artificial DNAs encoding poly-peptides represented by the general formula + (GlyAla)nGlyGlu]m— were constructed and cloned in Escherichia coli.Protein expression was monitored by in-vivo labeling with 3H-glycine. Protein products were isolated from the cell lysates by stepwise acidification in yields of 20–50 mg/L of culture, and fusion fragments derived from the cloning vectors were removed by cyanogen bromide cleavage. Amino acid sequence analysis verified the expected sequences through the first 40–50 N-terminal residues.

The 5′-untranslated leaders of mitochondrial mRNAs appear to localize translation within the organelle. VAR1 is the only yeast mitochondrial gene encoding a major soluble protein. A chimeric mRNA bearing the VAR1 untranslated regions and the coding sequence for pre-Cox2p appears to be translated at the inner membrane surface. We propose that translation of the ribosomal protein Var1p is also likely to occur in close proximity to the inner membrane.

The growing use of recombinant DNA methods for the preparation of artificial proteins has created a pressing need for techniques for the rapid analysis of macromolecular structure. We report herein the use of matrix-assisted laser desorption mass spectrometry to determine the structure and purity of an artificial copolypeptide prepared as part of our ongoing investigation of the crystallization behavior of periodic protein. The mass spectra demonstrate that SDS polyacrylamide gel electrophoresis (SDS-PAGE) is insensitive to the presence of degraded fragments of the artificial protein and that molecular masses estimated by SDS-PAGE may be in error by more than 100%. Furthermore, accurate mass determination by matrix-assisted laser desorption mass spectrometry allowed the discovery of two previously undetected mutations in the DNA code for the protein.

Three approaches to the synthesis of the repetitive copolypeptide [(GlyAla)3-GlyProGlu]n (1) are described. Direct chemical synthesis of 1 via classical solution methods required 18 steps and afforded a polydisperse product with an average molecular weight of less than 10,000. Two alternative genetic strategies were also explored. In the first, chemically synthesized DNA oligomers were self-ligated to produce a population of multimers, which were fitted with translational start and stop signals and inserted into an expression plasmid containing the lambda PL promoter and a synthetic ribosome binding site. Transformation of E. coli led to the isolation of a stable recombinant plasmid carrying an insert encoding 12 repeats of sequence 1. Attempts to identify polypeptide 1 after induction of transformed cultures were unsuccessful. A second strategy, generating a tripartite derivative of sequence 1 carrying short N- and C-terminal extensions, afforded excellent yields of product. The relative merits of chemical and genetic approaches to repetitive polypeptide materials are discussed.