News and Publications

Molecular Biology

Peter E. Wright, Ph.D., Chairman

he structure of biological macromolecules bythemselves and in assemblies continues to be one of the most compelling areas of research in molecular biology. Determining the three-dimensional structures of proteins and nucleic acids has led to a detailed understanding of the mechanisms by which these biological molecules function. This, in turn, provides important insights into the organism as a whole, disease states, and potential therapies to improve health.

Moreover, the physiological role of these molecules, as observed through molecular genetics, provides important insights into disease states and how these states can be blocked or modified to improve health. Not surprisingly, many of the researchers in the Department of Molecular Biology are interested in the entire range of biology at the molecular level -- from the most detailed structures to the broadest questions of molecular genetics.

A BREAKTHROUGH STRUCTURE

This year, Geoffrey Chang, Ph.D., solved an x-ray crystal structure that provides the first detailed glimpse of a membrane transporter protein. One of the ways that bacteria resist antibiotic drugs is by using membrane transporters, which are large proteins expressed in almost all organisms that sit in the cell membrane and move other molecules in and out. Harmful bacteria and certain cancer cells use these transporters to undermine the potency of antibiotics and chemotherapy drugs by pumping the drugs out before they have a chance to work.

Chang's structure is a breakthrough, opening the door for scientists to design a new class of drugs that patients would take in conjunction with antibiotic or chemotherapeutic agents to keep those drugs in the cells and increase their efficacy. The work is particularly impressive, because membrane protein structures have been notoriously difficult to solve as they do not form good crystals, an important first step in solving a structure.

One of the most compelling medical challenges today is to develop a vaccine that will provide complete prophylactic protection to someone who is exposed to the virus. An important part of such a vaccine will be an effective neutralizing antibody against HIV, which would circulate through the blood, and track down and kill the virus. Normally, the antibodies that the body produces to fight HIV are ineffective because much of the surface of the HIV virus is inaccessible due to a coat of sugar molecules. However, the antibody solved by Wilson has a long finger-like region on its surface that penetrates the surface of the main viral glycoprotein gp120 on the HIV virus. The antibody neutralizes the virus, making it unable to invade cells and demonstrating that the human immune system is capable of raising antibodies that are effective against HIV.

Wilson also recently received a structural genomics pilot grant from the National Institutes of Health to establish a large-scale, multi-institutional consortium for research in this new discipline. The grant will enable Wilson and others to develop new technologies for high-throughput structure determination and should accelerate therapeutic drug design based on the knowledge reaped from the human genome.

Another major new initiative is being funded by the National Institute of General Medical Sciences and led by James Paulson, Ph.D. The NIGMS awarded the grant to Paulson to establish large-scale, multi-institutional consortia for research in the field of functional glycomics, the scientific pursuit of identifying and studying all of the carbohydrate molecules produced by an organism.

Functional glycomics aims to untangle huge biomedical problems like teasing apart the roles carbohydrates and proteins play in cellular communication. Some carbohydrates carry zip code-like addresses to help cells know where to go in the body, but the precise interactions between carbohydrates and proteins continue to mystify scientists, mainly because carbohydrates have proven to be extremely difficult to study. Unlike proteins, which are produced for the most part from a single template -- an individual gene -- carbohydrates are made by a cascade of chemical reactions inside our bodies. Many of these reactions are difficult to replicate in the lab. Paulson's Consortium for Functional Glycomics promises to change the face of this field by bringing together a large group of scientists from leading academic medical centers across the country.

A NEW INSTRUMENT AND A NOVEL AMINO ACID

The department also is a leader in nuclear magnetic resonance spectroscopy, a technique that enables scientists to determine the structure-function relationships of molecules. This summer, the most powerful, high-resolution nuclear magnetic resonance (NMR) spectrometer ever constructed was delivered to TSRI. Referred to by the frequency at which it operates, 900 MHz, this instrument is the centerpiece of the NMR structural studies at TSRI. With ten instruments at or above 500 MHz, the institute now has one of the most impressive NMR facilities in the world.

Paul Schimmel, Ph.D., and his coworkers have developed a novel approach to incorporating unnatural amino acids into proteins. E. coli mutants with a modified enzyme called valyl-t-RNA synthetase were selected experimentally. This enzyme attaches valine to the tRNA, which incorporates it into a protein. Using mutagenesis and screening, they were able to find a valine tRNA synthetase with no proofreading mechanism. Proofreading mechanisms are normally highly specific, allowing fewer that one mistake in 100,000. These mutants are capable of replacing a high proportion of the amino acid valine in cellular proteins with the unnatural amino acid, aminobutyrate, which is not among the 20 amino acids used by nature. The proofreading mutants were so good at missing mistakes that Schimmel and his colleagues report in their paper that 24 percent of all the valines were replaced with aminobutyrates. The method provides a powerful new tool for studying protein function and creates new opportunities for protein engineering because by using it, novel proteins with unusual chemical composition can be evolved and expressed in abundance.

These new proteins can now be purified and studied in isolation, or left in vivo and used as a probe to study cellular functions. Furthermore, the proteins with novel amino acids may prove to have enhanced or emergent properties. Having a bacterial expression system will make them easy to produce on a massive scale.

IMPORTANT PLAYERS IN MOLECULAR GENETICS

Important advances have also been made in the elucidation of the fundamental molecular processes involved in control of eukaryotic cell division. Cyclin E plays a key role in the cell cycle, functioning together with Cdk2 to regulate DNA replication and duplication of the centrosome. Proper timing of cyclin E expression and degradation is crucial; dysregulation of cyclin E levels is associated with genome instability and tumor formation. Steven Reed, Ph.D., and his colleagues have now identified a critical protein, termed hCdc4, which regulates cyclin E turnover by directing it to the cellular degradation machinery. Mutant forms of this protein occur in several tumor cell lines, suggesting that it may function as a tumor suppressor.

Another advance in molecular genetics came this year when Paul Russell, Ph.D., and Clare H. McGowan, Ph.D., identified the "resolvase" enzyme Mus81 from the fission yeast Schizosaccharomyces pombe, and its human analog. This is one of the most important enzymes involved in genetic recombination, and it may be responsible for generating genetic diversity during sexual reproduction.

Genetic recombination occurs in the process of meiosis, when chromosomes from the mother and father become paired. Resolvase is essential for a crucial step in DNA recombination, because it is the molecule that allows two chromosomes to cross over. However, the DNA must at some point be uncrossed, which is the responsibility of resolvase enzymes.

The identification of a human resolvase may have a profound effect on cancer therapy because the enzyme also has an important role in cell replication. When cells are replicating their DNA prior to division, they have mechanisms to sense if the DNA is damaged. When the DNA is damaged, a cell's replication machinery will stop, spontaneously back up and form a Holliday junction. Resolvase recombines DNA strands at Holliday junctions and this allows the replication machinery to bypass the damaged DNA. Cancer cells are often defective in the mechanisms that sense damaged DNA. Russell and McGowan envision that treatment of tumors with chemotherapeutics that damage DNA, combined with rational targeting of resolvase activity, could be a highly potent cancer treatment.