Research sheds new light on protein behavior

Research sheds new light on protein behavior

August 31, 2010

August 31, 2010

 

 

 

 

 

 

 

Proteins play an essential role in virtually every cellular process. They provide actin and myosin for muscles, form the cytoskeleton that maintains a cell’s shape, and carry out innumerable duties critical for immune response, cell signaling and other central functions. A better understanding of protein complexity would be a significant advance in life sciences research, particularly for the study of the mechanisms underlying disease.

The need for speed Enzymes can speed up reactions in a variety of different ways. Dr. Sid Hecht and colleagues are working on a new project to understand in fine detail how enzymes work during normal cell growth and division, and go awry in diseases such as cancer.

Now, Dr. Sidney Hecht and his colleagues are pioneering new techniques to peer into the inner workings of proteins, hoping to learn how they perform their impressive feats.  Hecht is a professor of chemistry and director of the Center for BioEnergetics at the Biodesign Institute at Arizona State University. The group’s efforts may pave the way for more effective therapeutics for a host of major illnesses, among other advances.

The current project focuses on enzymes—a class of proteins that act to accelerate chemical reactions, through the process of catalysis. Hecht and his collaborator, Dr. Stephen J. Benkovic, professor of chemistry at Pennsylvania State University and a leading authority on mechanisms of enzymatic catalysis, are the recipients of a new four year, $1.3 million grant from the NIH. The group’s proposed research involves examining enzyme activity in fine-grained detail, particularly the folding, flexibility and dynamics of protein structure that make effective catalysis possible.

Proteins are formed from a 20-letter alphabet of amino acids, strung together like the beads of a necklace and then twisted into myriad three-dimensional forms. Both the amino acid sequence and the particular shape or conformation of the resulting protein molecule are responsible for protein function.

For living cells, catalysis is essential, due to the very low reaction rates of uncatalyzed reactions. In an enzyme-catalyzed reaction, a chemical substrate temporarily binds with the active site of an enzyme, reducing the energy required to initiate the reaction, and increasing the number of molecules able to react and form the product. The precise relationship between protein dynamics and resulting enzymatic function is not fully understood, though the new research promises to clarify some of this complexity.

One enzyme, in particular, has been the focus of considerable study—dihydrofolate reductase or DHFR. It is a particularly attractive candidate for research, as it is ubiquitous among known organisms. Further, DHFR acts to catalyze an important reaction, producing tetrahydrofolate or THF and helping to maintain pools of THF within cells.  THF is required for the synthesis of purine and thymidylate—chemicals necessary for cell growth and proliferation. For this reason, DHFR has been the target of several important anticancer and antibiotic drugs.

Hecht and Benkovic’s team will examine DHFR by means of fluorescent probes or fluorophores attached to specific sites on the enzyme. Once these probes have been attached to the protein, the enzyme may be studied in detail using a method known as fluorescence spectroscopy. The technique allows for fluorescently labeled enzymes to be observed at the single-molecule level and for ensembles, enabling detailed study of enzyme dynamics. Studying this rate of turnover with the aid of attached fluorophores will permit close scrutiny of the relationship between the processes of protein dynamics and catalysis.

Previously, changes in the protein conformation of DHFR had been studied through a variety of techniques including X-ray crystallography and nuclear magnetic resonance spectroscopy. As Hecht explains, however, such efforts have mostly been applied to studying the static structure of proteins, or else structural changes that occur on a relatively slow time scale. “The beauty of using fluorophores is that they allow us to closely examine the dynamics of protein structure under something approximating physiological conditions.” Although particular subdomains of the enzyme molecule have been implicated as central to the mechanism of catalysis, the team believes that the characteristics of the entire protein must be taken into account for a thorough understanding.

Hecht and Benkovic are exploring new means of introducing fluorescent probes. The development of a system enabling the study of DHFR through single-molecule fluorescence spectroscopy should help reveal features like protein deformation under physiological conditions. Further, the use of multiple fluorescent probes will allow protein dynamics and shape to be observed with enhanced clarity. The team believes that the project will contribute to a fundamental understanding of how enzymes achieve some of the highly accelerated, specific reactions essential for living systems.

 

 

Written by Richard Harth 
Biodesign Institute Science Writer 
richard.harth@asu.edu