Hunsicker-Wang Lab

Biochemistry & Bioinorganic Chemistry

Professor: Laura Hunsicker-Wang, Ph.D.

Overview: Research in the Hunsicker-Wang laboratory will focus on studying enzymes that utilize or bind metal ions, called metalloproteins. There are two major areas of interest:

Iron-sulfur proteins make up ~30% of all metalloproteins. These proteins utilize iron and sulfur atoms that are organized into clusters. These proteins are often involved in electron transfer reactions. Specifically, the Rieske protein, which is part of Complex III in the respiratory chain, contains a [2Fe-2S] cluster, which is ligated to the protein via 2 cysteine and 2 histidine residues. The reduction potential of this protein depends on the organism and the type of system that it was derived from. Previous studies have shown that the number of hydrogen bonds to the cluster, the solvent accessibility, and the type of charge residues near the cluster all affect the reduction potential. Research on this protein involves making site-specific mutations, purifying, crystallizing and solving the structure of the mutant enzymes. The reduction potentials of these mutants are also evaluated. This protein is also be chemically modified with reagents that alter the properties of specific amino acids. This approach allows a greater variety of chemical properties to explore. Chemical modification also allows study of how individual amino acids contribute to the electron transport function within the protein. Reactive oxygen species (ROS) are destructive and form from the reduction of molecular oxygen.

One hypothesis is that a mismatch in the potential of the Rieske protein with its partners within Complex III leads to the production of ROS and may lead to neurodegenerative diseases. This hypothesis is starting to be explored in the Hunsicker-Wang lab.

Cytochrome oxidase is complex IV in the respiratory system. Within this protein, there are 4 metal sites, 2 heme-iron sites and 2 copper binding sites. One of the copper sites is the CuA center, found in subunit II of cytochrome oxidase. This center may also be involved in H+ translocation within cytochrome oxiase. Subunit II can be expressed as an isolated protein. The Hunsicker-Wang lab is exploring how the histidines in this protein may function to pump H+ using chemical modification and site-directed mutagenesis. Additionally, the lab is also studying how the copper ions are incorporated into CuA when cytochrome oxidase is assembled. The protein, Sco, is thought to be involved in the process but its exact role is not well-defined. Thus, studies are aimed at understanding what role(s) that Sco plays in the assembly of the CuA site.


Maeder Lab

Biochemistry & Molecular Biology

Professor: Corina Maeder, Ph.D.

Overview: Research in the Maeder Lab centers on understanding the mechanisms involved in gene expression, specifically that of pre-messenger RNA splicing. In eukaryotes, initially, RNA transcribed from DNA may have intervening non-protein coding sequences, or introns. These sequences must be removed for accurate protein translation. The removal of these introns must be precisely coordinated to avoid inaccuracies that can result in many diseases, including cancer and retinitis pigmentosa. This process is known as premessenger RNA splicing.

Research focus: A large macromolecular complex of RNA and proteins called the spliceosome facilitates splicing. The mechanism of pre-mRNA splicing involves large-scale rearrangements of protein-RNA complexes, which must be regulated to ensure both splicing timing and accuracy. Our research focuses on understanding these largescale rearrangements within the spliceosome. The spliceosome is composed of five small nuclear ribonucleoprotein complexes (snRNPs). Dynamic rearrangements occur both within and between the snRNPs during the splicing cycle. These rearrangements are indicators that splicing is proceeding accurately. Our research aims to dissect the molecular interactions that stimulate spliceosome assembly and activation. Specifically, we are currently focused on the role of the Dib1 protein and its interactions in regulating these rearrangements. Dib1 is essential for cell viability and splicing and is conserved from yeast to humans. Dib1 (Figure 1) is a small protein with a thioredoxin-like fold and little is known about its function. Using site-directed mutagenesis and yeast growth assays, we have identified amino acids that are important for splicing. We are now trying to characterize the protein using biochemical and molecular biology techniques in order to understand the importance of these amino acids in splicing. For example, a change to an amino acid in the core of protein may cause the protein to be less stable overall, while a change to a amino acid in an external loop may be important for interactions with binding partners. Our works aims to determine the function of Dib1 through this characterization and whether it might be involved in the regulation of spliceosome assembly directly or indirectly.

Outcomes: Our studies on the spliceosome are quite interdisciplinary. In our lab, we use a variety of biochemical, molecular biological, and genetics techniques to dissect the importance of protein-nucleic acid and protein-protein interactions in the spliceosome. Students have opportunity to perform:

  1. Biochemistry assays, including gel based kinetic unwinding assays, binding assays for protein-RNA and protein-protein interactions, and structural studies using circular dichroism
  2. Bolecular biology, including protein purifications, DNA cloning, and RNA transcription and purification
  3. Genetic assays using Saccharomyces cerevisiae (Baker’s yeast) including gene deletion, tetrad analysis and growth assays. In addition, we have a collaborative project with Dr. Kelvin Cheng in Physics in which students perform computational modeling studies on the spliceosome. These theoretical studies inform and parallel our experimental studies.