Chemistry @ Colgate

Roger S. Rowlett

Research Projects Description

Research in my laboratory is focused on the elucidation of the structure and catalytic mechanism of enzymes. A summary of ongoing investigations in our laboratory is summarized below. A protein engineering protocol book in Adobe Acrobat (PDF) format is available describing routine experimental procedures used in our laboratory. A methods book for X-ray crystallography is now also available, also in PDF format.

Carbonic Anhydrase

Research in our laboratory is focused on understanding the mechanism of β-carbonic anhydrase, an enzyme that catalyzes the reaction:

CO2 + H2O = HCO3 + H+

 

Carbonic anhydrase may be unique among enzymes in that it has independently arisen during evolution on at least four separate occasions. The α-enzyme (Figure 2), which is found principally in animals and certain unicellular algae, is the most well known, and is a monomeric protein of 29 kD. It is thought to have arisen 200-300 million years ago. 

Figure 1. Cover of Archives of Biochemistry and Biophysics (May 1, 2004) featuring a figure from the work of Colgate undergraduates in my laboratory.

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Figure 2. Human α-Carbonic Anhydrase II

The γ-enzyme (Figure 3), which was discovered in an archaebacterium in 1994, is a trimeric protein of 69 kD, and has an unusual left-handed β-helix architecture. This carbonic anhydrase is though to have arisen 3.0-4.5 billion years ago. Both of these carbonic anhydrases are zinc-metalloenzymes, and appear to have similar His3-Zn-OH2 coordination spheres. Recently, yet another genetically distinct carbonic anhydrase has been noted in the marine diatom Thalassiosira weisflogii.
The β-enzyme, which is principally found in higher plants and some eubacteria, is also of ancient origins, perhaps as old as the g enzyme. β-carbonic anhydrase is also a zinc-metalloenzyme, but is typically much larger—100 to 200 kDa, corresponding to tetramers or octamers of 23-25 kD subunits—than the α- or g-enzymes. However, the coordination sphere of the zinc ion in b-carbonic anhydrase appears to be significantly different from that of the α-and γ-enzymes. A high-resolution X-ray diffraction structure has recently be reported for the b-carbonic anhydrases from the red microalgae Porhyridium purpureum and  from the green plant Pisium sativum (Figure 4), which confirms previous EXAFS data suggesting a coordination sphere consisting of two thiolate sulfurs from cysteine, a histidine nitrogen, an oxygen atom supplied by water (Figure 5).

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Figure 3. γ-Carbonic Anhydrase from Methanosarcina thermophila

 

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Figure 4. β-Carbonic Anhydrase from Pisium sativum.

Figure 5. Active Site of β-Carbonic Anhydrase 
from Pisium sativum.

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Figure 6. Arabidopsis thaliana.

The goal of our research is to understand the catalytic mechanism of β-carbonic anhydrase. Toward this end we have cloned a β-carbonic anhydrase from the molecular biologist's favorite plant, Arabidopsis thaliana (mouse-ear cress, Figure 6), a plant whose β-carbonic anhydrase is very closely related to that of P. sativum. To explore the role of specific amino acid residues in catalysis by A. thaliana carbonic anhydrase, we utilize protein engineering or site-directed mutagenesis. Using recombinant DNA technology, it is possible to alter the gene coding for β-carbonic anhydrase and introduce specific, known changes in its nucleotide sequence. Enzyme manufactured from the altered gene will contain specific changes in its amino acid sequence, potentially altering the structure and/or function of the enzyme. If such structural changes are introduced intelligently, it is possible to deduce information about the roles of specific amino acids in an enzyme by carefully examining the catalytic function of the original and structurally altered proteins.
Our laboratory is also involved in the structural characterization of β-carbonic anhydrases, including structurally modified variants, using X-ray crystallography. Bacterial β-carbonic anhydrases have strikingly different structural and functional characteristics compared to the plant β-carbonic anhydrases. One of our broad research goals is to understand the evolutionary differences between these two subclasses of β-carbonic anhydrase. X-ray crystallography affords a detailed structural view of proteins at near atomic resolution. The combination of enzyme kinetics and X-ray crystallographic structure determination are powerful and complementary tools for understanding protein function. While X-ray crystallographic data collection is currently carried out off-site, crystal preparation (Figure 7), data analysis (Figure 8), and model refinement (Figure 9) can be accomplished by undergraduates at Colgate. 

Figure 7. Crystals of a mutant bacterial b-carbonic anhydrase.

Figure 8. X-ray diffraction pattern for a bacterial b-carbonic anhydrase

Figure 9. Electron density map for a bacterial b-carbonic anhydrase

Students involved in research in this laboratory will gain practical experience in protein engineering, functional characterization, and/or protein crystallography of plant and bacterial β-carbonic anhydrases. Students will be expected to master a wide variety of experimental approaches, including microbiological techniques, polymerase chain reaction, site-directed mutagenesis, DNA sequencing, protein overexpression and purification, and protein crystallization. In addition, students will also gain practical experience characterizing the catalytic function of mutant enzymes using stopped-flow spectrophotometry, UV-visible spectroscopy, and inductively coupled plasma optical emission spectroscopy. Students engaged in crystallography projects will also gain experience in crystallographic data analysis and model refinement. Recent and current members of our laboratory research group are shown below:

Joy Chamberlin ('03) loading yet another sample on the ABI 310 DNA sequencer. 2003 was Joy's fourth year in the research lab.  (Fall 2002) Joey Lee ('04) conducting one of many kinetics runs for a mutant bacterial carbonic anhydrase on the SF61DX2 stopped-flow spectrophotometer. (Fall 2003)
Doug Chapnick ('04) injecting a mutant bacterial carbonic anhydrase sample for purification on the Akta FPLC system. (Fall 2003) Ariel Herman ('04) examining an electron density map and solving a protein structure in the Colgate Protein X-ray Crystallography Computing Facility. (Spring 2004)
Katherine Chewning ('05) 'cloning around' in the lab with a recombinant carbonic anhydrase core domain. (Summer 2004) Chelsea Glessner ('07) inoculating a culture with E. coli containing a recombinant bacterial b-carbonic anhydrase plasmid. (Summer 2004)

Selected Reading

Figures 1-4 were prepared using the program MOLMOL (Koradi, R., Billeter, M., and Wüthrich, K., J. Mol. Graphics 1996, 14, 51-55. "MOLMOL: a program for display and analysis of macromolecular structures.")

Figure 8 was prepared using the HKL program suite ( Z. Otwinowski and W. Minor, " Processing of X-ray Diffraction Data Collected in Oscillation Mode ", Methods in Enzymology, Volume 276: Macromolecular Crystallography, part A, p.307-326, 1997,C.W. Carter, Jr. & R. M. Sweet, Eds., Academic Press (New York))

Figure 9 was prepared using T. Alwyn Jones' excellent Program O.


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