January 28, 2013
Lauren Gadsby
219 W. Foster Ave.
Apartment #6
State College, PA 16801
Dr. Squire Booker
Associate Professor of Biochemistry and Molecular Biology
302 Chemistry Building
University Park, PA 16802
Dear Dr. Booker:
This proposal will present a plan for producing and investigating the characteristics of two
proteins, NosN and BlmOrf8, that catalyze the transfer of a methyl group from the compound Sadenosyl-methionine (SAM) to a substrate. The goal of this research will be to obtain
information about the chemistry of and mechanism through which these proteins operate. Thus,
this project will provide further understanding of the respective roles of NosN and BlmOrf8 in
the biological reactions they catalyze. In this proposal, I will outline the research problem that I
will address, my research plans, my qualifications, and a work schedule to complete the
research.
Proposed Program
The Problem:
Methyl transfer reactions are essential in the metabolism and modification of key biomolecules,
such as RNA, DNA, lipids, and proteins. Many of these crucial biological methylation reactions
involve S-adenosyl-methionine (SAM) as the methyl donor and proceed by an SN2 displacement
mechanism. However, researchers have discovered that some enzymes use SAM to add methyl
groups to molecules via a radical mechanism. These enzymes belong to the radical SAM
methyltransferase (RS) superfamily.
The structure of enzymes belonging the RS superfamily is distinct and important to their
function. Enzymes of the RS superfamily contain at least one iron-sulfur cluster, which consists
of four iron molecules and four sulfur molecules (commonly abbreviated [4Fe-4S]). This cluster
is coordinated by cysteine (C) amino acid residues. These residues are conserved throughout
members of the family in a CxxxCxxC motif. SAM binds the [4Fe-4S] cluster, and the enzymes
cleave SAM to yield S-adenosyl-homocysteine (SAH) and a 5’-deoxyadenosyl (5’dA) radical. The
5’dA radical then initiates a radical mechanism to transfer the methyl group from the radical to
the substrate. The enzymes that I will be working with, NosN and BlmOrf8, belong to the RS
family and have been identified to contain the conserved cysteine motif. Thus, they are good
candidates for mechanism determination and characterization.
Although the discovery that some methyl transfer reactions proceed by a radical mechanism is a
novel breakthrough, little is known about the enzymes of this RS family. The Booker lab has
succeeded in elucidating the structure and mechanism of two enzymes, RlmN and Cfr, that
belong to the RS family. The characterization of NosN and BlmOrf8 from the RS family would
augment the knowledge and diversity of methyl transfer reactions, and a publication detailing
findings would receive widespread attention from biological chemists.
Additionally, because NosN and BlmOrf8 catalyze reactions involving compounds important for
human health, researchers could use the discovery of more information about these enzymes to
develop medical therapies. NosN catalyzes a methyl transfer reaction in the indole side ring
during the biosynthesis of nosiheptide, an antibiotic important in the treatment of MRSA
(methilin-resistant Staphylococcus aureus). BlmOrf8 acts in the synthesis of bleomycin, a
clinically-used antitumor drug produced by Streptomyces verticillus. Because of a trend of
increasing antibiotic resistance, the development of drugs similar in structure and function to
nosiheptide and bleomycin could provide more effective treatments for bacterial infection.
Research Plans:
The research will consist of:
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Ordering the genes for NosN and BlmOrf8 from Invitrogen Life Technologies, who will
optimize the genes for expression in E. coli
Transformation of E. coli cells to insert the genes in the cells
Extraction and sequencing of DNA using Penn State’s sequencing facility to verify that E.
coli contains the desired genes
Growth and harvest of E. coli cells containing NosN and BlmOrf8
Purification of NosN and BlmOrf8 in two manners: apo (without the iron-sulfur cluster)
and holo (with the iron-sulfur cluster)
Characterization by various methods including: SDS-gel electrophoresis to assess purity,
iron and sulfur analysis to determine the number of iron-sulfur clusters the protein
contains, Mossbauer and EPR spectroscopy to determine the content and magnetic
properties of the cluster
Assays to test the enzymes for the ability to cleave SAM into SAH and 5’dA using mass
spectroscopy
Assays to assess the ability of the protein to bind SAM
Compilation of results in my thesis and in a publication for a scientific journal
Qualifications
I was trained in protein expression and purification techniques during an internship with Dr.
Jinhua Wu at Fox Chase Cancer Center in Philadelphia. During this internship, I worked to
produce proteins involved in cell adhesion using an E. coli expression system. I then harvested
the cells and purified the proteins using affinity, ion-exchange, and gel filtration
chromatography. The expression and purification techniques I performed are similar to those I
would employ to produce NosN and BlmOrf8.
During internships both in Dr. Philip Mason’s lab at Children’s Hospital of Philadelphia and Dr.
Eileen Jaffe’s lab at Fox Chase Cancer Center, I have been trained in various methods to
characterize proteins. Throughout these experiences, I have developed skills in SDS-gel
electrophoresis, protein concentration determination, and running assays, techniques I would
employ to characterize NosN and BlmOrf8.
Work Schedule
Note: Writing of my thesis will take place throughout this schedule as experiments are
completed.
Week of February 4:
Transformation of NosN and Blm8 into E. coli
Extraction of DNA
Sequencing and analyze results
Week of February 11:
Growth of E. coli cells containing genes for NosN: two cultures holo and two cultures apo
Harvest cells
Growth of E. coli cells containing genes for Blm8: two cultures holo and two cultures apo
Harvest cells
Week of February 18:
Purification of NosN holo and NosN apo using Ni column
Purification of Blm8 holo and Blm8 apo using Ni column
Week of February 28:
Additional purification of proteins using gel filtration
SDS gel electrophoresis to assess purification
Week of March 4:
Spring Break—no research plans
Week of March 11:
Iron and sulfur analysis for NosN holo and NosN apo
Iron and sulfur analysis for Blm8 holo and Blm8 apo
Week of March 18:
Mossbauer spectroscopy of all proteins
EPR spectroscopy of all proteins
Week of March 25:
Assays testing formation of SAH and 5’dA from SAM for NosN and Blm8
Week of April 1:
Assays testing binding ability of all NosN and Blm8 to bind SAM
Completion of draft of thesis
I have discussed the necessity of research into the characterization and mechanism of radical
SAM methyltransferases, particularly the representative enzymes NosN and BlmOrf8. I believe
that my research plans, qualifications, and work schedule will sufficiently address the research
problem. With your permission, I will be able to begin my thesis and fulfill this research need.
Sincerely,
Lauren Gadsby