Honors Thesis Proposal
Mutagenesis of the Leucine-binding protein
Kristin Wheeler
Dr. Linda Luck, advisor
Background and Significance
LIV-HP (leucine, isoleucine and valine binding protein) and LS-HP (leucine binding
protein) (see Figure 1) are both receptor proteins in E. Coli that bind hydrophobic amino acids,
and use the same type of transport system to deliver these amino acids into the cell. The two
proteins show about 80% homology, but have different specificities for the amino acids they will
bind and transport. Using NMR (nuclear magnetic resonance) spectroscopy, we hope to address
questions regarding the binding of hydrophobic amino acids to these receptors and gain
knowledge pertaining to the structure and function of receptor proteins in general.
Each of the aforementioned proteins can be biosynthetically labeled using fluorinated
amino acids at specific amino acid positions, namely tryptophan, which can in turn be analyzed
using 19F NMR. This method provides a relatively non-perturbing probe for mapping out
structural and functional features of proteins. One tryptophan in particular (Trp 18), is thought to
be responsible for the differing specificities of the LS- and LIV-HP proteins. Using NMR, we can
also study the structure of the binding clefts of these two proteins can also be mapped out. (See
Figure 1)
When a protein binds to a branched amino acid, certain structural changes take place within the
protein. NMR will be used to determine if the LIV -and LS-HP undergo the same structural
changes and whether different amino acids have an influence on the change within the protein,
since we can label a variety of areas within the protein structure. In addition, LS-HP has been
shown to have binding capacities for fluorinated, hydrophobic molecules. However, the
specificity of this binding has not been thoroughly investigated. Our lab will further investigate
fluorinated molecules and their ability to be recognized by the LS and LIV receptors. This
property may be useful in bioremediation efforts. Many pollutants are of a fluorinated nature, and
thus this protein receptor may be useful in binding and disposing of these pollutants.
Methods and Materials
To utilize 19F NMR, residues within the proteins, the residues of interest must be labeled
with fluorine. To label specific areas in the binding proteins, E. Coli cells with an overproducing
plasmid will be grown in a media with specific fluorinated amino acids, thus introducing the
fluorine to the desired areas of the proteins, allowing for future 19F NMR analysis. Initial studies
of LS and LIV are shown in Figure 2. These spectra illustrate the four tryptophan (Trp ) residues
in the LS receptor and the three Trp residues present in the LIV. The Trp -18, not present in the
LIV, is assigned to the large broad peak at 46 ppm in the LS spectrum shown in Figure 2. The
mutant LIV (WY18), which has a Trp in position 18 shows the same broad peak noted in the LS
spectrum.
The goal in this research is to unequivocally assign the NMR peaks in the LS protein. To
do this, each Trp residue will be switched one at a time to phenylalanine (Phe) residues. Each
mutant will be grown with F-Trp (fluorinated tryptophan) and an NMR analysis will be
performed. The missing peak in the NMR spectrum will assign the resonance to the specific
residue that was switched. To do this, site directed mutagenesis will be performed.
DNA consists of four nucleotide bases, adenine (a), guanine (g), thymine (t) and cytosine
(c). DNA is responsible for making RNA, a messenger that directs a cell in protein production.
Each sequence of three nucleotide bases in the original DNA indicates an amino acid that will be
present in a specific location of the protein that will ultimately result. By recognizing these
sequences, one can predict or confirm what amino acid will be present at a particular location in a
protein. Site-directed mutagenesis will change the code for a particular amino acid, and create a
mutant. We will make three mutants of the LS protein: Trp18 to Phe 18, Trp 336 to Phe 336, and
Trp 278 to Phe 278. By performing this specific mutation, one can observe the resultant absence
of an NMR peak when the residue is no longer present, thus allowing one to assign a specific
peak in the unaltered protein to the mutated residue. In this way, specific resonances can be
definitively attributed to residues within the protein. The participation of the residues can be
assigned in the same way, by watching for a difference in post-binding shifts in the NMR spectra
of the wild type (unaltered) and mutant protein.
Using the plasmid overproduced in the auxotrophic cells mentioned before, and two
oligonucleotide primers with the desired mutant in place of the labeled residue, one can perform
the mutagenesis. These primers each match one end of the DNA strand identically, except for the
desired mutation. When the primers are introduced, a mutated plasmid with spaces in it is
produced. The primers are thermo cycled, which allows for DNA reproduction, and then treated
with an enzyme that will digest only the wild type DNA, leaving the mutated product. The
mutated plasmid is then replaced into E. Coli cells, which repairs the spaces made in the plasmid
earlier. The DNA of the cells can then be sequenced, confirming a mutation through the
aforementioned nucleotide sequences. In this research, I need to design the correct primers for the
mutagenesis experiment. Colonies of the bacteria must be screened for mutants, and the mutants
must be confirmed by sequencing data.