Ten weeks with alpha-synuclein

I spent my summer working with alpha-synuclein, a somewhat poorly-understood protein found in the presynaptic terminals of human neurons. Its suggested functions include chaperoning SNARE complexes, regulating vesicle size in the presynaptic terminal and the release of neurotransmitters into the synapse.

The purpose of my research this summer was to use a thiocyanate vibrational probe group at several sites in relevant regions of alpha-synuclein to better characterize its structure. This builds on previous work by Alice Vienneau ’12 and Dan Konstantinovsky ’16, who had inserted the SCN probe group at 8 sites of interest. This process relies on site-directed mutagenesis to replace the original codon with a codon for cysteine, then expressing this monocysteine mutant and cyanylating to attach the CN moiety. The thiocyanate probe group has been the subject of previous and ongoing research in the Londergan lab and can be used in IR spectroscopy to report on whether the site is buried in a lipid bilayer, such as a vesicle membrane, or exposed to the aqueous solvent.

Recent research has discovered that codon 136 (TAC) of the alpha-synuclein plasmid used, which is typically translated into a tyrosine, can be mistranslated in Escherica coli to incorporate a cysteine; this happens roughly 20% of the time. This additional cysteine would likely be cyanylated along with the desired mutant cysteine at the probe-group site and could interfere with the IR spectrum results. By changing the codon to a degenerate codon for tyrosine (TAC to TAT), we can eliminate this problem, as E. coli will not express this as a cysteine.

The product of the site-directed mutagenesis reaction is transformed into bacteria and grown on a plate. Since it's unreliable, we tend to sequence three colonies at a time to check for the desired mutation.

The product of the site-directed mutagenesis reaction is transformed into bacteria and grown on a plate. Since it’s unreliable, we tend to sequence three colonies at a time to check for the desired mutation.

Consequently, my teammates and I spent part of our time in the lab using site-directed mutagenesis to alter the 8 original monocysteine mutants to incorporate the Y136tat change as well. Site-directed mutagenesis uses oligonucleotide primers of roughly 50 bp that contain the desired mutation—generally just one or two base pairs’ difference—but match the template in the surrounding base pairs. PCR creates the product DNA. This is a finicky process that requires a good bit of patience—some of our plasmids worked on the first try, but others required sequencing a dozen colonies before we saw the desired mutation. During the process we created three additional monocysteine mutant plasmids to study new sites. All the mutant alpha-synuclein plasmids were expressed in cultures of E. coli and induced with IPTG, since our plasmids use some of the same machinery as the lac operon.

Purification of the cell lysate took the form of a separation on the basis of charge (anion-exchange chromatography), followed by separation on the basis of size (size-exclusion chromatography). We encountered a major hiccup here: UV spectra of purified protein samples revealed very strong absorbance at 260 nm, which indicates the presence of nucleic acids. It seemed that DNA was eluting with alpha-synuclein on both columns. We experimented with salt cuts and DNases to try to eliminate the nucleotides, but the solution was to replace the DEAE anion-exchange column we were using with a similar column—a Q FF anion-exchange column. SDS-PAGE and UV spectra have suggested that this column separates the DNA from the protein.

Protein UV absorbance should peak at 280 nm (right vertical red marker), but this spectrum clearly shows the peak at 260 nm (left marker). This indicates the presence of nucleotides.

Protein UV absorbance should peak at 280 nm (right vertical red marker), but this spectrum clearly shows the peak at 260 nm (left marker). This indicates the presence of nucleotides.

With a reliable protocol finally in hand, the next step is to express and purify all the new, Y136tat mutants. These will then be cyanylated to create the SCN probe group at each of the 11 desired sites and studied with IR spectroscopy. We’re hoping to characterize the conformations alpha-synuclein takes when exposed to various sizes of lipid systems and to learn more about which regions and residues are involved in its aggregation process.