Graduate research

My thesis research was conducted in Ben Glick’s laboratory at the University of Chicago, Department of Molecular Genetics and Cell Biology. I had two main projects. The first was studying a red fluorescent protein called DsRed. The second project involved studying subdomains of the Endoplasmic Reticulum called transitional Endoplasmic Reticulum (tER) sites (also called ER exit sites) in the budding yeast Pichia pastoris.

DsRed

Introduction

adapted from Bevis and Glick NBT 2002

Live budding yeast expressing Green Fluorescent Protein fused to a Golgi protein and a red fluorescent protein, DsRed, targeted to mitochondria. Adapted from Bevis and Glick NBT 2002

Fluorescent proteins, as the name suggests, are proteins that fluoresce. When you shine one color of light on them, they absorb that light – some physical chemistry happens (e.g. electrons move to higher orbitals, then relax) – and light of a different color is emitted. The color difference between the absorbed light and the emitted light means that one can use optical filters to selectively pass only the emitted light to your eyes or an electronic detector (e.g. a camera). Because they are proteins, these fluorescent molecules can be expressed in cells and they can also be directly fused to other proteins you are interested in, rendering them a visible molecular tag.

The use of fluorescent proteins as reporters for gene expression and as fusion tags has revolutionized cell biology because now we can watch individual molecules in live cells. We don’t want the tags themselves to influence the cellular processes we are examining, therefore we want the fluorescent proteins to be as neutral as possible. The first fluorescent protein ever discovered is called GFP for Green Fluorescent Protein and it came from a jellyfish called Aquoria victoria. As luck would have it GFP is pretty neutral, specifically it is usually a monomer, meaning each copy of GFP stays by itself and doesn’t bind to other copies of GFP. However, there is a need for colors that will complement green, such as red. In 1999 Matz and colleagues cloned new fluorescent proteins from corals and identified a red protein they called DsRed. The [original] wild-type DsRed had a host of problems limiting it’s utility as a research tool. A couple of research groups including the Glick lab undertook projects to make it a better research tool using a technique called directed evolution to engineer improved variants. Directed evolution is just like natural evolution but sped up. Mutations are made on purpose and large libraries containing thousands of variants are screened in a high-throughput manner so that only improved variants are selected. Those improved variants are then mutated, screened, selected etc. When I started on my DsRed project, the Glick lab had just engineered the most advanced DsRed variant which they called DsRed.Express. There were two remaining problems with DsRed.Express, limiting its utility. The most significant was that it was a tetramer, which means that four copies of the protein bind together in a specific orientation (see figure). If tetrameric DsRed is used as a fusion tag it will drive the tertamerization of the protein its fused to which is likely to disrupt its funtion. Therefore my first goal was to engineer DsRed into a monomer (single copy). The second problem with DsRed.Express was that it aggregated a little bit, that is it non-specifically stuck to itself and other proteins in the cell, particularly at high concentrations and this too could interfere with the cells we are monitoring.

composite structuresDirected Evolution of Monomeric and Non-aggregating Mutants of DsRed

To make DsRed.Express into a monomer I first took advantage of other labs’ structural studies of DsRed. They identified the amino acids that made up the tetramerization interfaces on the surface of the protein, i.e. they identified which amino acids were acting like glue. I then used rational, site-directed mutagenesis to specifically disrupt those amino acids and change them to amino acids that were less likely to mediate binding. It so happens that after I made those mutations, that DsRed variant was no longer fluorescent. I therefore used several rounds of directed evolution, incorporating both random and rational mutations, to recover the fluorescence. The end result was a variant we called DsRed.M1 or DsRed.Monomer that had 45 mutations compared to the wild-type protein (there are 225 amino acids in the protein). In collaboration with Bob Keenan’s lab, also at the University of Chicago, we solved the x-ray crystal structure of both DsRed.Express and DsRed.Monomer and were able to rationalize several key properties of these proteins. These results are published HERE.

We then used a similar strategy, i.e. rational mutagenesis informed by structural data combined with random mutagenesis and a novel screening platform to engineer non-aggregating variants of DsRed.Express that we naturally called DsRed.Express2. This work, which I began, was moved forward in dramatic fashion by Rita Strack a new graduate student (at that time) in the lab. A more detailed description of the work can be found in the paper Rita and I wrote.

 

DsRed maturationMechanism of Chromophore Biogenesis in DsRed

While engineering DsRed I became interested in the chemical basis for chromophore formation. The chromophore is the part of the fluorescent protein that absorbs and emits light. A key feature of fluorescent proteins is that their chromophores form autocatallytically from amino acids within the protein. This means that a series of chemical reactions rearrange the bonds between atoms within amino acids inside the proteins without the help of additional enzymes or cofactors. Others had previously proposed that DsRed matures along a linear pathway that includes a GFP-like chromophore as an intermediate. There had been a puzzling result that DsRed tetramers occassionally contained copies of the protein with a green fluorescing chromophore instead of a red one and the linear model with GFP as an intermediate was initially an attractive one. However several pieces of data never quite fit this model, then one day we did a journal club on a paper by Rebekka Wachter’s lab about GFP maturation and I hypothesized that some of their findings might apply to DsRed and that the green and red fluorescing chromophores in DsRed might represent distinct endpoints of a branched maturation pathway. Again in collaboration with Rita, we then tested this branched model. A key prediction of the model was that there exists a kinetic competition between the green and red branches, meaning at the split point (3 to 4r or 3 to 4g, in the diagram above) whichever reaction happens first drives the chromophore irreversibly down that branch. We modified the chemical environment for chromophore maturation to push it down one path or the other and serendipitously all of our predictions were validated by the data. Thus we demonstrated that the DsRed chromophore matures along a branched pathway.

 

tER Sites

tER

Introduction to the biogenesis and maintenance of tER sites.

The secretory pathway carries proteins and lipids from the Endoplasmic Reticulum (ER), through the Golgi and eventually to stops along the secretory pathway or to the cell surface. Secretory proteins are inserted into the ER as they are being synthesized. In the ER, the proteins fold, and get a sugar group added to certain amino acids. Proteins then exit the ER towards the Golgi in a class of vesicles coated with the a group of proteins collectively called COPII, for coat protein class II. Vesicle budding and coat assembly require the 4 structural COPII proteins (Sec23, Sec24, Sec13 and Sec31), a small GTPase called Sar1, a GTP exchange factor (GEF) for Sar1 called Sec12. Several additional effector proteins and cargo receptors also facilitate vesicle formation. In the budding yeast Pichia pastoris as well as multicellular organisms, the COPII vesicles bud from distinct ribosome free regions on the ER membrane called transitional ER (tER) sites or ER exit sites. It is puzzling how these discrete regions can exist within the larger, contiguous ER. Brook Bevis, the grad student who mentored me when I arrived in the Glick lab, showed that tER sites were dynamic by imaging COPII proteins in live cells. She showed that they form de novo, diffuse, fuse with each other and maintain a preferred size, i.e. after forming de novo they initially grow then plateau at the preferred size and when two tER sites fuse the combined site shrinks to the preferred size. I wanted to investigate the mechanism underlying this dynamic activity. I hypothesized that tER sites were made up of proteins that could diffuse in and out of them or exit via vesicle budding (see figure) and that these transient interactions were sufficient to maintain a subdomain within the membrane.

FRAP idea

Diagram of the principle behind FRAP. 2 tER sites are showneach composed of GFP tagged tER site proteins. The one on the right is bleached by a laser pulse (blue). If the components diffuse among tER sites fluorescence will recover in the bleached site (bottom).

Dynamic proteins

To test my hypothesis that tER sites are made up of dynamically exchanging components, I used a technique called Fluorescence Recovery After Photobleaching (FRAP). The principle of FRAP is that one can permanently bleach the fluorescence from fluorescent protein fusions in one region of a cell, in our case a single tER site. If the molecules that make up the tER site are stably associated with it for a long time, the site will remain dark (bleached) indefinitely. However if the constituent molecules are mobile and exchange among other tER sites, fluorescence will recover at the bleached site (see figure).

I performed FRAP experiments with several different proteins known to localize to tER sites and all of them exhibited fluorescence recovery demonstrating that these proteins were diffusing among the tER sites, consistent with our model for tER site biogenesis and maintenance. An example with the protein Sec12p is shown below. The bleached tER site is indicated with an arrow. The figure and a quantitation of the fluorescence recovery not shown here is my contribution to Soderholm et al. 2004. The QuickTime movie that the gallery is derived from can be downloaded/viewed HERE.

Sec12p FRAP