For many years research on the dynamic behavior of proteins has focused on isolated proteins manipulated outside of the cellular environment (in vitro). There are only a few systems in which investigators have moved into more complex environments, including the living cell (in vivo). The proposed studies will develop Fast Relaxation Imaging, a technique that looks at biomolecular dynamics inside living cells, allowing connections to be made between what has been learned in vitro to how proteins behave in vivo. In the proposed experiments a small laser-induced temperature jump initiates protein folding kinetics, protein-protein interactions, or nucleic acid dynamics inside cells. A fluorescence microscope images the resulting dynamics as a function of time, making a "folding movie" inside the cell. The experiment will be applied to study the heat shock response of cells, interactions of the protein alpha-synuclein with cell membranes, and folding of metabolic and signal enzymes in various compartments of different cell types.
Broader Impacts: Because protein dynamics are critical to protein function, understanding how cells modulate the stability and kinetics of their constituent biomolecules is critical to understanding cell function and the regulation of cellular processes such as gene transcription, apoptosis and cell division. Defining protein behavior in the cell will also lead to determining what aspects of modulation are evolved and have been part of the selection process on protein structure. The research is complemented by several outreach programs, including recruitment of summer undergraduate research students, an exchange and teaching program with Hanoi University of Science to bring their teaching and research up to modern standards, and continuation of a K-12 outreach program that was set up by a biophysics student in the PIs lab, and is now available as a web-based tutorial in biophysics: a student, with guidance of a faculty mentor, collaborates with middle- and high school teachers to help them create educational materials employing biophysical visualization tools. The broader impacts also include development of the instrumentation for the proposed experiments. This instrumentation could be become widely used in other laboratories.
This project is jointly funded by Cellular Systems and Molecular Biophysics in MCB.
Proteins are the engines of life: they burn carbohydrates using the oxygen we breathe to produce energy, they receive signals on nerve cells that make us think, and they collect light to store energy in chemicals in plant leaves. Most proteins work inside cells, yet they have mostly been studied in the test tube. The NSF grant focused on how proteins fold inside cells, and comparing in-cell results with past test tube results. We found that some enzymes (such as the protein called PGK, which makes ATP, the source of energy in our cells and muscles) are actually more stable inside cells than in the test tube, whereas other proteins, such as the one called VlSE that Lyme disease bacteria use to attack hosts, become less stable in cells. As the cell cycle progresses through cell division, protein stability and the speed at which proteins fold crest and wane. Proteins also have different stabilities in different compartments of the cell, such as the nucleus or cytoplasm. Thus cells have the ability to regulate proteins to behave differently at different times and in different environments, unlike the homogeneous environment of a test tube. RNA is another ingredient in cells, and we also studied the folding of RNA molecules, first by test tube measurements, and then by large-scale computer simulations. We were able to pinpoint the chemical mechanism by which small RNA molecules fold into the shape of a hairpin, and how these hairpins bind together with a protein to make part of a cellular machine known as the spliceosome. As its name suggest, the spliceosome splits pieces off of messenger RNA. Splicing is an important process during translation of genetic information from DNA in the cell nucleus into proteins. These experiments led to our current and continuing research on how RNA and proteins interact inside living cells. We also studied a number of other biophysical processes in collaboration with other reearchers, such as the following: Poly(ethylene glycol), known in short as 'PEG,' is a substance that pharmaceutical companies attach to proteins so they become more stable and deliverable as drugs. Protein drugs hold great promise, and several are on the market today (e.g. insulin treatments against diabetes), but stable delivery is a challenge. No one really knows how PEG functions. We studied PEG attached to proteins by working together with Joshua Price at Brigham Young University. Our experiments and large-scale computer simulations painted a picture that contradicted our initial assumption: that PEG would form 'hydrogen bonds' with the surface of the protein. (A 'hydrogen bond' occurs when an oxygen atom in PEG weakly binds to a hydrogen atom on the protein surface.) Instead, the PEG actually changed the structure of the protein, and often stuck out into the surrounding water without coating the protein surface at all, yet stabilized the protein sometimes by a large amount. This is an example of many such collaborations we did during the grant period. Several graduate students and postdocs were trained with the aid of this grant. They have taken a variety of positions as professors and in industry, from universities such as Cal Poly San Luis Obispo to corporations such as Intel in Oregon. Their know-how keeps US companies and educational institutions competitive. Undergraduates were trained in hands-on research, and have gone on to graduate or medical school, where they have the know-how to enter MD-PhD programs and do medical research. The principal investigator, often together with students, also participated in a variety of outreach programs, such as developing research- and inquiry-based curriculum during the summer with high school teachers. The scientific results were presented a many conferences, other universities, and in publications of the principal investigator, graduate students, and undergraduates.
