Intellectual Merit. The folding of pure proteins in solution, starting from chemically or thermally generated unfolded states, has been studied intensively for almost 50 years. However, these unfolded states rarely occur in living cells and therefore we still know very little about how proteins actually fold in their native environments to achieve their active conformations. Proteins in all living cells are synthesized by ribosomes, complex molecular machines found in all living organisms. In living cells, a number of factors, including helper proteins called "chaperones," crowding by thousands of other molecules, as well as the ribosome itself, can profoundly affect how and whether a protein folds correctly once it is made. Least understood are the earliest stages of folding, when the protein first emerges from the ribosome where it is made.
This project explores the structural and dynamic differences between ribosome-bound and ribosome-released nascent proteins, and proposes to compare the timecourse leading to release of full-length protein chains from the ribosome ("protein birth"), in the presence and absence of chaperone proteins ("folding helpers"). Synergistic computational and experimental investigations will also be carried out on model nascent protein chains to assess the ability the ribosome's surface to spatially confine the nascent protein and promote folding. The following research objectives will be pursued:
#1: Equilibrium analysis of the conformation and dynamics of nascent single-domain proteins as they emerge from the ribosomal tunnel, before and after their release by the ribosome. These investigations will identify the most stable and most structured regions of nascent proteins by studying their degree of solvent exposure via H/D exchange followed by MALDI mass spectrometry.
#2: Kinetic measurements of the release of newly synthesized, single-domain proteins from the ribosome to follow the formation of protein 3D structure in real time.
#3: Equilibrium and kinetic analysis of incomplete and full-length nascent chains derived by multidomain proteins. The experiments described in specific aims #1 and 2 will be repeated for the simple multi-domain model protein Hmp. In addition, real-time translation studies will probe the effect of mRNA silent mutations on protein conformation.
#4: Computational analysis of ribosome-bound and ribosome-released nascent proteins. Synergistic computational and experimental studies will be carried out to assess the effect of the ribosome's surface on the nascent protein's conformation and spatial confinement.
BROADER IMPACTS. The project includes the pursuit of the following broader impacts: #1: Recruitment of underrepresented students to participate in research activities. The proposed studies will involve the mentoring and training of graduate and undergraduate students, especially women and students belonging to underrepresented minorities. Undergraduate students will routinely participate to advanced research in the PI's group. The PI will also participate as a lecturer to the Summer Course in Biophysics for Minority Students organized by the Biophysical Society and will serve as a mentor, recruiter and judge at the SACNAS conference, fostering participation of Chicanos and Latino Americans in science.
#2: Development of novel lecture demonstrations on protein folding and the effect of cell-relevant components on protein folding and stability. The PI will develop a number of classroom demos aimed at illustrating, with direct visual readouts, the reversibility of protein folding, its pH dependence, and the effect of osmolytes on protein stability.
#3: Presentation of lectures, videos and demonstrations on protein folding at the Middleton High School. This project will be done in collaboration with Kathryn Eilert, Teacher of Molecular Biology and Biotechnology at the Middleton (WI) High School.
In a nutshell, proteins are the machines of life. Living cells could not function without a myriad of proteins, each performing wonderful, highly specialized and diverse functions. Amazingly, protein activity is highly coordinated in both space and time, within each living organism. Much of this coordination is achieved, perhaps not surprisingly, via the help of other proteins! One of the most important requirements for proteins to be bioactive and perform their function is to achieve a highly organized, proper three-dimensional structure, in other words to achieve their natural "fold". There are hundreds of detailed structures or "folds", in Nature. While we know that the different folds are dictated by the amino acid sequence and by the specific environment experienced by the protein, we know very little about the actual mechanism for the "folding" of small and large proteins inside the living cell. The goal of this research was to unveil the way proteins achieve their natural fold in the cellular environment by using a variety of biochemical, spectroscopic and computational techniques. Because the main players that help protein fold in the cellular environment are the very machine that makes the protein, also known as the "ribosome", and other specialized proteins known as "molecular chaperones", our studies focused on the role of these biological players in the folding process. The investigations carried out in the Cavagnero group led to the discovery that the ribosome helps proteins properly sample conformational space while they are synthesized. The ribosome enables protein to try out folding by letting them become compact and partially structured for part of the time, while spending another portion of their early life unfolding back to ground zero, ready to start the folding trials again, until biosynthesis is complete. In summary, the Cavagnero group found that the ribosome is much more that a protein builder. It is also a protein folder! The group also discovered that proteins achieve the most important details of their structure, which is needed for function, at the very time they depart from the ribosomal machine. This structure formation comes along with stability, and the Cavagnero group learned how to approximately predict this stability on the computer, as the building blocks (or the "amino acids") composing the final biomolecule are added to the protein chain, one by one. The Cavagnero group made good progress on this research and hopes to be able to use its recent results as a sounding board for the training of new students, for fostering the emerging field of in vivo biophysics, and for encouraging students and scholars from underrepresented ethnical groups and disadvantaged backgrounds to pursue this exciting area of study. Finally, the group hopes to be able to exploit its past achievements and recently developed learning tools as a starting point to carry out additional informative experiments in the future.
