Silvia Cavagnero

Photo of Silvia Cavagnero
Professor (also Department of Chemistry)
B.S., University of Rome 'La Sapienza' (Italy)
M.S., University of Arizona
Ph.D., California Institute of Technology
Phone: (608) 262-5430
Email: cavagnero@chem.wisc.edu

Protein structure, folding, dynamics and aggregation in the cell;
role of ribosome and molecular chaperones

How do proteins fold in the cell, as they emerge out of the ribosome? The earliest stages of a protein's life in the cell are crucial for the achievement of a functional three-dimensional structure. This very structure is essential for biological activity and for the life of a healthy cell. The ribosome, which is well-known to catalyze protein biosynthesis, also serves as a key player in protein structure formation and in the prevention of protein aggregation. For instance, the bacterial ribosome is a nascent-protein solubilizing agent and it supports cotranslational protein folding. In addition, some specific ribosomal proteins interact directly with nascent chains during translation.

Cavagnero Figure 1 cartoon

Figure 1. Proteins (blue chains and image in the front) are synthesized
in the context of the ribosome (light green), in living cells.

Why is it important to understand protein folding and aggregation in the cell? The research carried out in the Cavagnero group focuses on understanding protein folding dynamics and aggregation in the cell. To help appreciate why this is important, it is helpful to discuss some practical implications. First, the large-scale production of soluble proteins is highly relevant to biotechnology and medicine. For instance, by exploiting the principles underlying protein folding in the cell it will be possible to enhance the yield of production of many protein-based drugs. Second, the role of molecular chaperones in the folding of client proteins and prions, once fully understood, can be harnessed to design novel antimicrobial agents targeting the protein folding machinery. Third, some of the fundamental principles learned in this research are likely to prove of general significance. As a result, these principles may prove to be crucial and ultimately inspire the design of strategies to control deleterious aggregation leading to a variety of deadly diseases, including prion-related, Parkinson’s and Alzheimer's disease, as well as Huntington's chorea.

Fundamental questions in protein folding and design. We are involved in the study of fundamental questions in protein folding, including kinetic trapping events across the folding energy landscape, the role of water and hydrophobic collapse, the role of the protein's C terminus in folding, the thermodynamic balance between protein folding and aggregation in regular proteins and prions, and the prediction of protein structure from amino acid sequence.

Cavagnero figure 2 cartoon

Figure 2. Many proteins are kinetically trapped relative to their aggregated states under physiologically relevant conditions. This means that several proteins have an intrinsic tendency to aggregate during their cellular lifetime, even in the absence of mutations or degradative processes. Aggregation is prevented by a crucial kinetic barrier that separates the native from the aggregates. The ribosome and molecular chaperones play a seminal role by ensuring that proteins land into the correct region of the free-energy landscape at birth, and stay there as long as possible.

The role of molecular chaperones in protein folding and disease. We are particularly interested understanding how cotranslationally- active molecular chaperones lead assist the generation of the protein native state, starting from unfolded, partially folded or misfolded conformations. Towards this end, we are exploring how the bacterial Hsp70 chaperone, known as DnaK, interacts with its client proteins and potentially affects their folding mechanism. This work is carried out primarily by multidimensional NMR on 15N- and 13C-enriched polypeptide substrates and involves both kinetic and structural analysis. We focus on characterizing the conformational and dynamic changes undergone by Hsp70 client proteins during different stages of the chaperone cycle.

Cavagnero figure 3 cartoon

Figure 3. The bacterial Hsp70 molecular chaperone, also known as DnaK, binds client proteins in a variety of conformational states. The structure and dynamics of several of these chaperone-bound states is yet to be discovered.

Our techniques. The Cavagnero group is engaged in the study of protein folding at the ribosomal exit tunnel by a combination of biochemical and biophysical approaches, including labeled-tRNA and cell-free technologies, time-resolved fluorescence and multidimensional nuclear magnetic resonance spectroscopy, including LED- and laser-assisted NMR. In addition, we employ single-molecule fluorescence to explore protein folding on the ribosome and interactions with molecular chaperones.