Principal Research Interests
The protein folding problem, namely how amino acid sequence determines the
three-dimensional structure of a protein, is not fully understood despite many years of effort. We are addressing this problem in a variety of ways in our laboratory. Methods we use in all of our folding work include circular dichroism, fluorescence, and nuclear magnetic resonance.
We are particularly interested in how a protein folds in vivo. There are many challenges presented to a newly synthesized protein as it navigates its energy landscape to the native state in the cell, including the co-translational emergence of the protein from the ribosome and potential for conformational search before the chain is complete, the extremely high concentration of macromolecules and consequent crowding of the cellular milieu, the heterogeneous and limited volumes accessible to a folding chain, and the numerous molecular chaperones that interact with partially folded states and modulate their conformational exploration. We are using both ‘top down’ approaches by developing methods to observe a folding chain in cells and to perturb the cellular environment through genetic manipulation or environmental influences, and ‘bottom up’ approaches, wherein we mimic the components of the cell and examine their influence on folding.
In addition to this effort to describe the folding environment of the cell, we are doing detailed mechanistic studies of major classes of molecular chaperones. Present work focuses on the Hsp70s, which are ubiquitous and play a wide array of roles in facilitating the folding, membrane translocation, assembly and disassembly of complexes, and degradation of proteins in nucleotide-regulate manner, and in partnership with a complex network of partner chaperones. The Hsp70s are two-domain proteins, in which nucleotide binding to one domain allosterically modulates substrate affinity in the other domain. We deploy a wide array of biophysical methods, including NMR, fluorescence, EPR, and others, to dissect in detail how the interdomain allostery works.
Lastly, we recognize that protein folding in the cell does not always succeed, with many pathological consequences associated with misfolding. Important among these is aggregation. We are using the systems we develop to observe folding in the cell to examine the origins and mechanisms of protein aggregation in vivo, with a goal of better understanding misfolding-based diseases such as the many neurodegenerative diseases (Alzheimer’s, Huntington’s, Parkinson’s).
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| Representative Publications
2007
“In-Cell Aggregation of a Polyglutamine-Containing Chimera is a Multi-step Process Initiated by the Flanking Sequence,” Z. Ignatova, A.K Thakur, R. Wetzel and L.M. Gierasch, J. Biol. Chem. 282(50), 36736-43 (2007).
“Use of Synthetic Signal Sequences to Explore the Protein Export Machinery,” E.M. Clerico, J.L. Maki and L.M. Gierasch, Biopolymers [epub] (2007).
“Effects of Osmolytes on Protein Folding and Aggregation in Cells,” Z. Ignatova and L.M. Gierasch, Methods Enzymol. 428, 355-72 (2007).
“Hsp70 Chaperone Ligands Control Domain Association via an Allosteric Mechanism Mediated by the Interdomain Linker,” J.F. Swain, G. Dinler, R. Sivendran, D.L. Montgomery, M. Stotz and L.M. Gierasch, Mol. Cell. 26, 27-39 (2007).
“Site-Specific Fluorescent Labeling of Poly-Histidine Sequences Using a Metal-Chelating Cysteine,” B. Krishnan, A. Szymanska, and L.M. Gierasch, Chem. Biol. Drug Des. 69, 31-40 (2007).
“From the Test Tube to the Cell: Exploring the Folding and Aggregation of a Beta-Clam Protein,” Z. Ignatova, B. Krishnan, J.P. Bombardier, A.M. Marcelino, J. Hong and L.M. Gierasch, Biopolymers 88, 157-63 (2007).
2006
Ignatova Z, Gierasch LM. Inhibition of protein aggregation in vitro and in vivo by a natural osmoprotectant. Proc Natl Acad Sci, 103, 13357-61 (2006).
Cavanaugh LF, Palmer AG 3rd, Gierasch LM, Hunt JF. Disorder Breathes Life into a DEAD Motor, Nat Struct Mol Biol, 13, 566-9 (2006).
I. L. Mainprize, D. R. Beniac, E. Falkovskaia, R. M. Cleverley, L. M. Gierasch, F. P. Ottensmeyer, and D. W. Andrews, The Structure of E. coli Signal Recognition Particle Revealed by Scanning Transmission Electron Microscopy, Mol. Biol. Cell, 17, 5063-74 (2006).
Z. Ignatova and L. M. Gierasch, Extended PolyQ Tracts Cause Aggregation and Structural Perturbation of a Neighboring β–Barrel Protein, J. Biol. Chem., 281, 12959-67 (2006).
A. C. Marcelino, R. G. Smock, and L. M. Gierasch, Evolutionary Coupling of Structural and Functional Sequence Information in the Intracellular Lipid-Binding Protein Family, Proteins: Structure Function Bioinformatics, 63, 373-384 (2006).
J. F. Swain and L. M. Gierasch, The Changing Landscape of Protein Allostery. Curr. Opin. Struct. Biol. 16, 102-108 (2006).
J. F. Swain, E. G. Schulz, and L. M. Gierasch, Direct Comparison of a Stable Isolated Hsp70 Substrate-binding Domain in the Empty and Substrate-bound States, J. Biol. Chem., 281, 1605-11 (2006).
K. S. Rotondi and L. M. Gierasch, Natural Polypeptide Scaffolds: β-Sheets, β-Turns, and β-Hairpins, Peptide Science, 84, 13-22 (2006).
2005
R. G. Smock and L. M. Gierasch, Finding the Fittest Fold: Using the Evolutionary Record to Design New Proteins, Cell, 122, 832-834 (2005).
J. F. Swain and L. M. Gierasch, First Glimpses of a Chaperonin-bound Folding Intermediate, Proc. Natl. Acad. Sci. USA, 102, 13715-13716 (2005).
Y.-T. Chou and L. M. Gierasch, The Conformation of a Signal Peptide Bound by Escherichia coli Preprotein Translocase SecA, J. Biol. Chem., 280, 32753-32760 (2005).
Z. Ignatova and L. M. Gierasch, Aggregation of a Slow-folding Mutant of a β-Clam Protein Proceeds through a Monomeric Nucleus, Biochemistry, 44, 7266-7274 (2005).
N. Sinha, C. V. Grant, K. S. Rotondi, L. Feduik-Rotondi, L. M. Gierasch, and S. J. Opella, Peptides and the Development of Double- and Triple- Resonance Solid-State NMR of Aligned Samples, J. Pept. Res., 65, 605-620 (2005).
K. S. Rotondi and L. M. Gierasch, A Well-Defined Amphipathic Conformation for the Calcium-Free Cyclic Lipopeptide Antibiotic, Daptomycin, in Aqueous Solution, Peptide Science, 80, 374-385 (2005).
2004
K. S. Rotondi and L. M. Gierasch, Solution Structure of Daptomycin, in Peptide Revolution: Genomics, Proteomics & Therapeutics, (M. Chorev and T. Sawyer, eds.), pp. 447-449 (2004).
J. J. Fak, A. Itkin, D. D. Ciobanu, E. C. Lin, X.-J. Song, Y.-T. Chou, L. M. Gierasch, and J. F. Hunt, Nucleotide Exchange from the High-Affinity ATP-Binding Site in SecA Is the Rate-Limiting Step in the ATPase Cycle of the Soluble Enzyme and Occurs through a Specialized Conformational State, Biochemistry, 43, 7307-27 (2004).
Z. Ignatova and L. M. Gierasch, Monitoring Protein Stability and Aggregation In Vivo by Real-Time Fluorescent Labeling, Proc. Natl. Acad. Sci. USA, 101, 523-528 (2004).
K. Gunasekaran, A. T. Hagler, and L. M. Gierasch, Sequence and Structural Analysis of Cellular Retinoic Acid Binding Proteins Reveals a Network of Conserved Hydrophobic Interactions, Proteins: Structure Function and Bioinformatics, 54, 179-194 (2004).
All Publications
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