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“NMR Investigation of protein folding and misfolding”

NMR folding of the collagen triple helix and misfolding in disease

Collagen, with a (Gly-X-Y)_n repetitive triple-helical motif, presents a rod-like folded form in which the Gly residues are all buried near a central axis, while the residues in the X and Y positions are largely exposed to solvent. The substitution of a single Gly by another amino acid breaks the characteristic repeating (Gly-X-Y)_n sequence pattern and results in connective tissue disease. Defective folding has been implicated in the etiology of the disease and the nature of the folding defect remains to be defined. Our laboratory has been investigating the NMR conformation, dynamics and folding of normal and mutant collagen-like triple helical peptides for many years in an effort to understand basic protein folding issues and to understand the basis of collagen folding diseases.

The folding of the collagen triple helix is a complex, multi step process that includes nucleation of monomer chains containing unusually high concentrations of proline and hydroxyproline, followed by propagation to the folded trimer form. Triple-helical model peptides offer an approach for isolating and characterizing these individual events and for studying the influence of specific Gly-X-Y triplets on these steps. We are designing triple-helical model peptides for biophysical characterization with selectively enriched ¹⁵N residues for NMR studies. Using these peptides, we have characterized the conformational heterogeneity of various unfolded monomer forms, the conformation and dynamics of various trimer forms and long lived transient kinetic intermediate states. Information about the NMR properties of these states have been combined with 2D NMR real time folding and real time diffusion experiments to obtain a detailed structural and kinetic picture of the folding pathway of the triple helix. Detailed structural and kinetic characterization of the folding pathway of the normal triple helix will serve as a basis for understanding the folding defect arising from Gly-->X mutations. We are pinpointing the stage in folding at which the defect is occurring by comparing NMR conformation, dynamics and folding of the unfolded, long-lived intermediate and trimer states of peptides with Gly-->X mutations along with real time folding experiments. Specifically, ¹⁵N labeled peptides are being designed to investigate the role of the local sequences surrounding the substitution site, the role of sequences N terminal to the substitution site and the role of the substituting residue.

Characterization of the unfolded forms of alpha synucleins and variants by NMR and by computational approaches

Defective folding has been implicated in the etiology of a number of degenerative diseases such as Parkinson's, Alzheimer's, type II diabetes, Huntington's disease and amyloidosis. a-Synuclein is a “natively unfolded” 14kD protein of unknown function that has been implicated in Parkinson's disease pathogenesis. Numerous studies have established the in vitro conversion of unfolded a-synuclein to a filamentous B-sheet aggregate. Despite exhaustive research efforts, the mechanism by which a-synuclein transforms, from a soluble unfolded protein to an insoluble aggregate, remain unclear.

We are comparing the equilibrium unfolded states of a-synuclein with the homologous proteins B, y-synuclein and mouse a-synuclein in order to study the relationship between amino acid sequence and protein misfolding. Although human a-synuclein is highly homologous in amino acid sequence to mouse a-synuclein, B and y-synuclein, it has been shown that the rate of fibrillization is highly dependent on the identity of the protein. We are characterizing the unfolded forms of human a-synuclein and variants by using both experimental NMR and computational approaches. Questions that will be addressed include: What are the conformation and dynamics of the different unfolded forms? What are the timescales of the fluctuations and the extent of residual secondary structure in the different species and how do these relate to the rate of fibril formation?

NMR investigations of de novo helical proteins

In collaboration with Professor Michael Hecht (Princeton University) we are focusing on the design and NMR characterization of novel proteins. Professor Hecht has pioneered an approach that goes beyond the residue-by-residue design of individual amino acid sequences, and enables construction of vast libraries of de novo proteins. Their approach is based on the premise that novel proteins need not be designed atom-by-atom but rather designed using polar/nonpolar patterning as a cornerstone for de novo protein design. The strategy uses a “binary code”, which specifies only whether a given position is hydrophobic or hydrophilic. Since the precise identity of each polar or nonpolar residue is not specified, the binary code facilitates the design and construction of libraries with enormous combinatorial diversity. Our group is using NMR to demonstrate that most of the proteins in a library of 102 residue sequences fold into a-helical structures. We have recently solved the solution structure of a protein from this library and have shown that the de novo protein forms a 4-helix bundle with nonpolar residues on the interior and polar residues on the surface. Further work is in progress to compare the structure and dynamics of other proteins in the library in order to understand how proteins with very high sequence homology can form species ranging from molten globules to well folded 4-helix bundle proteins.

Y. Li, S. Kim, B. Brodsky and J. Baum, “Identification of partially disordered peptide intermediates through residue specific NMR diffusion measurements”, J. Am. Chem. Soc. in press (2005).

A. Mohs, Y. Li, E. Doss-Pepe, J. Baum and B. Brodsky. “Stability junction at a common mutation site in the collagenous domain of the mannose binding lectin” Biochemistry 2005, 44, 1793.

A. Buevich, T. Silva, B. Brodsky and J. Baum. “Transformation of the mechanism of triple-helix peptide folding in the absence of a C-terminal nucleation domain and its implications for mutations in collagen disorders” J. Biol. Chem. 2004, 279, 46890.

S. Kim and J. Baum. “An on/off resonance rotating frame experiment to monitor millisecond to microsecond timescale dynamics” Journal of Biomolecular NMR, 2004, 30, 195.

Y. Xu, T. Hyde, X. Wang, M. Bhate, B. Brodsky, and Jean Baum. “NMR and CD Evidence for Efficient Folding of a Triple-Helical Peptide by Imino Acid Restriction of the Unfolded State”, Biochemistry 2003, 42, 8696.

Y. Wei, S. Kim, D. Fela, J. Baum, M. Hecht. “Solution structure of a de novo protein from a designed combinatorial library”, Proceedings of the National Academy of Science 2003, 100, 13270.

A. Buevich and J. Baum. “Defining the Aggregation States of Protein Folding Intermediates by Residue Specific Real Time NMR Diffusion Experiments”, J. Am. Chem. Soc. 2002, 124, 7156.