The development of multidimensional nuclear magnetic resonance (NMR) spectroscopy combined with modern molecular biology methods that enable stable, NMR active isotopes to be incorporated has increased the use of this technique for yielding structural and dynamic information on macromolecules such as proteins and nucleic acids.1,2 In spite of the numerous documented successes of solution-state NMR spectroscopy in generating atomic-resolution images of large molecules, a number of important biological systems cannot be analyzed directly using this approach because they are too large or exist in an inherently non-crystalline solid state environment. Examples of such systems include highly-ordered aggregates commonly referred to as amyloid fibrils.3,4
Amyloid fibrils are characterized by an elongated thread-like morphology, and generally believed to exhibit “cross-β” architecture with individual peptide strands arranged perpendicular to the fibril axis. Light-chain amyloidosis (AL) is the most prevalent systemic amyloidosis in the United States and is characterized by the deposition of Ig VL amyloid fibrils in organs such as kidneys or heart.5
Our initial studies, discussed in Chapter 2 of this thesis and published in 2009 in Biomolecular NMR Assignement,6 present the backbone resonance assignments for the LEN protein, which displays a high degree of sequence identity with another human immunoglobulin, SMA. In a subsequent series of studies, described in Chapter 3 and published in 2011 in Biochemistry,7 the conformational flexibility of LEN was investigated at physiological and acidic pH by multidimensional solution-state NMR methods. 15N Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR data show that LEN experiences considerable slower, millisecond time scale dynamics, confined primarily to three contiguous segments encompassing the N-terminal β-strand and complementarity determining loop regions (CDR) 2 and 3 in the vicinity of the dimer interface. Quantitative analysis of the CPMG relaxation dispersion data reveals that at physiological pH these slow backbone motions are associated with relatively low excited-state protein conformer populations. Upon acidification, the minor conformer populations increase significantly with most residues involved in stabilizing interactions across the dimer interface displaying increased flexibility. In Chapter 4, we extended the 15N-CMPG relaxation experiments to the pathogenic SMA protein as well as eight SMA-like point mutants of LEN to assess the impact of each mutation on the stability of the dimer interface.
In Chapter 5 the characterization of the amyloid core region of the LEN fibril, using DMSO-quenched hydrogen/deuterium (H/D) exchange combined with 2D solution-state NMR,9 and we compare these H/D exchange data to those recorded for LEN under native conditions.
Finally, in Chapter 6 we present some ongoing work on a different amyloid forming system, the Y145Stop mutant of the prion protein (PrP). Specifically, as part of a larger group effort to obtain structural information on the PrP fibrils, the mass-per-length (MPL) values, which serve as strong constraints on molecular structure,10 were calculated by quantification of intensities in dark-field electron microscope images obtained in the tilted-beam mode of a transmission electron microscope (TEM).