Solid state nuclear magnetic resonance methodology and applications to structure determination of peptides, proteins and amyloid fibrils

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2003.Vita.Includes bibliographical references.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Several methodological developments and applications of multidimensional solid-state nuclear magnetic resonance to biomolecular structure determination are presented. Studies are performed in uniformly 3C, 15N isotope labeled samples with magic-angle spinning for optimal resolution and sensitivity. Frequency selective rotational-echo double-resonance (FSR) and three-dimensional transferred-echo double-resonance (3D TEDOR) methods for carbon-nitrogen distance measurements in (U-'3C,S5N)-labeled peptides and proteins are described. FSR employs frequency selective Gaussian pulses in combination with broadband REDOR recoupling to measure dipolar couplings based on the isotropic chemical shifts of the selected 13C-15N spin pairs. The experiment is demonstrated in model peptides, N-acetyl-L-Val-L-Leu and N-formyl-L-Met-L-Leu-L-Phe, where multiple distances in the 3-6 A range are determined with high precision, and in a membrane protein, bacteriorhodopsin, where the distances between aspartic acids Asp-85 and Asp-212 and the retinal Schiff base nitrogen are measured in the active site. The 3D TEDOR methods employ 13C and 15N chemical shift dimensions for site-specific resolution and encode the distance information in the buildup of cross-peak intensities, allowing multiple distances to be measured simultaneously. The methods are demonstrated in N-acetyl-L-Val-L-Leu and N-formyl-L-Met-L-Leu-L-Phe, where 20 and 26 distances up to 6 A are determined, respectively. The molecular conformation of peptide fragment 105-115 of transthyretin in an amyloid fibril is investigated.(cont.) Complete sequence-specific 13C and 15N backbone and side- chain resonance assignments are obtained using two-dimensional 13C-13C and 15N-13C-3C chemical shift correlation experiments. Backbone torsion angles are measured directly using three-dimensional dipolar-chemical shift correlation experiments, which report on the relative orientations of 3C-15N, 3C-1H and 15N-'H dipolar tensors, and intramolecular 13C-15N distances in the 3-5 A range are determined using 3D TEDOR, resulting in about 60 constraints on the peptide structure. An atomic-resolution structure of the peptide consistent with the NMR constraints is calculated using simulated annealing molecular dynamics, and the results indicate that the peptide adopts an extended β-strand conformation in the fibril.by Christopher Peter Jaroniec.Ph.D

Hydrogen bonding in duplex DNA probed by DNP enhanced solid-state NMR N-H bond length measurements

Numerous biological processes and mechanisms depend on details of base pairing and hydrogen bonding in DNA. Hydrogen bonds are challenging to quantify by X-ray crystallography and cryo-EM due to difficulty of visualizing hydrogen atom locations but can be probed with site specificity by NMR spectroscopy in solution and the solid state with the latter particularly suited to large, slowly tumbling DNA complexes. Recently, we showed that low-temperature dynamic nuclear polarization (DNP) enhanced solid-state NMR is a valuable tool for distinguishing Hoogsteen base pairs (bps) from canonical Watson-Crick bps in various DNA systems under native-like conditions. Here, using a model 12-mer DNA duplex containing two central adenine-thymine (A-T) bps in either Watson-Crick or Hoogsteen confirmation, we demonstrate DNP solid-state NMR measurements of thymine N3-H3 bond lengths, which are sensitive to details of N-H···N hydrogen bonding and permit hydrogen bonds for the two bp conformers to be systematically compared within the same DNA sequence context. For this DNA duplex, effectively identical TN3-H3 bond lengths of 1.055 ± 0.011 Å and 1.060 ± 0.011 Å were found for Watson-Crick A-T and Hoogsteen A (syn)-T base pairs, respectively, relative to a reference amide bond length of 1.015 ± 0.010 Å determined for N-acetyl-valine under comparable experimental conditions. Considering that prior quantum chemical calculations which account for zero-point motions predict a somewhat longer effective peptide N-H bond length of 1.041 Å, in agreement with solution and solid-state NMR studies of peptides and proteins at ambient temperature, to facilitate direct comparisons with these earlier studies TN3-H3 bond lengths for the DNA samples can be readily scaled appropriately to yield 1.083 Å and 1.087 Å for Watson-Crick A-T and Hoogsteen A (syn)-T bps, respectively, relative to the 1.041 Å reference peptide N-H bond length. Remarkably, in the context of the model DNA duplex, these results indicate that there are no significant differences in N-H···N A-T hydrogen bonds between Watson-Crick and Hoogsteen bp conformers. More generally, high precision measurements of N-H bond lengths by low-temperature DNP solid-state NMR based methods are expected to facilitate detailed comparative analysis of hydrogen bonding for a range of DNA complexes and base pairing environments

Insights into the mode of action of a putative zinc transporter CzrB in thermus thermophilus

peer-reviewedThis paper was obtained through PEER (Publishing and the Ecology of European Research) http://www.peerproject.euThe crystal structures of the cytoplasmic domain of the putative zinc transporter CzrB in the apoand zinc-bound forms reported herein are consistent with the protein functioning in vivo as a homodimer. NMR, X-ray scattering and size exclusion chromatography provide support for dimer formation. Full-length variants of CzrB in the apo and zinc-loaded states were generated by homology modelling with the Zn2+ / H+ antiporter YiiP. The model suggests a way in which zinc binding to the cytoplasmic fragment creates a docking site to which a metallochaperone can bind for delivery and transport of its zinc cargo. Since the cytoplasmic domain may exist in the cell as an independent, soluble protein a proposal is advanced that it functions as a metallochaperone and that it regulates the zinc-transporting activity of the full-length protein. The latter requires that zinc binding becomes uncoupled from the creation of a metallochaperone-docking site on CzrB

Half a century of amyloids: past, present and future

Amyloid diseases are global epidemics with profound health, social and economic implications and yet remain without a cure. This dire situation calls for research into the origin and pathological manifestations of amyloidosis to stimulate continued development of new therapeutics. In basic science and engineering, the cross-ß architecture has been a constant thread underlying the structural characteristics of pathological and functional amyloids, and realizing that amyloid structures can be both pathological and functional in nature has fuelled innovations in artificial amyloids, whose use today ranges from water purification to 3D printing. At the conclusion of a half century since Eanes and Glenner's seminal study of amyloids in humans, this review commemorates the occasion by documenting the major milestones in amyloid research to date, from the perspectives of structural biology, biophysics, medicine, microbiology, engineering and nanotechnology. We also discuss new challenges and opportunities to drive this interdisciplinary field moving forward. This journal i

Rapid Quantitative Measurements of Paramagnetic Relaxation Enhancements in Cu(II)-Tagged Proteins by Proton-Detected Solid-State NMR Spectroscopy

We demonstrate rapid quantitative measurements of site-resolved paramagnetic relaxation enhancements (PREs), which are a source of valuable structural restraints corresponding to electron–nucleus distances in the ∼10–20 Å regime, in solid-state nuclear magnetic resonance (NMR) spectra of proteins containing covalent Cu²⁺-binding tags. Specifically, using protein GB1 K28C-EDTA-Cu²⁺ mutant as a model, we show the determination of backbone amide ¹⁵N longitudinal and ¹H transverse PREs within a few hours of experiment time based on proton-detected 2D or 3D correlation spectra recorded with magic-angle spinning frequencies ≥ ∼ 60 kHz for samples containing ∼10–50 nanomoles of ²H,¹³C,¹⁵N-labeled protein back-exchanged in H₂O. Additionally, we show that the electron relaxation time for the Cu²⁺ center, needed to convert PREs into distances, can be estimated directly from the experimental data. Altogether, these results are important for establishing solid-state NMR based on paramagnetic-tagging as a routine tool for structure determination of natively diamagnetic proteins