Nucleation of Huntingtin Aggregation Proceeds via Conformational Conversion of Pre‐Formed, Sparsely‐Populated Tetramers

Abstract Pathogenic huntingtin exon‐1 protein (httex1), characterized by an expanded polyglutamine tract located between the N‐terminal amphiphilic region and a C‐terminal polyproline‐rich domain, forms fibrils that accumulate in neuronal inclusion bodies, and is associated with a fatal, autosomal dominant neurodegenerative condition known as Huntington's disease. Here a complete kinetic model is described for aggregation/fibril formation of a httex1 construct with a 35‐residue polyglutamine repeat, httex1Q35. Using exchange NMR spectroscopy, it is previously shown that the reversible formation of a sparsely‐populated tetramer of the N‐terminal amphiphilic domain of httex1Q35, comprising a D2 symmetric four‐helix bundle, occurs on the microsecond time‐scale and is a prerequisite for subsequent nucleation and fibril formation on a time scale that is many orders of magnitude slower (hours). Here a unified kinetic model of httex1Q35 aggregation is developed in which fast, reversible tetramerization is directly linked to slow irreversible fibril formation via conversion of pre‐equilibrated tetrameric species to “active”, chain elongation‐capable nuclei by conformational re‐arrangement with a finite, monomer‐independent rate. The unified model permits global quantitative analysis of reversible tetramerization and irreversible fibril formation from a time series of 1H‐15N correlation spectra recorded during the course of httex1Q35 aggregation

Precision Measurements of Deuterium Isotope Effects on the Chemical Shifts of Backbone Nuclei in Proteins: Correlations with Secondary Structure

Precision NMR measurements of deuterium isotope effects on the chemical shifts of backbone nuclei in proteins (¹⁵N, ¹³CO, ¹³C_α, and ¹HN) arising from ¹H-to-²H substitutions at aliphatic carbon sites. Isolation of molecular species with a defined protonation/deuteration pattern at carbon-α/β positions allows distinguishing and accurately quantifying different isotope effects within the protein backbone. The isotope shifts measured in the partially deuterated protein ubiquitin are interpreted in terms of backbone geometry via empirical relationships describing the dependence of isotope shifts on (φ; ψ) backbone dihedral angles. Because of their relatively large magnitude and clear dependence on the protein secondary structure, the two- and three-bond backbone amide ¹⁵N isotope shifts, ²ΔN­(C_α,iD) and ³ΔN­(C_α,i‑1D), can find utility for NMR structural refinement of small-to-medium size proteins

Estimating Side-Chain Order in [U‑²H;¹³CH₃]‑Labeled High Molecular Weight Proteins from Analysis of HMQC/HSQC Spectra

A simple approach for quantification of methyl-containing side-chain mobility in high molecular weight methyl-protonated, uniformly deuterated proteins is described, based on the measurement of peak intensities in methyl ¹H–¹³C HMQC and HSQC correlation maps and relaxation rates of slowly decaying components of methyl ¹H–¹³C multiple-quantum coherences. A strength of the method is that [U-²H;¹³CH₃]-labeled protein samples are required that are typically available at an early stage of any analysis. The utility of the methodology is demonstrated with applications to three protein systems ranging in molecular weight from 82 to 670 kDa. Although the approach is only semiquantitative, a high correlation between order parameters extracted via this scheme and other more established methods is nevertheless demonstrated

Selective detection of 13CHD2 signals from a mixture of 13CH3/13CH2D/ 13CHD2 methyl isotopomers in proteins

In NMR spectra of partially deuterated proteins methyl correlations are commonly observed as a combination of signals from 13CH3, 13CH2D and 13CHD2 isotopomers. In a number of NMR applications, methyl groups of the 13CHD2 variety are targeted because of their AX-like character and concomitant simplification of the involved relaxation mechanisms. Although complete elimination of signals from 13CH2D methyl groups can be easily achieved in such applications, if the magnetization is not transferred through deuterium nuclei, efficient suppression of usually stronger 13CH3 peaks is more problematic. A pair of simple pulse-scheme elements are presented that achieve almost complete suppression of 13CH3 signals in the mixtures of 13CH 3/13CH2D/13CHD2 methyl isotopomers of small proteins at the expense of a moderate (～20-to-40%) reduction in intensities of the targeted 13CHD2 groups. The approaches described are based purely on scalar coupling (1J CH) evolution properties of different 13C and 1H transitions within 13CH3 spin-systems and are superior to magnetization transfer through deuterons with respect to sensitivity of the detected 13CHD2 methyl signals. ? 2010 Elsevier Inc. All rights reserved