Optimized co-solute paramagnetic relaxation enhancement for the rapid NMR analysis of a highly fibrillogenic peptide

Abstract

Co-solute paramagnetic relaxation enhancement (PRE) is an attractive way to speed up data acquisition in NMR spectroscopy by shortening the T 1 relaxation time of the nucleus of interest and thus the necessary recycle delay. Here, we present the rationale to utilize high-spin iron(III) as the optimal transition metal for this purpose and characterize the properties of its neutral chelate form Fe(DO3A) as a suitable PRE agent. Fe(DO3A) effectively reduces the T 1 values across the entire sequence of the intrinsically disordered protein α-synuclein with negligible impact on line width. The agent is better suited than currently used alternatives, shows no specific interaction with the polypeptide chain and, due to its high relaxivity, is effective at low concentrations and in 'proton-less' NMR experiments. By using Fe(DO3A) we were able to complete the backbone resonance assignment of a highly fibrillogenic peptide from α1-antitrypsin by acquiring the necessary suite of multidimensional NMR datasets in 3 h.

Speeding up NMR by in Situ Photo‐Induced Reversible Acceleration of T1‐Relaxation (PIRAT)

“…It is well established that the addition of inert paramagnetic compounds to an NMR sample enhances the longitudinal relaxation time, T 1 , causing higher signal intensities (Paramagnetic Relaxation Enhancement, PRE) . However, the application of PRE is limited because NMR lines may become substantially broadened . We have developed a concept showing that it is possible profiting from the PRE effect without suffering its downside.…”

“…Here we will follow as omehow alternative approach: We introduce the concept of accelerating the acquisitiono fN MR spectra by selectively manipulating the relaxation rates during the NMR experiment.It is well established that the addition of inert paramagnetic compounds to an NMR sample enhances the longitudinal relaxationt ime, T 1 ,c ausing higher signal intensities (Paramagnetic Relaxation Enhancement, PRE). [11][12][13][14] However,t he application of PRE is limited because NMR lines may become substantially broadened. [11] We have developed ac oncept showingt hat it is possible profiting from the PRE effect withouts uffering its downside.…”

“…[11][12][13][14] However,t he application of PRE is limited because NMR lines may become substantially broadened. [11] We have developed ac oncept showingt hat it is possible profiting from the PRE effect withouts uffering its downside. In multidimensionalN MR pulse sequences are ap-plied in ar epetitive manner.D uring the data acquisition period and the subsequentd elay (relaxationd elay) the z-magnetization relaxesb ack to its equilibrium value.…”

Critical Influence of Cosolutes and Surfaces on the Assembly of Serpin-Derived Amyloid Fibrils

“…In this study, we provide a comprehensive kinetic description of surface and solution effects on a proteolytically generated amyloid peptide (C-36) from the C-terminal region of the a1-antitrypsin (a 1 AT) serine protease inhibitor. C-36 is intrinsically disordered in solution (42), and has been found in various bodily fluids, in atherosclerotic plaques, and in alveolar fibrillar deposits (43,44). C-36 is bioactive (43) and is also represented in newly detected C-terminal transcripts of the SERPIN1A gene (45).…”

Solution NMR Experiment for Measurement of 15N–1H Residual Dipolar Couplings in Large Proteins and Supramolecular Complexes

“…Moreover, increased sensitivity offered by 1.0 – 1.2 GHz spectrometers close to the ‘magic TROSY field’, 1a along with sensitivity enhancement from paramagnetic relaxation agents 23 and possibly dynamic nuclear polarization, 24 promise to enable N-H RDC measurement up to τ c ~ 200 ns. For even longer τ c , residual 1 H density can be reduced by adding 50% 2 H 2 O, and cross-relaxation induced polarization transfer (CRIPT) 25 can be employed.…”