Dynamic Nuclear Polarization of Amyloidogenic Peptide Nanocrystals:  GNNQQNY, a Core Segment of the Yeast Prion Protein Sup35p

Abstract

Dynamic nuclear polarization (DNP) permits a approximately 10(2)-10(3) enhancement of the nuclear spin polarization and therefore increases sensitivity in nuclear magnetic resonance (NMR) experiments. Here, we demonstrate the efficient transfer of DNP-enhanced (1)H polarization from an aqueous, radical-containing solvent matrix into peptide crystals via (1)H-(1)H spin diffusion across the matrix-crystal interface. The samples consist of nanocrystals of the amyloid-forming peptide GNNQQNY(7-13), derived from the yeast prion protein Sup35p, dispersed in a glycerol-water matrix containing a biradical polarizing agent, TOTAPOL. These crystals have an average width of 100-200 nm, and their known crystal structure suggests that the size of the biradical precludes its penetration into the crystal lattice; therefore, intimate contact of the molecules in the nanocrystal core with the polarizing agent is unlikely. This is supported by the observed differences between the time-dependent growth of the enhanced polarization in the solvent versus the nanocrystals. Nevertheless, DNP-enhanced magic-angle spinning (MAS) spectra recorded at 5 T and 90 K exhibit an average signal enhancement epsilon approximately 120. This is slightly lower than the DNP enhancement of the solvent mixture surrounding the crystals (epsilon approximately 160), and we show that it is consistent with spin diffusion across the solvent-matrix interface. In particular, we correlate the expected DNP enhancement to several properties of the sample, such as crystal size, the nuclear T(1), and the average (1)H-(1)H spin diffusion constant. The enhanced (1)H polarization was subsequently transferred to (13)C and (15)N via cross-polarization, and allowed rapid acquisition of two-dimensional (13)C-(13)C correlation data.

Dynamic nuclear polarization of spherical nanoparticles

“…In a study performed on 100-200 nm thick GNNQQNY peptide crystals by van der Wel et al, 59 only a slight decrease (B25%) in the proton DNP enhancement was observed when the enhancement of the radical containing solvent and the isolated protein enhancements were compared. The solvent signals show the maximum proton enhancement since the radical polarizes this part first.…”

250GHz CW gyrotron oscillator for dynamic nuclear polarization in biological solid state NMR

“…Two research areas in which high resolution MAS experiments have proved especially successful are studies of amyloid fibrils [37,[51][52][53][54][55][56][57] and membrane proteins [42,[58][59][60][61][62][63][64][65][66][67][68][69][70][71][72][73][74][75]. However, in both of these cases, low sensitivity currently limits the information that can be gleaned from the spectra.…”

“…However, in both of these cases, low sensitivity currently limits the information that can be gleaned from the spectra. Accordingly, we recently demonstrated the use of DNP to enhance signal intensities in MAS spectra of amyloidiogenic nanocrystals [37] and we are currently utilizing DNP to improve the sensitivity of MAS spectra of the membrane protein bacteriorhodopsin (bR) [42,76,77]. In order to motivate the reader's interest in DNP, we devote this section to illustrating some of the scientific experiments that are possible with the increased sensitivity that is currently available, and the applicability of the DNP technique to scientific questions involving membrane proteins.…”

“…In the 140 GHz system mentioned above and the 250 GHz system considered here, the optimization is accomplished with a superconducting sweep coil, but in the future tunable gyrotrons [86] will likely become available. In the case of the 140 GHz/211 MHz system described previously, we generally employ long (~15-30 s) microwave pulses since the vacuum pumping efficiency in that system is insufficient for true CW operation [30,33] and the 1 H polarization appears in 3-5 T 1 's [34,37]. In contrast, the 250 GHz gyrotron operates in true CW mode and we apply between 4.0 and 4.5 watts of microwave power continuously to maintain a steady state 1 H polarization.…”

“…This system permitted us to demonstrate DNP at 5T fields and to explore many important features of the experiments. For example, we established that cross effect DNP using biradical polarizing agents is the optimal mechanism [34][35][36][37][38][39][40][41] for high field experiments involving CW microwave radiation. Traditional approaches, based on the solid effect and thermal mixing, yield enhancements that are an order of magnitude smaller [38] or require high concentrations of polarizing agents that lead to significant electron-nuclear dipolar broadening [42].…”