Recently developed solid state multiple-quantum NMR methods are applied to extended coupling networks, where direct dipole-dipole interactions can be used to create coherences of very high order ( -1(0). The progressive development of multiple-quantum coherence over time depends upon the formation of multiple-spin correlations, a phenomenon which also accompanies the normal decay to equilibrium of the free induction signal in a solid. Both the time development and the observed distributions of coherence can be approached statistically, with the spin system described by a time-dependent density operator whose elements are completely uncorrelated at sufficiently long times. With this point of view, we treat the distribution of coherence in a multiple-quantum spectrum as Gaussian, and characterize a spectrum obtained for a ~iven preparation time by its variance. The variance of the distribution is associated roughly wIth the number of coupled spins effectively interacting, and its steady growth with time reflects the continual expansion of the system under the action of the dipolar interactions. The increase in effective system "size" is calculated for a random walk model for the time development of the density operator. Experimental results are presented for hexamethylbenzene, adamantane, and squaric acid. The formation of coherence in systems containing physically isolated clusters is also investigated, and a simple method for estimating the number of spins involved is demonstrated.
NMR spectroscopy has been used to investigate the structure of a partially folded state of a protein, the molten globule or A-state of alpha-lactalbumin. The 1H NMR spectrum of this species differs substantially from those of both the native and fully unfolded states, reflecting the intermediate level of order. The resolution in the spectrum is limited by the widespread overlap and substantial line widths of many of the resonances. Methods have therefore been developed that exploit the well-resolved spectrum of the native protein to probe indirectly the A-state. A number of resonances of the A-state have been found to be substantially shifted from their positions in the spectrum of the unfolded state and have been identified through magnetization transfer with the native state, under conditions where the two states are interconverting. The most strongly perturbed residues in the A-state were found to be among those that form a hydrophobic core to the native structure. A number of amides were found to be highly protected from solvent exchange in the A-state. These have been identified through pH-jump experiments, which label them in the spectrum of the native protein. They were found to occur mainly in segments that are helical in the native structure. These results enable a model of the A-state to be proposed in which significant conformational freedom exists but where specific elements of native-like structure are preserved.
We describe a class of continuously phase modulated radiation pulses that result in coherent population inversion on resonance as well as over a large range of transition frequencies and radiation field strengths. This is a population inversion analogy to Self Induced Transparency.Simulations of the inversion properties of the modulated inversion pulse (MIP) are presented. It is shown that the inversion behavior can be explained by treating the MIP as a highly efficient adiabatic sweep.Criteria for establishing adiabaticity are discussed in detail . Finally, a method is presented for generating a sequence of phase shifted radio frequency pulses, from the continously modulated pulse, which can be implemented on modern NMR and coherent optical spectrometers; experimental confirmation is given. measurements, spin or photon echoes' and spin decoupling. The simplest way to coherently invert populations is with a single n pulse,i.e. a pulse of radiation such that the product of amplitude in angular frequency units and the time in seconds equals n. For good population inversion to be achieved, the difference between the radiation frequency and the resonant frequency of the transition for which the populations are to be inverted must be much smaller than the radiation amplitude. In other words, the inversion bandwidth of a single n pulse is quite limited.Often it is the case experimentally that the bandwidth of resonant frequencies is comparable to or greater than the available radiation amplitude. In NMR, the bandwidth may result from static magnetic field gradients, chemical shifts or spin couplings. In coherent optics, this may be due to inhomogeneous broadening from crystal strains or Doppler shifts.An established technique in NMR for inverting spin populations over a large bandwidth is Adiabatic Rapid Passage 5 , in which the frequency of applied radio frequency (rf) radiation is swept through the resonances at a constant rate that is small compared to the rf amplitude but large compared to the inverse of the relaxation times. Adiabatic sweeps have beenemp oye ln co erent optlCS as we • An alternative approach to broadband inversion in NMR was proposed some time ago by Levitt and 12 Freeman • They suggested using a sequence of phase-shifted pulses, collectively called a compostie n pulse, to produce inversion over a broad The relation to a composite n pulse arises from considering a composite n pulse as a single phase-modulated pulse, with a piecewise-constant phase function. A composite n pulse may then be regarded as an approximation of a continously phase-modulated pulse. One way to generate composite n pulses would be by approximating the continously varying phase function of a pulse similar to that of Equations (1) and (2) by a piecewise-constant function. Procedures for generating composite n pulses from continously phase modulated pulses are developed below. B. OrganizationIn Section II, the class of phase-modulated, constant-amplitude pulses first presented in reference 26 is derived from considerat...
The conformational properties of soluble a-synuclein, the primary protein found in patients with Parkinson's disease, are thought to play a key role in the structural transition to amyloid fibrils. In this work, we report that recombinant 100% N-terminal acetylated a-synuclein purified under mild physiological conditions presents as a primarily monomeric protein, and that the N-terminal acetyl group affects the transient secondary structure and fibril assembly rates of the protein. Residue-specific NMR chemical shift analysis indicates substantial increase in transient helical propensity in the first 9 N-terminal residues, as well as smaller long-range changes in residues 28-31, 43-46, and 50-66: regions in which the three familial mutations currently known to be causative of early onset disease are found. In addition, we show that the N-terminal acetylated protein forms fibrils that are morphologically similar to those formed from nonacetylated a-synuclein, but that their growth rates are slower. Our results highlight that N-terminal acetylation does not form significant numbers of dimers, tetramers, or higher molecular Abbreviations: asyn, alpha synuclein; Ac-asyn, acetylated asyn; BOG, b-octyl glucoside; EM, fluorescence electron microscopy; ESI-IMS-MS, ion mobility spectrometry combined with ESI-MS; ESI-MS, noncovalent electrospray ionization mass spectrometry; IDP, intrinsically disordered protein; NatB, N-acetyltransferase B; SEC, analytical size-exclusion chromatography; ThT, Thioflavin T.Additional Supporting Information may be found in the online version of this article. † Lijuan Kang and Gina M. Moriarty contributed equally to this work. weight species, but does alter the conformational distributions of monomeric a-synuclein species in regions known to be important in metal binding, in association with membranes, and in regions known to affect fibril formation rates.
Summaryα-synuclein is an intrinsically disordered protein that appears in aggregated forms in the brains of patients with Parkinson's Disease. The conversion from monomer to aggregate is complex and aggregation rates are sensitive to changes in amino acid sequence and environmental conditions. It has previously been observed that α-synuclein aggregates faster at low pH than at neutral pH. Here, we combine NMR spectroscopy and molecular simulations to characterize α-synuclein conformational ensembles at both neutral and low pH in order to understand how the altered charge distribution at low pH changes the structural properties of these ensembles and leads to an increase in aggregation rate. The N-terminus, which has a small positive charge at neutral pH due to a balance of positively and negatively charged amino acid residues, is very positively charged at low pH. Conversely, the acidic C-terminus is highly negatively charged at neutral pH and becomes essentially neutral and hydrophobic at low pH. Our NMR experiments and REMD simulations indicate that there is a significant structural reorganization within the low pH ensemble relative to that at neutral pH in terms of long range contacts, hydrodynamic radius, and the amount of heterogeneity within the conformational ensembles. At neutral pH there is a very heterogeneous ensemble with transient contacts between the N-terminus and the NAC, however at low pH there is a more homogeneous ensemble which exhibits strong contacts between the NAC and the C-terminus. At both pHs, transient contacts between the N-and C-termini are observed, the NAC region shows similar exposure to solvent, and the entire protein shows similar propensities to secondary structure. Based on the comparison of the neutral and low pH conformational ensembles, we propose that exposure of the NAC region to solvent and the secondary structure propensity are not factors that account for differences in propensity to aggregate in this context. Instead, the comparison of the neutral and low pH ensembles suggests that the change in long-range interactions between the low and neutral pH ensembles, the compaction of the C-terminal region at low pH and the uneven distribution of charges across the sequence are key to faster aggregation.
Two-dimensional 1H-NMR spectroscopy has been used to study the acid-denatured molten globule (A-state) of alpha-lactalbumin. The NMR spectra show that chemical shift dispersion is limited but significantly greater than that expected for a random coil conformation. The small chemical shift dispersion of side-chain resonances in the A-state together with line broadening associated with conformational averaging indicates that most of the long-range tertiary structure in the A-state is likely to be nonspecific. Side-chain resonances in the A-state are generally shifted somewhat upfield of random coil values; this and the observation of a large number of interresidue NOEs, however, indicate that some side-chain interactions, at least at the level of hydrophobic clustering, exist in the A-state. Analysis of NOESY spectra shows no evidence for an ordered structure for either of the two major clusters of aromatic residues which in the native structure make up part of the hydrophobic core of the helical domain of the native protein. A new aromatic cluster in the A-state which results from rearrangement of the side chains of Tyr103, Trp104, and His107 from their native state positions was, however, detected by a number of well-defined interresidue NOE effects. Similar NOE patterns are observed in a peptide corresponding to residues 101-110 of alpha-lactalbumin in trifluoroethanol, suggesting that the non-native structure in the 101-110 region of the A-state is not dependent on specific interactions with the rest of the chain. Trapping experiments indicate that amide protons from regions of the sequence which in the native state are helical are among those strongly protected from solvent exchange in the A-state; those from one of the helices (the C helix) were specifically identified. Taken together, these results reinforce a model of the A-state which has stable regions of localized secondary structure but a largely disordered tertiary structure.
Homonuclear and heteronuclear 2D NMR methods are used to study two triple-helical peptides. One peptide, (POG)10, is considered to be the most stable prototype of a triple helix. The second peptide, (POG)3ITGARGLAGPOG(POG)3 (denoted T3-785), was designed to model an imino acid poor region of collagen and contains 12 residues from near the unique collagenase cleavage site in type III collagen. Both peptides associated as trimers, with melting temperatures of 60 degrees C for (POG)10 and 25 degrees C for the T3-785 peptide. Sequence-specific assignments were made for a tripeptide unit POG in (POG)10, and 80% of the POG triplets are found to be in an equivalent environment. In T3-785, with nonrepeating X-Y-Gly units incorporated in the sequence, the three chains of the homotrimer can be distinguished from one another by NMR. The solution conformation of (POG)10 is very similar to the model derived from X-ray fiber diffraction data, although the peptide contains less ordered regions at the peptide ends. In the trimer from of T3-785, the central residues of the three chains are closely packed, and the data are consistent with a triple-helical model with a one-residue stagger of three parallel chains. For T3-785, in contrast to (POG)10, there are also resonances from a less ordered form, which are probably due to the presence of a small amount of monomer. The similarity of the backbone conformations of T3-785 and (POG)10 suggests that an alternative conformation is not present in the imino acid poor region.
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