Chemical exchange saturation transfer (CEST) is a magnetization transfer (MT) technique to indirectly detect pools of exchangeable protons through the water signal. CEST MRI has focused predominantly on signals from exchangeable protons downfield (higher frequency) from water in the CEST spectrum. Low power radiofrequency (RF) pulses can slowly saturate protons with minimal interference of conventional semi-solid based MT contrast (MTC). When doing so, saturation-transfer signals are revealed upfield from water, which is the frequency range of non-exchangeable aliphatic and olefinic protons. The visibility of such signals indicates the presence of a relayed transfer mechanism to the water signal, while their finite width reflects that these signals are likely due to mobile solutes. It is shown here in protein phantoms and the human brain that these signals build up slower than conventional CEST, at a rate typical for intramolecular nuclear Overhauser enhancement (NOE) effects in mobile macromolecules such as proteins/peptides and lipids. These NOE-based saturation transfer signals show a pH dependence, suggesting that this process is the inverse of the well-known exchange-relayed NOEs in high resolution NMR protein studies, thus an relayed-NOE CEST process. When studying 6 normal volunteers with a low-power pulsed CEST approach, the relayed-NOE CEST effect was about twice as large as the CEST effects downfield and larger in white matter than gray matter. This NOE contrast upfield from water provides a way to study mobile macromolecules in tissue. First data on a tumor patient show reduction in both relayed NOE and CEST amide proton signals leading to an increase in magnetization transfer ratio asymmetry, providing insight into previously reported amide proton transfer (APT) effects in tumors.

Amyloid fibrils are self-assembled filamentous structures associated with protein deposition conditions including Alzheimer's disease and the transmissible spongiform encephalopathies. Despite the immense medical importance of amyloid fibrils, no atomic-resolution structures are available for these materials, because the intact fibrils are insoluble and do not form diffraction-quality 3D crystals. Here we report the high-resolution structure of a peptide fragment of the amyloidogenic protein transthyretin, TTR(105-115), in its fibrillar form, determined by magic angle spinning NMR spectroscopy. The structure resolves not only the backbone fold but also the precise conformation of the side chains. Nearly complete 13 C and 15 N resonance assignments for TTR(105-115) formed the basis for the extraction of a set of distance and dihedral angle restraints. A total of 76 self-consistent experimental measurements, including 41 restraints on 19 backbone dihedral angles and 35 13 C-15 N distances between 3 and 6 Å were obtained from 2D and 3D NMR spectra recorded on three fibril samples uniformly 13 C, 15 N-labeled in consecutive stretches of four amino acids and used to calculate an ensemble of peptide structures. Our results indicate that TTR(105-115) adopts an extended ␤-strand conformation in the amyloid fibrils such that both the main-and side-chain torsion angles are close to their optimal values. Moreover, the structure of this peptide in the fibrillar form has a degree of long-range order that is generally associated only with crystalline materials. These findings provide an explanation of the unusual stability and characteristic properties of this form of polypeptide assembly.

The ability to measure proton exchange rates in tissue using MRI would be very useful for quantitative assessment of magnetization transfer properties, both in conventional MT imaging and in the more recent chemical exchange saturation transfer (CEST) approach. CEST is a new MR contrast mechanism that depends on several factors, including the exchange rate of labile protons in the agent in a pH-dependent manner. Two new methods to monitor local exchange rate based on CEST are introduced. The two MRI-compatible approaches to measure exchange are quantifying exchange using saturation time (QUEST) dependence and quantifying exchange using saturation power (QUESP) dependence. These techniques were applied to poly-L-lysine (PLL) and a generation-5 polyamidoamine dendrimer (SPD-5) to measure the pH dependence of amide proton exchange rates in the physiologic range. Data were fit both to an analytical expression and to numerical solutions to the Bloch equations. Results were validated by comparison with exchange rates determined by two established spectroscopic methods. The exchange rates determined using the four methods were pooled for the pH-calibration curve of the agents consisting of contributions from spontaneous (k 0 ) acid catalyzed (k a ), and base catalyzed (k b ) exchange rate constants. These constants were k 0 ‫؍‬ 68.9 Hz, k a ‫؍‬ 1 Chemical exchange saturation transfer (CEST) has recently been proposed as a new imaging contrast mechanism (1) in which the radiofrequency-induced saturation of labile protons in the agent is transferred to water protons. The resulting MRI signal intensity depends on a multitude of parameters, including agent concentration, number of exchangeable protons, proton exchange rate, T 1 , T 2 , saturation time, and saturation efficiency (1-8). Of these, the chemical exchange rate is often the parameter of interest that reflects tissue pH and the molecular environment, such as salt or metal content. During saturation, labile protons of the lowconcentration solute are saturated and exchange multiple times with unsaturated protons of the large water pool, resulting in a fractional reduction of the water line. If this exchange rate and the T 1 of water are sufficiently large, there is an amplification of the MR sensitivity with respect to the agent concentration (1). Recently, we showed that macromolecules with multiple amide groups (2) or imino groups (5) can give a CEST effect within the micromolar range. The exchange rates of these proton types have a strong pH dependence in the physiologic range (9,10) and may be useful as pH reporters. Measurement of these rates would be a powerful means for pH calibration (3,4,11-15), but existing technologies for this, such as the water exchange (WEX) sequence (16) and measurement of linewidths, are spectroscopy (MRS) based. As a consequence, they are time consuming for the lower rates because of the low contrast agent concentration and not suitable for measuring the faster rates at high pH because of signal loss due to line broadening. If such ...