Ritu Khurana

A General Model for Amyloid Fibril Assembly Based on Morphological Studies Using Atomic Force Microscopy

Based on atomic force microscopy analysis of the morphology of fibrillar species formed during fibrillation of alpha-synuclein, insulin, and the B1 domain of protein G, a previously described model for the assembly of amyloid fibrils of immunoglobulin light-chain variable domains is proposed as a general model for the assembly of protein fibrils. For all of the proteins studied, we observed two or three fibrillar species that vary in diameter. The smallest, protofilaments, have a uniform height, whereas the larger species, protofibrils and fibrils, have morphologies that are indicative of multiple protofilaments intertwining. In all cases, protofilaments intertwine to form protofibrils, and protofibrils intertwine to form fibrils. We propose that the hierarchical assembly model describes a general mechanism of assembly for all amyloid fibrils.

FOURIER TRANSFORM INFRARED SPECTROSCOPY IN ANALYSIS OF PROTEIN DEPOSITS

Publisher Summary The intrinsic insoluble nature of protein deposits (as in amorphous aggregates, inclusion bodies, amyloid fibrils, etc.) places severe restrictions on the availability of methods required for ascertaining the structure of the material. Fourier transform infrared (FTIR) spectroscopy is well suited for determining structural features of proteins, both in solution and as deposits. Proteins in the form of solutions, thin films (hydrated or dry), solids (including lyophilized or spray-dried powders), or suspensions of precipitates in various solvents can be used for FTIR analysis. The most commonly used methods for FTIR analysis of protein deposits are KBr pellets (in which the dry solid is dispersed in a KBr disk) and diffuse reflectance for dry samples, thin films (using transmission or attenuated total reflectance, or ATR modes) for samples in solution or suspension, and attenuated total reflectance mode for solid, liquid, or suspended samples. For dry solid samples there are several sampling techniques, including powder, mull, alkali halide pellet or disk, and film. Powdered samples can be difficult to analyze because of the high incidence of scattered light, which results in a loss of energy transmitted to the detector. To reduce scattering, samples should be ground to a powder of 5 μm particle size or less, smaller than the wavelength of the radiation.

Do Parallel β-Helix Proteins Have a Unique Fourier Transform Infrared Spectrum?

Abstract Several polypeptides have been found to adopt an unusual domain structure known as the parallel β -helix. These domains are characterized by parallel β -strands, three of which form a single parallel β -helix coil, and lead to long, extended β -sheets. We have used ATR-FTIR (attenuated total reflectance-fourier transform infrared spectroscopy) to analyze the secondary structure of representative examples of this class of protein. Because the three-dimensional structures of parallel β -helix proteins are unique, we initiated this study to determine if there was a corresponding unique FTIR signal associated with the parallel β -helix conformation. Analysis of the amide I region, emanating from the carbonyl stretch vibration, reveals a strong absorbance band at 1638cm −1 in each of the parallel β -helix proteins. This band is assigned to the parallel β -sheet structure. However, components at this frequency are also commonly observed for β -sheets in many classes of globular proteins. Thus we conclude that there is no unique infrared signature for parallel β -helix structure. Additional contributions in the 1638cm −1 region, and at lower frequencies, were ascribed to hydrogen bonding between the coils in the loop/turn regions and amide side-chain interactions, respectively. A 13-residue peptide that forms fibrils and has been proposed to form β -helical structure was also examined, and its FTIR spectrum was compared to that of the parallel β -helix proteins.

Elucidation of the Molecular Mechanism During the Early Events in Immunoglobulin Light Chain Amyloid Fibrillation: Evidence for an Off-pathway Oligomer at Acidic pH

Light chain amyloidosis involves the systemic pathologic deposition of monoclonal light chain variable domains of immunoglobulins as insoluble fibrils. The variable domain LEN was obtained from a patient who had no overt amyloidosis; however, LEN forms fibrils in vitro, under mildly destabilizing conditions. The in vitro kinetics of fibrillation were investigated using a wide variety of probes. The rate of fibril formation was highly dependent on the initial protein concentration. In contrast to most amyloid systems, the kinetics became slower with increasing LEN concentrations. At high protein concentrations a significant lag in time was observed between the conformational changes and the formation of fibrils, consistent with the formation of soluble off-pathway oligomeric species and a branched pathway. The presence of off-pathway species was confirmed by small angle x-ray scattering. At low protein concentrations the structural rearrangements were concurrent with fibril formation, indicating the absence of formation of the off-pathway species. The data are consistent with a model for fibrillation in which a dimeric form of LEN (at high protein concentration) inhibits fibril formation by interaction with an intermediate on the fibrillation pathway and leads to formation of the off-pathway intermediate.

Effect of association state and conformational stability on the kinetics of immunoglobulin light chain amyloid fibril formation at physiological pH

Light chain amyloidosis involves the systemic deposition of fibrils in patients overproducing monoclonal immunoglobulin light chains. The kinetics of fibril formation of LEN, a benign light chain variable domain, were investigated at physiological pH in the presence of urea. Despite the lack of in vivofibril formation, LEN readily forms fibrils in vitro under mildly destabilizing conditions. The effect of low to moderate concentrations of urea on the conformation, association state, stability, and kinetics of fibrillation of LEN were investigated. The conformation of LEN was only slightly affected by the addition of up to 4 m urea. The fibrillation kinetics were highly dependent on protein and urea concentrations, becoming faster with decreasing protein concentration and increasing urea concentration. Changes in spectral probes were concomitant to fibril formation throughout the protein and urea concentration ranges, indicating the absence of off-pathway oligomeric species or amorphous aggregates prior to fibril formation. Reducing the amount of dimers initially present in solution by either decreasing the protein concentration or adding urea resulted in faster fibril formation. Thus, increasing concentrations of urea, by triggering dissociation of dimeric LEN, lead to increased rates of fibrillation.

A model for amyloid fibril formation in immunoglobulin light chains based on comparison of amyloidogenic and benign proteins and specific antibody binding.

In an attempt to understand the mechanism of amyloid fibril formation in light chain amyloidosis, the properties of amyloidogenic (SMA) and benign (LEN) immunoglobulin light chain variable domains (VL) were compared. The conformations of LEN and SMA were measured using secondary and tertiary structural probes over the pH range from 2 and 8. At all pH values, LEN was more stable than SMA. The CD spectra of LEN at pH 2 were comparable to those of SMA atpH 7.5, indicating that the low pH conformation of LEN closely resembles that of SMA at physiological pH. At low pH, a relatively unfolded intermediate conformation is populated for SMA and rapidly leads to amyloid fibrils. The luck of such an intermediate with LEN, is attributed to sequence differences and accounts for the lack of LEN fibrils in the absence of agitation. A κIV-specific monoclonal antibody that recognizes the N-terminal of SMA caused unraveling of the fibrils to the protofilaments and was observed to bind to one end of SMA protofilaments by atomic force microscopy. The antibody result indicates that each protofilament is asymmetric with different ends. A model for the formation of fibrils by SMA is proposed.