K.K.M. Sweers

Nanomechanical properties of α-synuclein amyloid fibrils: a comparative study by nanoindentation, harmonic force microscopy, and Peakforce QNM

We report on the use of three different atomic force spectroscopy modalities to determine the nanomechanical properties of amyloid fibrils of the human α-synuclein protein. α-Synuclein forms fibrillar nanostructures of approximately 10 nm diameter and lengths ranging from 100 nm to several microns, which have been associated with Parkinsons disease. Atomic force microscopy (AFM) has been used to image the morphology of these protein fibrils deposited on a flat surface. For nanomechanical measurements, we used single-point nanoindentation, in which the AFM tip as the indenter is moved vertically to the fibril surface and back while the force is being recorded. We also used two recently developed AFM surface property mapping techniques: Harmonic force microscopy (HarmoniX) and Peakforce QNM. These modalities allow extraction of mechanical parameters of the surface with a lateral resolution and speed comparable to tapping-mode AFM imaging. Based on this phenomenological study, the elastic moduli of the α-synuclein fibrils determined using these three different modalities are within the range 1.3-2.1 GPa. We discuss the relative merits of these three methods for the determination of the elastic properties of protein fibrils, particularly considering the differences and difficulties of each method.

Atomic force microscopy under controlled conditions reveals structure of C-terminal region of alpha-synuclein in amyloid fibrils

Atomic force microscopy (AFM) is widely used to measure morphological and mechanical properties of biological materials at the nanoscale. AFM is able to visualize and measure these properties in different environmental conditions. However, these conditions can influence the results considerably, rendering their interpretation a matter of some subtlety. We demonstrate this by imaging ~10 nm diameter α-synuclein amyloid fibrils, focusing specifically on the structure of the C-terminal part of the protein monomers incorporated into fibrils. Despite these influences leading to variations in fibril heights, we have shown that by maintaining careful control of AFM settings we can quantitatively compare the morphological parameters of fibrils imaged in air or in buffer conditions. From this comparison we were able to deduce the semiflexible character of this C-terminal region. Fibril height differences measured in air and liquid indicate that the C-terminal region collapses onto the fibril core upon drying. The fibril heights decrease upon increasing ion concentration in solution, suggesting that the C-terminal tails collapse into more compact structures as a result of charge screening. Finally, PeakForce QNM measurements show an apparent heterogeneity of C-terminal packing along the fibril length.

Nanomechanical properties of single amyloid fibrils

Amyloid fibrils are traditionally associated with neurodegenerative diseases like Alzheimers disease, Parkinsons disease or Creutzfeldt-Jakob disease. However, the ability to form amyloid fibrils appears to be a more generic property of proteins. While disease-related, or pathological, amyloid fibrils are relevant for understanding the pathology and course of the disease, functional amyloids are involved, for example, in the exceptionally strong adhesive properties of natural adhesives. Amyloid fibrils are thus becoming increasingly interesting as versatile nanobiomaterials for applications in biotechnology. In the last decade a number of studies have reported on the intriguing mechanical characteristics of amyloid fibrils. In most of these studies atomic force microscopy (AFM) and atomic force spectroscopy play a central role. AFM techniques make it possible to probe, at nanometer length scales, and with exquisite control over the applied forces, biological samples in different environmental conditions. In this review we describe the different AFM techniques used for probing mechanical properties of single amyloid fibrils on the nanoscale. An overview is given of the existing mechanical studies on amyloid. We discuss the difficulties encountered with respect to the small fibril sizes and polymorphic behavior of amyloid fibrils. In particular, the different conformational packing of monomers within the fibrils leads to a heterogeneity in mechanical properties. We conclude with a brief outlook on how our knowledge of these mechanical properties of the amyloid fibrils can be exploited in the construction of nanomaterials from amyloid fibrils.

Spatially resolved frequency-dependent elasticity measured with pulsed force microscopy and nanoindentation

Recently several atomic force microscopy (AFM)-based surface property mapping techniques like pulsed force microscopy (PFM), harmonic force microscopy or Peakforce QNM® have been introduced to measure the nano- and micro-mechanical properties of materials. These modes all work at different operating frequencies. However, complex materials are known to display viscoelastic behavior, a combination of solid and fluid-like responses, depending on the frequency at which the sample is probed. In this report, we show that the frequency-dependent mechanical behavior of complex materials, such as polymer blends that are frequently used as calibration samples, is clearly measurable with AFM. Although this frequency-dependent mechanical behavior is an established observation, we demonstrate that the new high frequency mapping techniques enable AFM-based rheology with nanoscale spatial resolution over a much broader frequency range compared to previous AFM-based studies. We further highlight that it is essential to account for the frequency-dependent variation in mechanical properties when using these thin polymer samples as calibration materials for elasticity measurements by high-frequency surface property mapping techniques. These results have significant implications for the accurate interpretation of the nanomechanical properties of polymers or complex biological samples. The calibration sample is composed of a blend of soft and hard polymers, consisting of low-density polyethylene (LDPE) islands in a polystyrene (PS) surrounding, with a stiffness of 0.2 GPa and 2 GPa respectively. The spring constant of the AFM cantilever was selected to match the stiffness of LDPE. From 260 Hz to 1100 Hz the sample was imaged with the PFM method. At low frequencies (0.5-35 Hz), single-point nanoindentation was performed. In addition to the materials stiffness, the relative heights of the LDPE islands (with respect to the PS) were determined as a function of the frequency. At the lower operation frequencies for PFM, the islands exhibited lower heights than when measured with tapping mode at 120 kHz. Both spring constants and heights at the different frequencies clearly show a frequency-dependent behavior.

Characterizing nanoscale morphologic and mechanical properties of α-synuclein amyloid fibrils with atomic force microscopy

We have used atomic force microscopy (AFM) to image wild-type and disease-related mutant α-synuclein (aS) amyloid fibrils deposited on various hard and soft surfaces, ranging from freshly cleaved mica to two different supported lipid bilayers: phosphatidylcholine (POPC) and a mixture of POPC and phosphatidylglycerol (POPG). Quantitative morphologic analyses show that fibrils deposited on freshly cleaved mica, gold and glass substrates appeared to have similar heights and lengths, suggesting that the surface–fibril interaction in these cases does not influence the fibril morphology. When the same fibril sample is deposited on HOPG or quartz, the aS fibrils appear shorter in length and completely distorted, respectively, indicating that the interaction with these surfaces severely affects the fibril morphology. Sequentially recorded AFM images of fibrils on POPC bilayers clearly revealed that the amyloid fibrils were mobile on the bilayer, indicating that the fibrils are in thermal equilibrium on the surface. Persistence lengths of the fibrils on mica and POPC have been determined using two different methods, and reveal no significant differences, indicating that, although the fibrils appear immobile on mica, they do thermally equilibrate in 2D before they finally attach to the substrate. The persistence length of aS fibrils is found to be between 3.3 and 7.1 μm, which is within the expected range for amyloid fibrils. We observe no fibril movement on the mixed POPC/POPG bilayer, suggesting a stronger interaction with the lipids. Furthermore, the reduced heights of the fibrils on top of the mixed bilayer suggest that the fibrils are partly embedded within the bilayer.