Following the Aggregation of Human Prion Protein on Heparin Functionalized Gold Surface in Real Time

Prion diseases are fatal neurodegenerative disorders caused by prion protein (PrP) misfolding and aggregation. PrP can exist in two conformations: PrPC and PrPSc. PrPSc can convert PrPC into PrPSc through a template-directed mechanism. PrP aggregation is an autocatalytic process with a high kinetic barrier. It involves several steps: nucleation, elongation, and fibril formation. Nucleation is the rate-limiting step that requires a critical concentration of PrP molecules. Elongation is characterized by the growth of protofibrils and short fibers. Fibril formation is marked by the lateral association of protofibrils and short fibers. Several factors can influence PrP aggregation, such as pH, temperature, salt concentration, metal ions, and molecular chaperones. Among these factors, sulfated glycosaminoglycans (GAGs) have a significant role in PrP misfolding and aggregation. GAGs are linear polysaccharides that are attached to proteins or lipids on the cell surface or in the extracellular matrix. They can interact with PrP through electrostatic and hydrophobic interactions. Heparin is a highly sulfated GAG that has been widely used as a model GAG to study its effects on PrP aggregation. Heparin can bind to PrP and induce its conformational changes, leading to the formation of oligomers and fibrils. Heparin has also been found to be associated with PrPSc deposits in prion-infected brains, suggesting that it may have a catalytic role in PrP aggregation processes. However, the exact role and details of heparin in PrP aggregation are not clear and need a thorough perusal. there is a need for a novel approach to study the PrP aggregation process on a heparin functionalized surface under physiological conditions by using single molecule techniques. Single molecule techniques can provide real-time and atomic scale information on the whole aggregation process.

Single molecule techniques are experimental methods that enable the study of individual proteins, rather than bulk measurements that represent an average of many molecules. These techniques have revolutionized the field of biophysics and molecular biology, allowing scientists to observe the behavior of individual molecules and study the underlying mechanisms that drive biological processes. In the context of prion protein aggregation, single molecule techniques are particularly important because they allow scientists to investigate the early stages of protein aggregation at a level of detail that was previously impossible. Some common single molecule techniques used in the study of protein aggregation include single molecule fluorescence microscopy, atomic force microscopy, and nanopore sensing. Single molecule fluorescence microscopy involves labeling individual proteins with fluorescent probes and tracking their movements and interactions in real time. Atomic force microscopy uses a small, sharp probe to scan the surface of individual proteins and generate high-resolution images. Nanopore sensing involves threading individual proteins through a tiny pore and measuring changes in electrical current as the protein interacts with the pore.

In a new research work published in the peer-reviewed Journal ACS Applied Bio Materials, PhD candidate Tong Zhang, Dr. Yangang Pan, PhD candidates Sneha Kandapal, Xin Sun, and led by Professor Bingqian Xu from the University of Georgia used in situ time-lapse atomic force microscopy (AFM) to examine the PrP aggregation process on the heparin modified gold surface. Surprisingly, they discovered that heparin only accelerates the creation of oligomers nuclei during PrP aggregation, which is the rate-limiting phase in the nucleation-dependent aggregation process. Heparin is not directly engaged in the PrP assembly. The authors used single molecule AFM to monitor the PrP aggregation process on a heparin modified gold surface in situ and in real time. They combined simultaneous topographic and recognition (TREC) imaging and single molecule force spectroscopy (SMFS) to obtain atomic scale details of the whole aggregation process. They used full-length human recombinant PrP (23-231) as the model protein. They observed the whole aggregation process from the formation of oligomers, to the assembly of protofibrils and short fibers, and the formation of elongated mature fibers. They found that heparin promoted the PrP aggregation by facilitating the formation of oligomers during the early nucleation stage. They also found that heparin increased the binding affinity between PrP molecules and enhanced the stability of PrP aggregates. They revealed that PrP aggregates had different morphologies and mechanical properties depending on their size and structure. They suggested that heparin could act as a template or scaffold for PrP aggregation by providing binding sites and inducing conformational changes. They provided new insights into the molecular mechanisms and kinetics of PrP aggregation on GAGs and its implications for prion diseases.

In summary, the study by Professor Bingqian Xu and his research group provides insights into the role of GAGs in PrP aggregation and the molecular mechanism driving the process in prion disease. It also demonstrates the power of AFM techniques to monitor protein aggregation at the single molecule level. These insights may ultimately lead to the development of new therapies for diseases associated with protein aggregation.