Researchers from the Hong Kong University of Science and Technology (HKUST) developed optical tweezers-coupled Raman spectroscopy that can directly probe the structural features of alpha-synuclein, an intrinsically disordered protein (IDP) that is closely linked to Parkinson’s disease. Through focus on individual protein molecules, the approach probes the IDP at the physiological concentration. It is a challenge to analyze proteins at low concentrations, especially for those in a mixture of various conformations, such as IDPs, which play an important role in biological processes — many IDPs are associated with incurable neurodegenerative diseases. Alpha-synuclein is a typical IDP, in that it lacks a stable 3D architecture, known as “secondary structures.” The IDP spontaneously undergoes conversions from one secondary structure to another, which could eventually result in the buildup of protein aggregates involved in the pathology of Parkinson’s disease. The optical tweezers-controlled hot spot for the protein structural characterization by surface-enhanced Raman spectroscopy. Courtesy of Vince St. Dollente Mesias and Jinqing Huang, Hong Kong University of Science and Technology. The transient species during the convergent process possess various structures and exist in low population among a dynamic equilibrium mixture. As a result, their structural features are usually buried under the detection results obtained by traditional measurement techniques that average the signals detected from large sample quantities and long detection times. In the new study, the research team led by assistant chemistry professor Huang Jinqing integrated optical tweezers and surface-enhanced Raman spectroscopy (SERS) to generate SERS enhancements that were tunable and reproducible with single-molecule level sensitivity in aqueous environments. The novel platform the team designed allowed it to characterize IDPs and, at the same time, maintain the IDP’s intrinsic heterogeneity with biological significance. Specifically, the researchers showed they could use optical tweezers to visualize and control a “hot spot” to allow proteins to go through in a microfluidic flow chamber. This made it easier to adjust the measurement parameters in real time to enable in situ spectroscopic characterizations. It directly identified the structural features of the transient species of alpha-synuclein among its predominant monomers at a physiological concentration of 1 μM by reducing the ensemble averaging in quantity and time. In its ability to deliver insight into the initiation of amyloid protein aggregation, the team’s SERS platform, members said, exhibited strong potential to be used to reveal structural information of IDPs in certain dynamic, heterogeneous, and complex biological systems. The researchers used micrometer-size silver nanoparticle-coated silica beads in the work; the strategy enabled precise control of the hot spot between two trapped beads to improve the SERS efficiency and reproducibility in aqueous detections, Jinqing said. “Except for the tunable SERS enhancement, the integrated optical tweezers also offer subnanometer spatial resolution and subpiconewton force sensitivity to monitor light-matter interactions in the plasmonic hot spot for extra physical insight,” she said. The researchers also said the method opens a new door in the biophysics community, to characterize the transient species of IDPs in dilute solutions, which remains a significant challenge. “Ultimately, it will be exciting to fully exploit the precise force manipulation of the integrated optical tweezers to unfold a single protein inside the controllable hot spot and resolve its structural dynamics from the endogenous molecular vibrations by the integrated Raman spectroscopy,” Jinqing said. The research was published in Nature Communications (www.doi.org/10.1038/s41467-021-21543-3).