🔬Biophysics Unit 14 – Single-Molecule Biophysics & Nanophenomena

Introduction to Single-Molecule Biophysics

• Focuses on studying biological systems at the level of individual molecules and their interactions
• Aims to understand the behavior, dynamics, and function of biomolecules in their native environment
• Provides insights into the heterogeneity and stochasticity of biological processes
• Reveals mechanisms that are often obscured in ensemble-averaged measurements
• Enables the investigation of rare events and transient intermediates in biological pathways
• Complements traditional bulk biochemical and biophysical techniques
• Requires specialized instrumentation and techniques to detect and manipulate single molecules
• Has applications in various fields, including molecular biology, biophysics, and biomedical research

Key Concepts and Terminology

• Single-molecule resolution refers to the ability to detect and study individual molecules
• Fluorescence refers to the emission of light by a substance after absorbing light or other electromagnetic radiation
  • Commonly used in single-molecule studies due to its high sensitivity and specificity
• Förster Resonance Energy Transfer (FRET) is a mechanism of energy transfer between two fluorophores
  • Allows for the measurement of distances and conformational changes at the nanoscale
• Optical tweezers use focused laser beams to trap and manipulate small objects, such as beads attached to biomolecules
• Atomic Force Microscopy (AFM) uses a sharp tip to probe the surface of a sample and measure forces at the nanoscale
• Stochastic processes describe random events that occur in biological systems, such as the binding and unbinding of molecules
• Molecular motors are proteins that convert chemical energy into mechanical work, such as kinesin and myosin
• Mechanotransduction refers to the conversion of mechanical stimuli into biochemical signals in cells

Experimental Techniques and Instrumentation

• Total Internal Reflection Fluorescence (TIRF) microscopy uses an evanescent wave to selectively illuminate fluorophores near a surface
  • Reduces background fluorescence and improves signal-to-noise ratio
• Confocal microscopy uses a pinhole to reject out-of-focus light and achieve high spatial resolution
• Super-resolution techniques, such as STORM and PALM, allow for imaging beyond the diffraction limit of light
• Microfluidic devices enable the precise control and manipulation of small volumes of liquids
  • Used for single-molecule experiments, such as trapping and sorting of molecules
• Surface immobilization techniques, such as biotin-streptavidin binding, allow for the attachment of molecules to surfaces
• Fluorescent labeling strategies, such as using genetically encoded tags or small molecule dyes, enable the visualization of specific molecules
• Optical trapping systems use focused laser beams to manipulate and measure forces on single molecules
• Magnetic tweezers use magnetic fields to apply forces and torques on magnetic beads attached to biomolecules

Single-Molecule Detection Methods

• Fluorescence correlation spectroscopy (FCS) measures fluctuations in fluorescence intensity to determine diffusion coefficients and concentrations
• Fluorescence resonance energy transfer (FRET) measures the distance-dependent energy transfer between two fluorophores
  • Used to study conformational changes and interactions between molecules
• Tethered particle motion (TPM) tracks the Brownian motion of a bead attached to a DNA molecule to study DNA-protein interactions
• Optical trapping can measure forces and displacements of single molecules, such as the stepping of molecular motors
• Magnetic tweezers can apply constant forces and measure the extension of molecules, such as the unzipping of DNA
• Atomic force microscopy (AFM) can image and manipulate single molecules on surfaces
• Nanopore sensing uses changes in ionic current to detect the passage of single molecules through a nanoscale pore
• Single-molecule FRET (smFRET) can measure conformational dynamics and interactions of biomolecules in real-time

Nanoscale Phenomena in Biological Systems

• Brownian motion describes the random motion of particles due to thermal fluctuations
  • Plays a crucial role in the diffusion and transport of molecules in cells
• Molecular crowding refers to the high concentration of macromolecules in cellular environments
  • Affects the behavior and interactions of biomolecules
• Protein folding involves the self-assembly of amino acid chains into functional three-dimensional structures
  • Single-molecule studies have revealed the presence of multiple folding pathways and intermediates
• DNA supercoiling refers to the twisting and writhing of DNA molecules
  • Affects DNA replication, transcription, and packaging
• Membrane dynamics, such as lipid diffusion and domain formation, play important roles in cell signaling and organization
• Molecular recognition involves the specific binding of molecules through non-covalent interactions, such as hydrogen bonding and van der Waals forces
• Enzyme catalysis involves the acceleration of chemical reactions by lowering the activation energy barrier
  • Single-molecule studies have revealed the presence of multiple conformational states and dynamic fluctuations in enzyme activity
• Ion channels are membrane proteins that selectively allow the passage of ions across cell membranes
  • Single-molecule studies have provided insights into the gating mechanisms and conductance properties of ion channels

Data Analysis and Interpretation

• Signal processing techniques, such as filtering and denoising, are used to extract meaningful information from single-molecule data
• Statistical analysis, such as hypothesis testing and parameter estimation, is used to quantify the significance and reliability of single-molecule measurements
• Hidden Markov models (HMMs) are used to analyze single-molecule trajectories and identify discrete states and transitions
• Dwell time analysis involves measuring the duration of specific states or events in single-molecule traces
  • Used to determine kinetic rates and mechanistic details of biological processes
• Correlation analysis, such as autocorrelation and cross-correlation, is used to identify temporal and spatial relationships in single-molecule data
• Machine learning approaches, such as clustering and classification, are used to identify patterns and extract features from large single-molecule datasets
• Molecular dynamics simulations are used to model the behavior of biomolecules at the atomic level and complement single-molecule experiments
• Data visualization techniques, such as heat maps and kymographs, are used to represent single-molecule data in a clear and informative manner

Applications in Research and Medicine

• Studying protein-protein interactions and complex formation at the single-molecule level
  • Provides insights into the assembly and regulation of biological machines, such as the replisome and spliceosome
• Investigating the mechanisms of DNA replication, transcription, and repair
  • Single-molecule studies have revealed the dynamics and coordination of enzymes involved in these processes
• Characterizing the mechanical properties of biomolecules, such as the elasticity of DNA and the force generation of molecular motors
• Developing new diagnostic tools based on single-molecule detection, such as for the early detection of cancer biomarkers and infectious agents
• Designing targeted drug delivery systems that can release therapeutic agents in response to specific molecular triggers
• Studying the effects of mutations and disease-associated variants on the function and stability of biomolecules
• Investigating the mechanisms of viral infection and host-pathogen interactions at the single-molecule level
• Developing new imaging and sensing technologies based on single-molecule principles, such as nanopore sequencing and super-resolution microscopy

Challenges and Future Directions

• Improving the spatial and temporal resolution of single-molecule techniques to capture faster and smaller-scale events
• Developing new labeling strategies that are less invasive and more specific to the molecules of interest
• Integrating single-molecule techniques with other approaches, such as cryo-electron microscopy and mass spectrometry, to obtain a more comprehensive understanding of biological systems
• Extending single-molecule studies to more complex environments, such as living cells and tissues
• Developing standardized protocols and data analysis pipelines to ensure reproducibility and comparability of single-molecule experiments across different labs
• Addressing the limitations of current single-molecule techniques, such as the short observation times and the need for surface immobilization
• Exploring the potential of single-molecule techniques for personalized medicine, such as for the identification of disease subtypes and the prediction of drug responses
• Investigating the role of dynamic heterogeneity and stochasticity in the emergence of complex biological behaviors, such as cell differentiation and adaptation