Full Answer Section

Among the numerous proteins orchestrating FA assembly and function, talin and vinculin stand out as key players. Talin is a large, modular protein that directly binds to integrin receptors in the plasma membrane and to actin filaments in the cytoskeleton. This crucial linkage provides the primary mechanical connection between the ECM and the cell’s internal machinery. Vinculin, on the other hand, is a peripheral membrane protein known for its ability to bind to talin, actin, and other FA components like paxillin. The interaction between talin and vinculin is particularly significant as it is believed to strengthen the integrin-actin linkage and regulate FA stability and turnover. Vinculin binding to talin is thought to occur at specific sites within the talin rod domain and is crucial for the mechanical reinforcement of FAs under force. Disruptions in the expression or function of either talin or vinculin have been implicated in various pathological conditions, including cancer metastasis, cardiovascular diseases, and developmental abnormalities, highlighting the critical biological importance of understanding their interaction.

2) What do you propose to study, and why?

This research proposal aims to investigate the specific molecular interactions between talin and vinculin and to determine the structural characteristics of their complex. While it is established that these two proteins interact, the precise binding sites, the stoichiometry of the complex, the conformational changes induced upon binding, and the overall three-dimensional architecture remain incompletely understood.

We propose to utilize a combination of biophysical techniques learned in this class – Fluorescence Spectroscopy, Light Scattering, and Nuclear Magnetic Resonance (NMR) Spectroscopy – to gain a comprehensive understanding of the talin-vinculin interaction. Fluorescence spectroscopy will be employed to probe the binding affinity and conformational changes upon interaction. Light scattering will be used to determine the size and oligomeric state of the complex. Finally, NMR spectroscopy will provide detailed atomic-level information about the binding interfaces and the structural dynamics of the interacting domains. Understanding the intricacies of this interaction at a molecular level is crucial for elucidating the mechanisms underlying FA assembly, stability, and mechanotransduction.

3) Potential Significance of Your Proposed Research

This research has the potential to significantly advance our understanding of the fundamental mechanisms governing cell-matrix adhesion. By elucidating the specific interactions and the complex structure formed by talin and vinculin, we can:

• Gain deeper insights into FA dynamics: Understanding how these key proteins interact will shed light on the processes of FA assembly, maturation, reinforcement under mechanical stress, and disassembly.
• Uncover regulatory mechanisms: Identifying the binding sites and conformational changes could reveal potential targets for endogenous regulatory factors or for the development of therapeutic interventions.
• Contribute to the understanding of disease pathogenesis: Given the involvement of talin and vinculin in various diseases, a detailed understanding of their interaction could provide crucial insights into the molecular basis of these conditions and potentially identify novel therapeutic targets.
• Inform the development of biomaterials and tissue engineering strategies: A thorough understanding of cell-matrix interactions is essential for designing biomaterials that can effectively promote cell adhesion and tissue integration.

Ultimately, this research will contribute to a more complete picture of the molecular machinery driving cell-matrix adhesion, with broad implications for basic biology, disease research, and biomedical engineering.

4) Experimental Design

To achieve the research objectives, we propose the following experimental design utilizing Fluorescence Spectroscopy, Light Scattering, and NMR Spectroscopy:

a) Protein Expression and Purification:

• Recombinant expression of specific domains of human talin known or predicted to interact with vinculin (e.g., the VBS – Vinculin Binding Site – containing regions within the talin rod domain) and the vinculin head and tail domains will be performed in E. coli.
• The proteins will be purified to homogeneity using a combination of affinity chromatography (e.g., His-tag purification), ion exchange chromatography, and size exclusion chromatography.
• Protein purity will be assessed by SDS-PAGE, and concentration will be determined by UV-Vis spectroscopy.

b) Fluorescence Spectroscopy:

• Binding Affinity Determination: We will use fluorescence titration experiments to determine the binding affinity (Kd) between the purified talin and vinculin domains. This will involve labeling one of the proteins (e.g., vinculin head domain) with a fluorescent probe (e.g., FITC) and titrating it with increasing concentrations of the unlabeled partner (e.g., talin VBS-containing fragment). Changes in fluorescence intensity or anisotropy will be monitored as a function of the concentration of the unlabeled protein. The resulting data will be fitted to appropriate binding models to determine the Kd.
• Conformational Changes Upon Binding: We will employ Förster Resonance Energy Transfer (FRET) to investigate conformational changes upon complex formation. This will involve labeling different regions of talin and vinculin with donor and acceptor fluorophores. Changes in FRET efficiency upon interaction will indicate changes in the distance and/or orientation between the labeled sites, providing insights into conformational rearrangements.

c) Light Scattering:

• Static Light Scattering (SLS): SLS will be used to determine the molecular weight and potentially the radius of gyration (Rg) of the individual proteins and their complex. By analyzing the intensity of scattered light as a function of angle and concentration, we can determine the stoichiometry of the complex formation (e.g., 1:1, 1:2).
• Dynamic Light Scattering (DLS): DLS will be employed to measure the hydrodynamic radius (Rh) of the proteins and their complex. Changes in Rh upon interaction can provide information about the overall size and shape of the complex. Combining SLS and DLS data can yield insights into the shape and oligomeric state of the talin-vinculin complex in solution.

d) Nuclear Magnetic Resonance (NMR) Spectroscopy:

• Sample Preparation: Isotopically labeled (¹⁵N and/or ¹³C) samples of the interacting domains of talin and vinculin will be prepared for NMR studies.
• Chemical Shift Perturbation (CSP) Mapping: Heteronuclear Single Quantum Coherence (HSQC) experiments will be performed on the labeled protein while titrating with the unlabeled partner. Changes in the chemical shifts of specific amino acid residues in the HSQC spectra will identify the residues involved in the binding interface on both proteins.
• Intermolecular NOEs (Nuclear Overhauser Effects): Experiments such as Heteronuclear Overhauser Spectroscopy (HOESY) or Saturation Transfer Difference (STD) NMR will be used to detect intermolecular NOEs, which provide direct evidence of close spatial proximity (within ~5 Å) between protons of the interacting proteins, further defining the binding interface.
• Structural Characterization: Advanced multi-dimensional NMR experiments can be used to determine the three-dimensional structure of the individual domains and potentially the structure of the complex, especially if smaller interacting fragments are used. This would provide atomic-level details of the binding interface and any conformational changes upon interaction.

5) Expected Results of Your Proposed Experiments

We anticipate the following results from our proposed experiments:

• Fluorescence Spectroscopy: We expect to observe a measurable change in fluorescence intensity or anisotropy upon titration of the labeled protein with its unlabeled partner, allowing us to determine the binding affinity (Kd) between specific talin and vinculin domains. FRET experiments are expected to reveal changes in the distance between labeled sites upon complex formation, indicating conformational rearrangements within the proteins or at the interface.
• Light Scattering: SLS experiments should provide the molecular weight of the individual proteins and the complex, allowing us to determine the stoichiometry of the interaction (e.g., whether talin and vinculin form a 1:1 or other ratio complex). DLS measurements are expected to show an increase in the hydrodynamic radius upon complex formation, reflecting the formation of a larger molecular assembly. The combined SLS and DLS data will provide insights into the overall shape of the complex.
• NMR Spectroscopy: CSP mapping is expected to identify specific amino acid residues on both talin and vinculin that are directly involved in the interaction, pinpointing the binding interface. Intermolecular NOE experiments should provide direct evidence of physical proximity between these residues. If successful, high-resolution NMR structures of the individual domains and potentially the complex will reveal the detailed atomic interactions and the three-dimensional architecture of the binding interface.

Collectively, these expected results will provide a comprehensive understanding of the molecular interactions between talin and vinculin, including their binding affinity, stoichiometry, conformational changes upon binding, and the structural details of their complex. This information will significantly advance our knowledge of the fundamental mechanisms governing cell-matrix adhesion.