Kamil Górecki

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Method Development

In my research, I have developed robust and innovative assays for detecting and quantifying various biochemical activities, with particular focus on measuring flux over biological membranes. Here are some examples of the assays I developed:

Measuring Cu Flux: Microfluidics meet Fluorescent Microscopy and Giant Unilamellar Vesicles

From: Microfluidic-Derived Detection of Protein-Facilitated Copper Flux Across Lipid Membranes, Górecki et al, Anal Chem 2023.

The measurement of copper (Cu) flux across biological membranes presents several challenges. These include difficulties with protein reconstitution into stable membranes, issues with leakage and vesicle bursting, and the low solubility of Cu at neutral pH. Additionally, biologically relevant Cu levels are in the micro and sub-micro molar range, and fluorescent methods for Cu detection are underdeveloped.

To investigate the Cu conducting properties of a bacterial Cu channel, we combined several innovative approaches:

  1. Protein Reconstitution in Giant Unilamellar Vesicles (GUVs): The protein was reconstituted into GUVs, which are large enough to be visualized under a standard optical microscope. GUVs provide a stable environment for the protein and facilitate easier observation and measurement. The incorporation of the protein can be tracked with fluorescent labels, and the lipids can be doped with a fluorescent group, allowing for real-time inspection of vesicle stability. Faulty vesicles can be discarded from the analysis, ensuring only functional vesicles are considered.
  2. Copper Detection Using FluoZin-3: While some Cu sensors exist, their use showed suboptimal results. Cu detection was therefore achieved by utilizing a zinc sensor, FluoZin-3. The fluorescence of FluoZin-3 is deactivated by Cu in a concentration-dependent linear fashion, allowing for accurate measurement of Cu levels. This reverse fashion in which Cu is measured allowed for more precision and for exclusion of vesicles that broke during the measurement.
  3. Microfluidic Setup with Fluorescent Microscopy: We employed a microfluidic device equipped with a fluorescent microscope. The setup included a channel slide with vesicles attached via biotin-avidin anchors, enabling continuous measurement of Cu flux under a constant supply of low Cu concentration buffer. This method overcomes the solubility issues by gentle “pushing” the solution through the microfluidic channels, ensuring efficient and consistent exposure of the vesicles to Cu.

These combined techniques allowed for precise and real-time monitoring of Cu flux, providing valuable insights into the function of the bacterial Cu channel. This innovative approach demonstrates the potential for microfluidic systems in overcoming traditional limitations in metal ion transport studies, paving the way for advancements in biosensing and drug development applications.

Publications:

  1. K. Górecki, J. S. Hansen, P. Li, N. Nayeri, K. Lindkvist-Petersson, P. Gourdon, Microfluidic-Derived Detection of Protein-Facilitated Copper Flux Across Lipid Membranes, (2022), Analytical Chemistry 94, 34, 11831–11837. Read
  2. P. Li, N. Nayeri, K. Górecki, E. Ramos Becares, K. Wang, D. Ram Mahato, M. Andersson, S.Abeyrathna, K. Lindkvist-Petersson, G. Meloni, J. Winkel Missel, P. Gourdon, PcoB is a defense outer membrane protein that facilitates cellular uptake of copper, (2022), Protein Science 31(7), e4364. Read (published earlier as a preprint on bioRxiv: Read)

NMR-Based Assays for in vivo Sodium Transport

Overview of the 23Na NMR method.

While studying the ion-transport functions of the respiratory chain complex I and the related sodium/proton antiporter Mrp, we observed that impairing the wild-type functions was detrimental to cells grown in the presence of sodium. However, we could not definitively prove that these effects were due to sodium accumulation inside the cells. Measuring sodium (Na) within biological systems is challenging due to sodium’s ubiquitous presence and the small concentration differences that can significantly impact cellular health. Traditional methods are laborious and often lack the precision required to detect these subtle changes, which can be lethal to cells. Fluorescent Na sensors used in eukaryotic cells could not be successfully employed for bacterial cells, so we developed a new method for measuring the Na content in bacterial cells.

To investigate the sodium transport properties of bacterial antiporters, we employed several innovative approaches:

  1. 23Na NMR Spectroscopy: We adapted the 23Na NMR method to measure internal sodium concentrations in Bacillus subtilis. Sodium plays a critical role in cellular function, and proteins involved in its transport must be accurately characterized. Impairing these proteins can lead to sodium accumulation and cell death. Traditional measurement methods are inadequate because they cannot achieve the necessary precision, where even a few mM differences can be lethal.
  2. Lanthanide Shifting Agents: We utilized a cell-impermeable lanthanide shifting agent to split the sodium NMR signal into intracellular and extracellular signals, allowing for accurate estimation of sodium contents.
  3. Quantitative Analysis: Being able to measure the Na levels both inside and outside of the cells in real time, we could quantify the flux and distinguish between healthy and unhealthy cells. This method allowed us to identify transport deficiencies in mutant strains and validate the function of sodium/proton antiporters. Accurate measurement of sodium flux was essential for understanding the protein’s impact on cellular sodium levels and the overall health of the cell.

These combined techniques enabled us to monitor sodium transport with high precision, offering valuable insights into the mechanisms of bacterial sodium/proton antiporters. This innovative approach demonstrates the potential of NMR spectroscopy in overcoming traditional limitations in ion transport studies, paving the way for advancements in understanding membrane protein functions and developing new therapeutic strategies.

Publications:

  1. K. Górecki, C. Hägerhäll, T. Drakenberg, The Na+ transport in Gram-positive bacteria defect in the Mrp antiporter complex measured with 23Na-NMR, (2014) Analytical Biochemistry 445: 80-86. Read (open access at PubMed)
  2. E. Sperling, K. Górecki, T. Drakenberg, C. Hägerhäll, Functional Differentiation of Antiporter-Like Polypeptides in Complex I; a Site-Directed Mutagenesis Study of Residues Conserved in MrpA and NuoL but Not in MrpD, NuoM, and NuoN, (2016), PLOS ONE 11, 7. Read
  3. V. K. Moparthi, B. Kumar, Y. Al-Eryani, E. Sperling, K. Górecki, T. Drakenberg, C. Hägerhäll, Functional role of the MrpA- and MrpD-homologous protein subunits in enzyme complexes evolutionary related to respiratory chain complex I, (2014), BBA – Bioenergetics 1837: 178-185. Read