How does fluorescent labeling affect the binding kinetics of proteins with intact cells?

Abstract

Fluorescent labeling is a mainstream technology for detecting molecular binding. Despite the importance, few studies have been devoted to quantitatively examine the effect of labeling on the molecular binding processes. Here we present a quantitative study on the binding kinetics of fluorescent-labeled and un-labeled molecules (lectin proteins) with glycoproteins on the membrane of cells using surface plasmon resonance imaging (SPRi) technique. The study shows that fluorescent labeling has a significant influence on the binding behaviors of proteins, especially the association processes, and the influence depends sensitively on the charge of fluorescent labels. It further shows that the labels also affect the local distribution of probe proteins, due to the inhomogeneous surface charge distribution of the cell membrane. Our work indicates that fluorescent labeling in general affects the binding behaviors, but proper design of the label will help to minimize its effect.

Structural Determinants of the Interactions Between Influenza A Virus Matrix Protein M1 and Lipid Membranes

“…6C) show that interactions between lipids and full-length M1 might involve residues belonging to N-terminal domain (and therefore to M1-N as well) that possess aliphatic amines able to react with the succinimidyl ester group in the fluorescent label, such as Gln75, Gln81, Arg76 and Arg77. Notably, a recent SPR study has shown that labeling of wheat germ agglutinin with fluorescent groups reduces significantly binding affinity of this lectin [62]. Unfortunately, we could not study binding of labeled M1-N via SPR since, due to the rather low labeling efficiency (see above), SPR would report essentially on the non-labeled protein fraction.…”

Optimisation of multimodal coherent anti-Stokes Raman scattering microscopy for the detection of isotope-labelled molecules

“…Nonnegative matrix factorisation applied to the imaginary part and the average real part of the susceptibility with an additional concentration constraint (FSC 3 method) was used to obtain susceptibility spectra of independently varying chemical components and their volume concentrations. The FSC 3 is unsupervised and thus does not require prior knowledge of the spectra of chemical components and instead uses random initial guesses. A detailed overview of the data analysis procedure is described in ref 15.…”

“…Whilst fluorescence microscopy remains the most widely used optical tool for imaging cellular components with high spatial resolution, with technologies such as super-resolution light microscopy achieving resolution in the submicrometre range, the technology is limited by a necessity for exogenous label incorporation to enable chemical specificity 1 . The majority of these fluorescent labels share a similar molecular weight with many small biological molecules, consequently affecting their physical and chemical properties [2][3][4][5] . Fluorophores are also susceptible to photobleaching with prolonged excitation and cells can become damaged by light at the necessary wavelengths, leading to difficulties in observing samples over longer timescales 6 .…”

“…Through acquisition of hyperspectral CARS images and subsequent FSC 3 analysis, we can identify an exogenously applied, deuterium-labelled molecule within a cell, which offers potential for studying molecule distribution, accumulation and metabolism. Hyperspectral CARS images of cytosolic lipid droplets were acquired from HeLa cells fed with deuterated and non-deuterated oleic acid, and unsupervised FSC 3 analysis was performed to produce concentration maps of chemical components, thus enabling direct comparison of the chemical composition of lipid droplets.…”

Label‐Free Optical Single‐Molecule Micro‐ and Nanosensors

“…However, this technique requires the labeling of the molecules of interest that can be a drawback in certain investigations. In some cases, it might not be possible to attach a label, or the label might affect the kinetics of the molecules under study.…”