Diffusion Coefficients Of Proteins Research Articles

Electrostatic complex of neurotrophin 4 with dendrimer nanoparticles: controlled release of protein in vitro and in vivo.

Background: NT4 has been regarded as a promising therapeutic protein for treatment of damaged retinal pigment epithelium cells.Purpose: Here, we studied physicochemical parameters of an NT4–polyamidoamine (PAMAM) electrostatic complex, which can provide a sustained concentration of protein in intraocular space over an extended period after delivery. Adsorption/desorption of NT4 molecules to/from positively charged PAMAM dendrimers were precisely determined to control the concentration of bounded/unbounded protein molecules, diffusion coefficient, and size of a protein-laden dendrimer structure. We determined kinetics of NT4 desorption in PBS, vitreous, and damaged retina.Methods: Initially, adsorption of NT4 molecules on PAMAM dendrimers was studied in PBS using dynamic light scattering, electrophoresis, solution depletion, ELISA, and atomic force microscopy. This allowed us precisely to determine desorption of NT4 from nanoparticles under in situ conditions. The maximum coverage of irreversibly adsorbed NT4 determined by ELISA allowed us to devise a robust procedure for preparing stable and well-controlled coverage of NT4 on PAMAM nanoparticles. Thereafter, we studied diffusion of nanospheres containing NT4 molecules by injecting them into vitreous cavities of mice exposed to intravenous injections of sodium iodate and evaluated their intraocular desorption kinetics from drug carriers in vivo.Results: Our measurements revealed NT4–dendrimer nanoparticles can be used for continuous neurotrophic factor delivery, enhancing its distribution into mouse vitreous, as well as damaged retina over 28 days of postinjury observation.Conclusion: Understanding of polyvalent neurotrophin interactions with dendrimer nanoparticles might be useful to obtain well-ordered protein layers, targeting future development of drug-delivery systems, especially for neuroprotection of damaged retinal neurons.

Characterization of Single-Protein Dynamics in Polymer-Cushioned Lipid Bilayers Derived from Cell Plasma Membranes.

Native cell-membrane-derived supported lipid bilayers (SLBs) are an emerging platform with broad applications ranging from fundamental research to next-generation biosensors. Central to the success of the platform is the proper accommodation of membrane proteins so that their dynamics and functions are preserved. Polymer cushions have been commonly employed to avoid direct contact between the bilayer membrane and the supporting substrate, and thus, the mobility of the transmembrane proteins is maintained. However, little is known about how the polymer cushion affects the absolute mobility of membrane molecules. Here, we characterized the dynamics of single membrane proteins in polymer-cushioned lipid bilayers derived from cell plasma membranes and investigated the effects of polymer length. Three membrane proteins with distinct structures, i.e., a GPI-anchored protein, single-pass transmembrane protein CD98 heavy chain, and seven-pass transmembrane protein SSTR3, were fused with green fluorescent protein (GFP), and their dynamics were measured by fluorescent single-molecule tracking. An automated data acquisition was implemented to study the effects of PEG polymer length on protein dynamics with large statistics. Our data showed that increasing the PEG polymer length (molecular weight from 1000 to 5000) enhanced the mobile fraction of the membrane proteins. Moreover, the diffusion coefficients of transmembrane proteins were augmented with the polymer length, whereas the diffusion coefficient of the GPI-anchored protein remained almost identical for different polymer lengths. Importantly, the diffusion coefficients of the three membrane proteins became identical (2.5 μm2/s approximately) for the cushioned membrane with the longest polymer length (molecular weight of 5000), indicating that at the microscopic length scale, the SLBs were fully suspended from the substrate by the polymer cushion. Transient confinements were observed for all three proteins, and increasing the polymer length reduced the tendency of transient confinement. The measured dynamics of membrane proteins were found to be nearly unchanged after the depletion of cholesterol, suggesting that the observed immobilization and transient confinement were not due to cholesterol-enriched membrane nanodomains (lipid rafts). Our single-molecule dynamics elucidate the biophysical properties of polymer-cushioned plasma membrane bilayers that are potentially useful for the future developments of membrane-based biosensors and analytical assays.

Physicochemical Properties of Nucleoli in Live Cells Analyzed by Label-Free Optical Diffraction Tomography.

The cell nucleus is three-dimensionally and dynamically organized by nuclear components with high molecular density, such as chromatin and nuclear bodies. The structure and functions of these components are represented by the diffusion and interaction of related factors. Recent studies suggest that the nucleolus can be assessed using various protein probes, as the probes are highly mobile in this organelle, although it is known that they have a densely packed structure. However, physicochemical properties of the nucleolus itself, such as molecular density and volume when cellular conditions are changed, are not yet fully understood. In this study, physical parameters such as the refractive index (RI) and volume of the nucleoli in addition to the diffusion coefficient (D) of fluorescent probe protein inside the nucleolus are quantified and compared by combining label-free optical diffraction tomography (ODT) with confocal laser scanning microscopy (CLSM)-based fluorescence correlation spectroscopy (FCS). 3D evaluation of RI values and corresponding RI images of nucleoli in live HeLa cells successfully demonstrated varying various physiological conditions. Our complimentary method suggests that physical property of the nucleolus in live cell is sensitive to ATP depletion and transcriptional inhibition, while it is insensitive to hyper osmotic pressure when compared with the cytoplasm and nucleoplasm. The result demonstrates that the nucleolus has unique physicochemical properties when compared with other cellular components.

Finite-Size-Corrected Rotational Diffusion Coefficients of Membrane Proteins and Carbon Nanotubes from Molecular Dynamics Simulations.

We investigate system-size effects on the rotational diffusion of membrane proteins and other membrane-embedded molecules in molecular dynamics simulations. We find that the rotational diffusion coefficient slows down relative to the infinite-system value by a factor of one minus the ratio of protein and box areas. This correction factor follows from the hydrodynamics of rotational flows under periodic boundary conditions and is rationalized in terms of Taylor–Couette flow. For membrane proteins like transporters, channels, or receptors in typical simulation setups, the protein-covered area tends to be relatively large, requiring a significant finite-size correction. Molecular dynamics simulations of the protein adenine nucleotide translocase (ANT1) and of a carbon nanotube porin in lipid membranes show that the hydrodynamic finite-size correction for rotational diffusion is accurate in standard-use cases. The dependence of the rotational diffusion on box size can be used to determine the membrane viscosity.

Determination of δ-opioid receptor molecules mobility in living cells plasma membrane by novel method of FRAP analysis

Fluorescence recovery after photobleaching (FRAP) is the preferred method for analyzing the lateral mobility of fluorescently-tagged proteins in the plasma membranes (PMs) of live cells. FRAP experiments are described as being easy to perform; however, the analysis of the acquired data can be difficult. The evaluation procedure must be properly combined with the imaging setup of the confocal microscope to provide unbiased results.With the aim of increasing the accuracy of determining the diffusion coefficient (D) and mobile fraction (Mf) of PM proteins, we developed a novel method for FRAP analysis in the equatorial plane of the cell. This method is based on the calculation of photobleaching characteristics, derived from the light intensity profile and optical parameters of the confocal microscope, and on the model of fluorescent molecule diffusion in PM regions outside of the focal plane. Furthermore, cell movement artifacts in the FRAP data are ameliorated by using a region of interest, which is not fixed but instead moves adaptively in coordination with the movement of cells.When this method was used to determine the mobility of the δ-opioid receptor-eYFP in HEK293 cells, a highly significant decrease in receptor mobility was detected in cholesterol-depleted cells. This decrease was fully reversible by the replenishment of cholesterol levels. Our results demonstrate the crucial role played by cholesterol in the dynamic organization of δ-opioid receptors in the PM under in vivo conditions. Our method may be applied for the determination of the D and Mf values of other PM proteins.

Protein diffusion from microwells with contrasting hydrogel domains.

Understanding and controlling molecular transport in hydrogel materials is important for biomedical tools, including engineered tissues and drug delivery, as well as life sciences tools for single-cell analysis. Here, we scrutinize the ability of microwells—micromolded in hydrogel slabs—to compartmentalize lysate from single cells. We consider both (i) microwells that are “open” to a large fluid (i.e., liquid) reservoir and (ii) microwells that are “closed,” having been capped with either a slab of high-density polyacrylamide gel or an impermeable glass slide. We use numerical modeling to gain insight into the sensitivity of time-dependent protein concentration distributions on hydrogel partition and protein diffusion coefficients and open and closed microwell configurations. We are primarily concerned with diffusion-driven protein loss from the microwell cavity. Even for closed microwells, confocal fluorescence microscopy reports that a fluid (i.e., liquid) film forms between the hydrogel slabs (median thickness of 1.7 μm). Proteins diffuse from the microwells and into the fluid (i.e., liquid) layer, yet concentration distributions are sensitive to the lid layer partition coefficients and the protein diffusion coefficient. The application of a glass lid or a dense hydrogel retains protein in the microwell, increasing the protein solute concentration in the microwell by ∼7-fold for the first 15 s. Using triggered release of Protein G from microparticles, we validate our simulations by characterizing protein diffusion in a microwell capped with a high-density polyacrylamide gel lid (p > 0.05, Kolmogorov-Smirnov test). Here, we establish and validate a numerical model useful for understanding protein transport in and losses from a hydrogel microwell across a range of boundary conditions.

Dynamical Signatures of Local DNA Damage in Live Cells

The dynamic organization of chromatin inside the eukaryotic cell nucleus is essential to the regulation of the genome such as maintaining its integrity, gene expression and replication. However, the physical picture of chromatin structure and dynamics remains unclear and requires identifying the dynamical and structural signatures of specific biological processes. In this work, we study the chromatin dynamics in interphase, time between two cell divisions, in response to DNA damage. As shown previously, chromatin dynamics across the whole nucleus change dramatically after DNA damage; chromatin's coherent motion is eliminated, while its local mobility increases and its compaction decreases [1]. Here, we explore the local chromatin dynamics and compaction in response to local DNA damage in human cells. To do so, we induce double stranded DNA breaks (DSBs), which we visualize using 53BP1-mCherry, and observe the spatiotemporal evolution of their dynamics using single particle tracking. Simultaneously, we use histone H2B-GFP as a reporter on the chromatin compaction, to compare DSB dynamics inside the heterochromatin and euchromatin. To elucidate the dynamic signature of the local DNA damage response, we vary the number of DSBs as well as explore the ATP-dependence of these processes. Our preliminary results show the local subdiffusive dynamics of the DSBs changes with an increasing DSB count inside the cell nucleus. We also explore the relationship between the protein aggregation, diffusion coefficient and chromatin compaction at a DSB. [1] Zidovska A, Weitz DA & Mitchison TJ, Proc. Natl. Acad. Sci., 110(39), 15555-60, 2013.

The Investigation of Protein Diffusion via H-Cell Microfluidics

In this study, we developed a microfluidics method, using a so-called H-cell microfluidics device, for the determination of protein diffusion coefficients at different concentrations, pHs, ionic strengths, and solvent viscosities. Protein transfer takes place in the H-cell channels between two laminarly flowing streams with each containing a different initial protein concentration. The protein diffusion coefficients are calculated based on the measured protein mass transfer, the channel dimensions, and the contact time between the two streams. The diffusion rates of lysozyme, cytochrome c, myoglobin, ovalbumin, bovine serum albumin, and etanercept were investigated. The accuracy of the presented methodology was demonstrated by comparing the measured diffusion coefficients with literature values measured under similar solvent conditions using other techniques. At low pH and ionic strength, the measured lysozyme diffusion coefficient increased with the protein concentration gradient, suggesting stronger and more frequent intermolecular interactions. At comparable concentration gradients, the measured lysozyme diffusion coefficient decreased drastically as a function of increasing ionic strength (from zero onwards) and increasing medium viscosity. Additionally, a particle tracing numerical simulation was performed to achieve a better understanding of the macromolecular displacement in the H-cell microchannels. It was found that particle transfer between the two channels tends to speed up at low ionic strength and high concentration gradient. This confirms the corresponding experimental observation of protein diffusion measured via the H-cell microfluidics.

Microdomain formation is a general property of bacterial membrane proteins and induces heterogeneity of diffusion patterns

BackgroundProteins within the cytoplasmic membrane display distinct localization patterns and arrangements. While multiple models exist describing the dynamics of membrane proteins, to date, there have been few systematic studies, particularly in bacteria, to evaluate how protein size, number of transmembrane domains, and temperature affect their diffusion, and if conserved localization patterns exist.ResultsWe have used fluorescence microscopy, single-molecule tracking (SMT), and computer-aided visualization methods to obtain a better understanding of the three-dimensional organization of bacterial membrane proteins, using the model bacterium Bacillus subtilis. First, we carried out a systematic study of the localization of over 200 B. subtilis membrane proteins, tagged with monomeric mVenus-YFP at their original gene locus. Their subcellular localization could be discriminated in polar, septal, patchy, and punctate patterns. Almost 20% of membrane proteins specifically localized to the cell poles, and a vast majority of all proteins localized in distinct structures, which we term microdomains. Dynamics were analyzed for selected membrane proteins, using SMT. Diffusion coefficients of the analyzed transmembrane proteins did not correlate with protein molecular weight, but correlated inversely with the number of transmembrane helices, i.e., transmembrane radius. We observed that temperature can strongly influence diffusion on the membrane, in that upon growth temperature upshift, diffusion coefficients of membrane proteins increased and still correlated inversely to the number of transmembrane domains, following the Saffman–Delbrück relation.ConclusionsThe vast majority of membrane proteins localized to distinct multimeric assemblies. Diffusion of membrane proteins can be suitably described by discriminating diffusion coefficients into two protein populations, one mobile and one immobile, the latter likely constituting microdomains. Our results show there is high heterogeneity and yet structural order in the cell membrane, and provide a roadmap for our understanding of membrane organization in prokaryotes.

A rapid method for determining protein diffusion through hydrogels for regenerative medicine applications.

Hydrogels present versatile platforms for the encapsulation and delivery of proteins and cells for regenerative medicine applications. However, differences in hydrogel cross-linking density, polymer weight content, and affinity for proteins all contribute to diverse diffusion rates of proteins through hydrogel networks. Here, we describe a simple method to accurately measure protein diffusion through hydrogels, within a few hours and without the use of large amounts of protein. We tracked the diffusion of several proteins of varying molecular weights along the axial direction of capillary tubes filled with alginate, collagen, or poly(ethylene glycol) hydrogels. The rate of protein diffusion decreased with increasing molecular weight. A computational model of protein diffusion through capillary tubes was also created to predict and verify experimental protein diffusion coefficients. This in vitro capillary tube-based method of measuring protein diffusion represents a simple strategy to interrogate protein diffusion through natural and synthetic hydrogels and aid in the design of better biomaterial-based delivery vehicles that can effectively modulate protein release.

Measuring translational diffusion of 15N-enriched biomolecules in complex solutions with a simplified 1H-15N HMQC-filtered BEST sequence.

Pulsed-field gradient nuclear magnetic resonance has seen an increase in applications spanning a broad range of disciplines where molecular translational diffusion properties are of interest. The current study introduces and experimentally evaluates the measurement of translational diffusion coefficients of 15N-enriched biomolecules using a 1H-15N HMQC-filtered band-selective excitation short transient (BEST) sequence as an alternative to the previously described SOFAST-XSTE sequence. The results demonstrate that accurate translational diffusion coefficients of 15N-labelled peptides and proteins can be obtained using this alternative 1H-15N HMQC-filtered BEST sequence which is implementable on NMR spectrometers equipped with probes fitted with a single-axis field gradient, including most cryoprobes dedicated to bio-NMR. The sequence is of potential use for direct quantification of protein or peptide translational diffusion within complex systems, such as in mixtures of macromolecules, crowded solutions, membrane-mimicking media and in bicontinuous cubic phases, where conventional sequences may not be readily applicable due to the presence of intense signals arising from sources other than the protein or peptide under investigation.

Computational Signaling Protein Dynamics and Geometric Mass Relations in Biomolecular Diffusion.

We present an atomistic level computational investigation of the dynamics of a signaling protein, monocyte chemoattractant protein-1 (MCP-1), that explores how simulation geometry and solution ionic strength affect the calculated diffusion coefficient. Using a simple extension of noncubic finite size diffusion correction expressions, it is possible to calculate experimentally comparable diffusion coefficients that are fully consistent with those determined from cubic box simulations. Additionally, increasing the concentration of salt in the solvent environment leads to changes in protein dynamics that are not explainable through changes in solvent viscosity alone. This work in accurate computational determination of protein diffusion coefficients led us to investigate molecular-weight-based predictors for biomolecular diffusion. By introducing protein volume- and protein surface-area-based extensions of traditional statistical relations connecting particle molecular weight to diffusion, we find that protein solvent-excluded surface area rather than volume works as a better geometric property for estimating biomolecule Stokes radii. This work highlights the considerations necessary for accurate computational determination of biomolecule diffusivity and presents insight into molecular weight relations for diffusion that could lead to new routes for estimating protein diffusion beyond the traditional approaches.

Diffusion of Proteins and Lipids in Membranes Corrected for Finite-Size Effects

Translational diffusion coefficients are primary reporters on the dynamics in lipid membranes. However, diffusion coefficients of lipids and membrane proteins calculated in molecular dynamics (MD) simulations suffer from severe finite-size effects. We show that these finite-size effects can be quantified in terms of hydrodynamic theory and develop accurate correction formulas. Using large-scale coarse-grained MD simulations of up to 132 million particles, we assess the effects of box size and shape on translational diffusion in lipid membrane simulations. For the flat periodic boxes commonly used for membrane simulations, we find that the diffusion coefficients of lipids and membrane proteins increase logarithmically (and therefore without bound) with the box width. Only if the box size is increased also normal to the membrane surface, the diffusion coefficient converges. This dependence on both the size and the shape of the simulation box is captured quantitatively by a hydrodynamic theory. The corresponding correction formulas make it possible to perform meaningful comparisons of diffusion coefficients from simulations with different box sizes, and facilitate the comparison to experiment.

Nanopore-Based, Multi-Parametric Characterization of Single, Unlabeled Proteins in Solution

Most methods for characterizing proteins - including electron microscopy, NMR spectroscopy, and X-Ray crystallography - require either some sort of physical modification such as purifying, drying, freezing, or crystallizing proteins, or they require chemical modification such as fluorescently labeling or immobilizing proteins. Resistive pulse-based nanopore sensing of proteins is emerging as a powerful analysis technique; it has the potential to overcome these requirements while characterizing individual, unlabeled proteins in aqueous solution. We recently used lipid-coated synthetic nanopores to determine the volume, shape, charge, dipole moment, and rotational diffusion coefficient of single proteins that we tethered to the fluid lipid bilayer to slow their passage through the pore. Here, we compare two different non-adsorptive nanopore coatings combined with high bandwidth recordings to determine the shape and volume of freely translocating proteins in solution. The difficulty of this approach lies in the lateral diffusion of untethered proteins transiting a nanopore, which facilitates position-dependent, asymmetric disruptions in the electric field as proteins approach the nanopore walls. These ‘off-axis’ effects pose a challenge to multi-parametric protein characterization because they distort the observed resistive pulse magnitudes, leading to analysis artifacts. Using lipid-coated nanopores to minimize non-specific protein adhesion, we mitigated these off-axis effects by reducing the residence time of proteins at the pore wall. This approach made it possible to estimate shape and volume values for nine different native proteins free in solution. In contrast, pores coated with the detergent Tween-20 were not suitable for protein shape and volume characterization likely because of significant adhesion to the pore wall and concomitant off-axis effects. By examining unlabeled proteins in an aqueous sample on a single molecule level in lipid bilayer-coated nanopores, we enable new applications for nanopore-based protein sensing, especially those involving transient protein complexes or macromolecules.

Interphase Chromatin Dynamics in Response to Double Stranded DNA Breaks

The dynamic organization of chromatin inside the eukaryotic cell nucleus is essential to the regulation of the genome such as maintaining its integrity, gene expression and replication. However, the physical picture of chromatin structure and dynamics remains unclear and requires identifying the dynamical and structural signatures of specific biological processes. In this work, we study the chromatin dynamics in interphase, time between two cell divisions, in response to DNA damage. As shown previously, chromatin dynamics across the whole nucleus change dramatically after DNA damage; chromatin's coherent motion is eliminated, while its local mobility increases and its compaction decreases [1]. Here, we explore the local chromatin dynamics and compaction in response to local DNA damage in human cells. To do so, we induce double stranded DNA breaks (DSBs), which we visualize using 53BP1-mCherry, and observe the spatiotemporal evolution of their dynamics using single particle tracking. Simultaneously, we use histone H2B-GFP as a reporter on the chromatin compaction, to compare DSB dynamics inside the heterochromatin and euchromatin. To elucidate the dynamic signature of the local DNA damage response, we vary the number of DSBs as well as explore the ATP-dependence of these processes. Our preliminary results show that with an increasing DSB count the local subdiffusive dynamics of the DSBs remains unchanged. We also explore the relationship between the protein aggregation, diffusion coefficient and chromatin compaction at a DSB. [1] Zidovska A, Weitz DA, Mitchison TJ, Proc. Natl. Acad. Sci. USA, 110(39), 1555560, 2013.

Modelling non-Markovian fluctuations in intracellular biomolecular transport

To model non-Markovian fluctuations arising in biomolecular transport, we introduce a stochastic process with memory where Brownian motion is modulated sinusoidally. The probability density function and moments of this non-Markovian process are evaluated analytically as Hida stochastic functional integrals. Comparison of graphs of computed variance vis-á-vis empirical data for protein diffusion coefficients closely match with both exhibiting emergent superdiffusive then subdiffusive behavior for longer proteins.

Determination of diffusion coefficients in live cells using fluorescence recovery after photobleaching with wide-field fluorescence microscopy.

Fluorescence recovery after photobleaching (FRAP) enables characterization of quantitative dynamic properties such as diffusion coefficients of fluorescent molecules in living cells by analyzing the recovery of fluorescence intensity after photobleaching in a specific cellular compartment or area. To quantitatively determine high intracellular diffusion coefficients, a suitable optical system as well as an appropriate model for fast diffusion analysis is necessary. Here, we propose a procedure to quantify the diffusion coefficient of rapidly-diffusing fluorescent molecules that makes use of an epi-fluorescence microscope with a photobleaching laser in combination with established models for diffusion analysis. Analysis for the diffusion coefficients of tandemly oligomerized green flurescent proteins (GFPs) in living cells when changing the photobleaching times showed that photobleaching with shorter times than the diffusion speed indicated not the only way to obtain appropriate diffusion coefficients of fast-moving molecules. Our results also showed that the apparent spreading of the effective radius of the photobleached area works as a correction factor for determining appropriate diffusion coefficients of fast-moving molecules like monomeric GFPs. Our procedure provides a useful approach for quantitative measurement of diffusion coefficients in living cells. This procedure is relevant for characterizing dynamic molecular interactions, especially of fast-moving molecules, and is relevant for studies in many biological fields.

Diffusion of cytokines in live lymph node tissue using microfluidic integrated optical imaging

Communication and drug efficacy in the immune system rely heavily on diffusion of proteins such as cytokines through the tissue matrix. Available methods to analyze diffusion in tissue require microinjection or saturating the tissue in protein, which may alter local transport properties due to damage or rapid cellular responses. Here, we developed a novel, user-friendly method – Microfluidic Integrated Optical Imaging (micro-IOI) – to quantify the effective diffusion coefficient of bioactive proteins in live tissue samples ex vivo. A microfluidic platform was used to deliver picograms of fluorescently labelled cytokines to microscale regions within slices of murine lymph node, and diffusion was monitored by widefield fluorescence microscopy. Micro-IOI was validated against theory and existing methods. Free diffusion coefficients were within 8% and 24% of Stokes-Einstein predictions for dextrans and cytokines, respectively. Furthermore, diffusion coefficients for dextrans and proteins in a model matrix were within 1.5-fold of reported results from fluorescence recovery after photobleaching (FRAP). We used micro-IOI to quantify the effective diffusion of three cytokines from different structural classes and two different expression systems – tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), and interleukin-2 (IL-2), from human and mouse – through live lymph node tissue. This is the first method to directly measure cytokine transport in live tissue slices, and in the future, it should promote a deeper understanding of the dynamics of cell-cell communication and enable targeted immunotherapy design.

Diffusion of Integral Membrane Proteins in Protein-Rich Membranes.

The lateral diffusion of embedded proteins along lipid membranes in protein-poor conditions has been successfully described in terms of the Saffman-Delbrück (SD) model, which predicts that the protein diffusion coefficient D is weakly dependent on its radius R as D ∝ ln(1/R). However, instead of being protein-poor, native cell membranes are extremely crowded with proteins. On the basis of extensive molecular simulations, we here demonstrate that protein crowding of the membrane at physiological levels leads to deviations from the SD relation and to the emergence of a stronger Stokes-like dependence D ∝ 1/R. We propose that this 1/R law mainly arises due to geometrical factors: smaller proteins are able to avoid confinement effects much better than their larger counterparts. The results highlight that the lateral dynamics in the crowded setting found in native membranes is radically different from protein-poor conditions and plays a significant role in formation of functional multiprotein complexes.

Measurement of Rapid Protein Diffusion in the Cytoplasm by Photo-Converted Intensity Profile Expansion

SummaryThe fluorescence microscopy methods presently used to characterize protein motion in cells infer protein motion from indirect observables, rather than measuring protein motion directly. Operationalizing these methods requires expertise that can constitute a barrier to their broad utilization. Here, we have developed PIPE (photo-converted intensity profile expansion) to directly measure the motion of tagged proteins and quantify it using an effective diffusion coefficient. PIPE works by pulsing photo-convertible fluorescent proteins, generating a peaked fluorescence signal at the pulsed region, and analyzing the spatial expansion of the signal. We demonstrate PIPE’s success in measuring accurate diffusion coefficients in silico and in vitro and compare effective diffusion coefficients of native cellular proteins and free fluorophores in vivo. We apply PIPE to measure diffusion anomality in the cell and use it to distinguish free fluorophores from native cellular proteins. PIPE’s direct measurement and ease of use make it appealing for cell biologists.