Macromolecular crowding

The aim of this publication is to present a general strategy to engineer more efficient photoluminescent dendritic molecules based on polyhedral oligomeric silsesquioxane (POSS) cores. A series of chromophores were grafted
on POSS cores to form dendritic molecules for which steric hindrance was used as a trigger to tune their photophysical properties. For fluorescence in the blue/near-UV spectral ranges, 4-vinylbiphenyl molecules were chosen as model chromophores to present a general approach based on stable chemistry and bulky groups grafted to the chromophores to enhance photoluminescence efficiency of the dendritic molecules.
Photoluminescence quantum yields as well as steady-state and time-resolved solution spectroscopy along with molecular dynamics investigation and electronic structure calculations on a family of new materials are reported. We highlight an apparent contrast between free chromophore and dendritic molecules photophysical properties and show that chromophores’ engineering and confinement around an inorganic core allows the design of more efficient photoluminescent dendritic molecules relevant to sensors and hybrid light-emitting diodes.

A range of dendritic molecules was made using the monodentate SEMI-ESPHOS phosphine oxide ligand, which was derivatised with a series of functional groups including bromide, vinyl, allyl and terminal alkyne. Several methods to attach the resulting precursors onto octavinylsilsesquioxane (OVS), ranging from hydrosilylation, Suzuki, Heck, Grubbs or Sonogashira coupling reactions, have been investigated. Cross-metathesis of SEMI-ESPHOS oxide dendrons containing vinyl end groups with OVS catalyzed by Grubbs’ catalyst was proven to be effective in the formation of precursors for dendritic molecules based on OVS.

Highlights:
Dynamic nature is a crucial factor for optimal enzyme activity.
Due to enormous complexity, it is difficult to directly correlate enzyme dynamics and activity.
This question is addressed here taking an industrially crucial proteolytic enzyme, bromelain.
We contemplated and correlated the structure, dynamics and activity of bromelain in versatile chemicals.
In all the cases, a robust relationship between active-site dynamics and activity emerges.
Microsecond active-site dynamics is probably a key factor for activity.


Abstract
Proteins are dynamic entity with various molecular motions at different timescale and length scale. Molecular motions are crucial for the optimal function of an enzyme. It seems intuitive that these motions are crucial for optimal enzyme activity. However, it is not easy to directly correlate an enzyme's dynamics and activity due to biosystems' enormous complexity. amongst many factors, structure and dynamics are two prime aspects that combinedly control the activity. Therefore, having a direct correlation between protein dynamics and activity is not straightforward. Herein, we observed and correlated the structural, functional, and dynamical responses of an industrially crucial proteolytic enzyme, bromelain with three versatile classes of chemicals: GnHCl (protein denaturant), sucrose (protein stabilizer), and Ficoll-70 (macromolecular crowder). The only free cysteine (Cys-25 at the active-site) of bromelain has been tagged with a cysteine-specific dye to unveil the structural and dynamical changes through various spectroscopic studies both at bulk and at the single molecular level. Proteolytic activity is carried out using casein as the substrate. GnHCl and sucrose shows remarkable structure-dynamics-activity relationships. Interestingly, with Ficoll-70, structure and activity are not correlated. However, microsecond dynamics and activity are beautifully correlated in this case also. Overall, our result demonstrates that bromelain dynamics in the microsecond timescale around the active-site is probably a key factor in controlling its proteolytic activity.

Beim Formensehen höherer Säugetiere und Primaten spielt der über das Geniculatum laterale und den primären visuellen Kortex führende Signalweg die wichtigste Rolle, und dementsprechend finden sich die Projektionseigenschaften des retinokortikalen Pfades in den psychophysischen Ergebnissen wieder. In vielen Fällen sind sie sogar ausreichend zur Erklärung der Leistungsunterschiede zwischen verschiedenen Ge-sichtsfeldorten. Im einfachsten Fall nimmt man, wie in Kap. 1.2.1 ausgeführt, an, daß die Struktur der Verschaltung an verschiedenen Gesichtsfeldorten gleichartig ist und daher nur die Volumen, oder auch − durch die flächige Struktur − die Flächen der beteiligten Nervengewebe eine Rolle spielen. Man bestimmt deshalb den Quotient zwischen beteiligter kortikaler und retinaler Fläche. Vermittels der optischen Abbil-dungeigenschaften des Auges entspricht einer Fläche der Netzhaut in eindeutiger Weise ein gewisser Raumwinkel des Gesichtsfeldes, und so gelangt man zur Definition des flächigen kortikalen Vergrößerungsfaktors als derjenigen Fläche Kortexge-webes, auf die 1×1 Grad des Gesichtsfeldes projizieren. Entsprechend ist der lineare kortikale Vergrößerungsfaktor als der Durchmesser des gleichen Areals definiert; er wird mit dem Buchstaben M bezeichnet. Über die Größe von M als Funktion des Gesichtsfeldortes ist heute sehr viel be-kannt, s. Kap. 1.2.1. Trotzdem sind, wie oben bemerkt, wichtige Fragen bis heute kontrovers und Gegenstand aktueller Forschung: • Wie groß ist M im Zentrum der Fovea? Auf Grund der lateralen anatomischen Verschiebung zwischen Rezeptorzellen und zugehörigen Ganglienzellen ist man hier gegenwärtig auf Schätzungen angewiesen. • Ist M unterschiedlich für Subpopulationen? Von P-und M-Zellen weiß man, daß sie unterschiedliche Übertragungseigenschaften besitzen, so daß ihr zahlenmäßi-ges Verhältnis zueinander Auswirkungen auf die Übertragungseigenschaften des Gesamtkanals hat.

Hsp16.3, a small heat shock protein from Mycobacterium tuberculosis proposed to form specific trimer-of-trimers structures, acts as a molecular chaperone in vitro. The assembly and re-assembly mechanisms of this oligomeric protein were studied and compared using in vitro transcription/translation and denaturization/renaturization systems. Analysis using a combination of non-denaturing pore gradient polyacrylamide gel electrophoresis, chemical cross-linking, and size-exclusion chromatography demonstrate that the predominant form of Hsp16.3 produced in the in vitro transcription/translation system is the trimer, which can be further assembled into a nonameric structure via a hexamer intermediate in the presence of purified exogenous Hsp16.3 proteins. Meanwhile, an "inert" Hsp16.3 dimer, which does not seem to participate in nonamer assembly but may be involved in forming other forms of Hsp16.3, was also detected in the in vitro expression system. On the other hand, our current data clearly show that the re-assembly of Hsp16.3 nonamers also occurs via a very similar mechanism, with the formation of trimers and hexamers. The presence of high levels of macromolecular crowding protein agent in the in vitro expression system promoted the formation of the nonamers to a very limited degree, indicating that the assembly of proteins like Hsp16.3 may depend mainly on its own concentration instead of those of the macromolecules in the environment.

Electron transfer in riboflavin binding protein in cellular environments Picosecond-resolved electron transfer dynamics of Vitamin B 2 in protein Ligand binding in micellar crowding Tertiary structure of riboflavin binding protein a b s t r a c t

The excluded volume effect (EVE) rules all life processes. It is created by macromolecules that occupy a given volume thereby confining other molecules to the remaining space with large consequences on reaction kinetics and molecular assembly. Implementing EVE in fibroblast culture accelerated conversion of procollagen to collagen by procollagen C-proteinase (PCP/BMP-1) and proteolytic modification of its allosteric regulator, PCOLCE1. This led to a 20-30-and 3-6-fold increased collagen deposition in two-and three-dimensional cultures, respectively, and creation of crosslinked collagen footprints beneath cells. Important parameters correlating with accelerated deposition were hydrodynamic radius of macromolecules and their negative charge density.

New diazaphospholidine POSS macromolecules have been synthesised and tested in styrene and vinyl acetate hydroformylation. Whilst some of them have shown activity, others precipitated upon mixing with the rhodium precursor preventing its efficient use. Molecular dynamics has been used to help understand these observations. Rigid and compact dendritic structures with phosphine groups engineered to have low mobility but high probability of sitting at distances favouring bidentate coordination with the rhodium precursors are necessary for the macromolecular ligands to be active.
More flexible structures having lower probability of phosphine separations suitable for bidentate complex formation are more prone to form oligomeric dendritic species and hence to crosslink the macromolecules and precipitate.

One of the main differences in the intercellular environment compared to the laboratory condition is the presence of macromolecular crowders of various compositions, sizes, and shapes. In this article, we have contemplated a systematic shape dependency of macromolecular crowders on the thermodynamics and microsecond conformational fluctuation dynamics of protein unfolding by taking human serum albumin (HSA) as the model protein and similar-sized crowders, namely, dextran-40, ficoll-70, and PEG-35 as macromolecular crowders of different shapes, to mimic the cell environment. We observed that dextran-40 and ficoll-70 counteract the thermal denaturation and PEG-35 assists it. A complete thermodynamic analysis suggests that the stabilization by dextran-40 and ficoll-70 occurs mainly through stabilizing entropic effect, which is somewhat counteracted by the destabilizing enthalpic effect, in line with what is expected from the traditional interpretation of excluded volume and soft interaction. Surprisingly, the destabilizing effect of PEG-35 is not through unfavorable interaction but through a destabilizing entropic effect, which is opposite to the excluded volume prediction. Our speculation is that the modulation of the associated water structure due to crowder-induced distortion plays a crucial role in modulating the entropic component. Moreover, while a two-state model can approximate the overall thermal denaturation of HSA in the absence and presence of various crowders, the thermal denaturation profile of domain III of HSA involves a distinct intermediate state. The active-site dynamics of HSA are altered significantly in the presence of all the three differently shaped crowders used in the study. Rod-shaped dextran-40 and spherical ficoll-70 hinder the microsecond conformational dynamics of domain III of HSA in all states except the intermediate state. Mesh-like PEG-35 in addition to hindering the microsecond conformational dynamics shifts the intermediate state from 40 to 30°C. Overall, our results provide new insight into deciphering the mechanism of crowder-induced changes in protein. Through our interpretation, we not only explain the unfavorable entropic contribution but also provide a physical basis to explain the entropy−enthalpy compensation.

The polymerase chain reaction's (PCR) phenomenal success in advancing fields as diverse as Medicine, Agriculture, Conservation, or Paleontology is based on the ability of using isolated prokaryotic thermostable DNA polymerases in vitro to copy DNA irrespective of origin. This process occurs intracellularly and has evolved to function efficiently under crowded conditions, namely in an environment packed with macromolecules. However, current in vitro practice ignores this important biophysical parameter of life. In order to more closely emulate conditions of intracellular biochemistry in vitro we added inert macromolecules into reverse transcription (RT) and PCR. We show dramatic improvements in all parameters of RT-PCR including 8-to 10-fold greater sensitivity, enhanced polymerase processivity, higher specific amplicon yield, greater primer annealing and specificity, and enhanced DNA polymerase thermal stability. The faster and more efficient reaction kinetics was a consequence of the cumulative molecular and thermodynamic effects of the excluded volume effect created by macromolecular crowding.

Macromolecular crowding is known to affect protein folding, binding of small molecules, interaction with nucleic acids, enzymatic activity, protein-protein interactions, and protein aggregation. Although for a long time it was believed that the major mechanism of the action of crowded environments on structure, folding, thermodynamics, and function of a protein can be described in terms of the excluded volume effects, it is getting clear now that other factors originating from the presence of high concentrations of "inert" macromolecules in crowded solution should definitely be taken into account to draw a more complete picture of a protein in a crowded milieu. This review shows that in addition to the excluded volume effects important players of the crowded environments are viscosity, perturbed diffusion, direct physical interactions between the crowding agents and proteins, soft interactions, and, most importantly, the effects of crowders on solvent properties.

In the present study we have investigated the thermal stability of the globular transport protein human serum albumin (HSA), in the presence of two small chain polyethylene glycols (namely PEG 200 and PEG 400). Both near-and far-UV circular dichroism (CD) study reveal that addition of PEG moderately increases the a-helical content of the protein without abruptly changing its tertiary structure. The hy-dration structure at the protein surface experiences a notable change at 30% PEG (v/v) concentration as evidenced from compressibility and dynamic light scattering (DLS) measurements. Thermal denaturation of HSA in the presence of PEG has been studied by CD and fluorescence spectroscopy using the intrinsic fluorophore tryptophan and it has been found that addition of PEG makes the protein more prone towards unfolding, which is in contrary to what has been observed in case of larger molecular weight polymers. The energetics of the thermal unfolding process has been obtained using differential scanning calorimetry (DSC) measurements. Our study concludes that both the indirect excluded volume principle as well as interaction of the polymer at the protein surface is responsible for the observed change of the unfolding process.

Fluorescence correlation spectroscopy (FCS) was used to measure the translational diffusion of labeled apomyoglobin (tracer) in concentrated solutions of ribonuclease A and human serum albumin (crowders), as a quantitative model system of protein diffusive motions in crowded physiological environments. The ratio of the diffusion coefficient of the tracer protein in the protein crowded solutions and its diffusion coefficient in aqueous solution has been interpreted in terms of local apparent viscosities, a molecular parameter characteristic for each tracer-crowder system. In all protein solutions studied in this work, local translational viscosity values were larger than the solution bulk viscosity, and larger than rotational viscosities estimated for apomyoglobin in the same crowding solutions. Here we propose a method to estimate local apparent viscosities for the tracer translational and rotational diffusion directly from the bulk viscosity of the concentrated protein solutions. As a result of this study, the identification of protein species and the study of hydrodynamic changes and interactions in model crowded protein solutions by means of FCS and time-resolved fluorescence depolarization techniques may be expected to be greatly simplified.

Tissue engineering by self-assembly hypothesises that optimal repair and regeneration can be achieved best by using the cells’ inherent ability to create organs with proficiency still unmatched by currently available scaffold fabrication technologies. However, the prolonged culture time required to develop an implantable device jeopardises clinical translation and commercialisation of such techniques. Herein, we report that macromolecular crowding, a biophysical in vitro microenvironment modulator, dramatically accelerates extracellular matrix deposition in cultured human corneal, lung and dermal fibroblasts and human bone marrow mesenchymal stem cells. In fact, an almost 5 to 30 fold increase in collagen type I deposition was recorded as early as 48 hours in culture, without any negative effect in cell phenotype and function.

Biological reactions in the cellular environment differ physicochemically from those performed in dilute buffer solutions due to, in part, slower diffusion of various components in the cellular milieu, increase in their chemical activities, and modulation of their binding affinities and conformational stabilities. In vivo transcription is therefore expected to be strongly influenced by the 'crowdedness' of the cell. Previous studies of transcription under macromolecular crowding conditions have focused mainly on multiple cycles of RNAP-Promoter associations, assuming that the association is the rate-determining step of the entire transcription process. However, recent reports demonstrated that late initiation and promoter escape could be the rate-determining steps for some promoter DNA sequences. The investigation of crowding effects on these steps under single-round conditions is therefore crucial for better understanding of transcription initiation in vivo. Here, we have implemented an in vitro transcription quenched-kinetics single-molecule assay to investigate the dependence of transcription reaction rates on the sizes and concentrations of crowders. Our results demonstrate an expected slowdown of transcription kinetics due to increased viscosity, and an unexpected enhancement in transcription kinetics by large crowding agents (at a given viscosity). More importantly, the enhancement's dependence on crowder size significantly deviates from hard-sphere model (scaled-particle theory) predictions, commonly used for description of crowding effects. Our findings shed new light on how enzymatic reactions are affected by crowding conditions in the cellular milieu.

The cellular environment is crowded with different kinds of molecules with varying sizes, shapes and compositions. Most of the experiments studying the nature and behaviour of a protein have been done on the isolated protein in dilute buffer solutions which actually do not imitate the in vivo situation. To understand the consequences of such crowded environment, we investigated the effect of macromolecular crowding on the stability and activity of hen egg white lysozyme. Two crowding agents, dextran 70 and ficoll 70 which have different shapes and composition, have been employed in this study. To mimic the cellular condition from physiological point of view, the effect of mixtures of both the crowding agents has been also studied. The results indicate that owing to volume exclusion, lysozyme is stabilized while its activity decays with the increasing concentration of both the crowders elucidating the hypothesis of stability-activity trade-off. Mixed macromolecular crowding exerts greater effect than the sum of constituent crowding agents (dextran 70 and ficoll 70).

One of the main differences of intercellular environment compared to the laboratory condition is the presence of macromolecular crowder of various compositions, sizes, and shapes. In this contribution, we have contemplated a systematic shape dependency of macromolecular crowder on the thermodynamics and microsecond (µs) conformational fluctuation dynamics of protein unfolding taking human serum albumin (HSA) as the model protein and almost similar sized crowders namely dextran-40, ficoll-70 and PEG-35 as macromolecular crowders of different shapes, to mimic the cell environment. We observed that dextran-40 and ficoll-70 counteract the thermal denaturation and PEG-35 assists it. A complete thermodynamic analysis suggests that the stabilisation by dextran-40 and ficoll-70 occurs mainly through stabilising entropic effect, which is somewhat counteracted by destabilising enthalpic effect, in line with what is expected from the traditional interpretation of excluded volume and soft interaction. Surprisingly, the destabilising effect of PEG-35 is not through unfavourable interaction but through destabilising entropic effect, which is opposite to the excluded volume prediction. Our speculation is that the modulation of associated water structure due to crowder induced distortion plays a crucial role in modulating the entropic component. Moreover, while a two-state model can approximate the overall thermal denaturation of HSA in the absence and presence of various crowders, the thermal denaturation profile of domain-III of HSA involves a distinct intermediate state. The active site dynamics of HSA gets altered significantly in the presence of all the three differently shaped crowders used in the study. Rod shaped dextran-40 and spherical ficoll-70 hinder the µs conformational dynamics of domain-III of HSA in all the states except in the intermediate state. Mesh-like PEG-35 in addition to hindering the µs conformational dynamics shifts the intermediate state from 40 ⁰C to 30 ⁰C. Overall, our result provides a new insight to decipher the mechanism of crowder induced changes of protein. Through our interpretation, we not only explain the unfavourable entropic contribution but also provides a physical basis to explain entropy-enthalpy compensation.

In the present study we have investigated the thermal stability of the globular transport protein human serum albumin (HSA), in the presence of two small chain polyethylene glycols (namely PEG 200 and PEG 400). Both near- and far-UV circular dichroism (CD) study reveal that addition of PEG moderately increases the α-helical content of the protein without abruptly changing its tertiary structure. The hydration structure at the protein surface experiences a notable change at 30% PEG (v/v) concentration as evidenced from compressibility and dynamic light scattering (DLS) measurements. Thermal denaturation of HSA in the presence of PEG has been studied by CD and fluorescence spectroscopy using the intrinsic fluorophore tryptophan and it has been found that addition of PEG makes the protein more prone towards unfolding, which is in contrary to what has been observed in case of larger molecular weight polymers. The energetics of the thermal unfolding process has been obtained using differential scanning calorimetry (DSC) measurements. Our study concludes that both the indirect excluded volume principle as well as interaction of the polymer at the protein surface is responsible for the observed change of the unfolding process.

DnaA(L366K), in concert with a wild-type DnaA (wtDnaA) protein, restores the growth of Escherichia coli cells arrested in the absence of adequate levels of cellular acidic phospholipids. In vitro and in vivo studies showed that DnaA(L366K) alone does not induce the initiation of replication, and wtDnaA must also be present. Hitherto the different behavior of wt and mutant DnaA were not understood. We now demonstrate that this mutant may be activated at significantly lower concentrations of acidic phospholipids than the wild-type protein, and this may explain the observed growth restoration in vivo.

Traditionally, biochemical studies are performed in dilute homogenous solutions, which are very different from the dense mixture of molecules found in cells. Thus, the physiological relevance of these studies is in question. This recognition motivated scientists to formulate the effect of crowded solutions in general, and excluded volume in particular, on biochemical processes. Using polymers or proteins as crowders, it was shown that while crowding tends to significantly enhance the formation of complexes containing many subunits, dimerizations are only mildly affected. Computer simulations, together with experimental evidence, indicate soft interactions and diffusion as critical factors that operate in a concerted manner with excluded volume to modulate protein binding. Yet, these approaches do not truly mimic the cellular environment. In vivo studies may overcome this shortfall. The few studies conducted thus far suggest that in cells, binding and folding occur at rates close to those determined in dilute solutions. Obtaining quantitative biochemical information on reactions inside living cells is currently a main challenge of the field, as the complexity of the intracellular milieu was what motivated crowding research to begin with.

Macromolecular crowding can alter the structure and function of biological macromolecules. We used small-angle scattering to measure the effects of macromolecular crowding on the size of a protein complex, SOD (superoxide dismutase). Crowding was induced using 400 MW PEG (polyethylene glycol),TEG (triethylene glycol), a-MG (methyl-a-glucoside), and TMAO (trimethylamine n-oxide). Parallel small-angle neutron scattering and small-angle x-ray scattering allowed us to unam-biguously attribute apparent changes in radius of gyration to changes in the structure of SOD. For a 40% PEG solution, we find that the volume of SOD was reduced by 9%. Considering the osmotic pressure due to PEG, this deformation corresponds to a highly compressible structure. Small-angle x-ray scattering done in the presence of TEG suggests that for further deforma-tion—beyond a 9% decrease in volume—the resistance to deformation may increase dramatically.

We review recent evidence illustrating the fundamental difference between cytoplasmic and test tube biochemical kinetics and thermodynamics, and showing the breakdown of the law of mass action and power-law approximation in in vivo conditions. Simulations of biochemical reactions in non-homogeneous media show that as a result of anomalous diffusion and mixing of the biochemical species, reactions follow a fractal-like kinetics. Consequently, the conventional equations for biochemical pathways fail to describe the reactions in in vivo conditions. We present a modification to fractal-like kinetics following the Zipf-Mandelbrot distribution which will enable the modelling and analysis of biochemical reactions occurring in crowded intracellular environments. r

In this contribution, we study the effect of confinement on the ultrafast electron transfer (ET) dynamics of riboflavin binding protein (RBP) to the bound cofactor riboflavin (Rf, vitamin B2), an important metabolic process, in anionic sodium bis(2-ethylhexyl) sulfosuccinate reverse micelles (AOT-RMs) of various hydration levels. Notably, in addition to excluded volume effect, various nonspecific interactions like ionic charge of the confining surface can influence the biochemical reactions in the confined environment of the cell. To this end, we have also studied the ET dynamics of RBP-Rf complex under the confinement of a cationic hexadecyltrimethylammonium bromide (CTAB) RMs with similar water pool size to the anionic AOT-RMs towards simulating equal restricted volume effect. It has been found that the spatial confinement of RBP in the AOT-RM of w 0 = 10 leads to the loss of its tertiary structure and hence vitamin binding capacity. Although, RBP regains its binding capacity and tertiary structure in AOT-RMs of w 0 ≥ 20 due to its complete hydration, the ultrafast ET from RBP to Rf merely occurs in such systems. However, to our surprise, the ET process is found to occur in cationic CTAB-RMs of similar volume restriction. It is found that under the spatial confinement of anionic AOT-RM, the isoalloxazine ring of Rf is improperly placed in the protein nanospace so that ET between RBP and Rf is not permitted. This anomaly in the binding behaviour of Rf to RBP in AOT-RMs is believed to be the influence of repulsive potential of the anionic AOT-RM surface to the protein. Our finding thus suggests that under similar size restriction, both the hydration and surface charge of the confining volume could have major implication in the intraprotein ET dynamics in real cellular environments.

The cellular environment is crowded by macromolecules of various sizes, shapes, and charges, which modulate protein structure, function and dynamics. Herein, we contemplated the effect of three different macromolecular crowders: dextran-40, ficoll-70 and PEG-35 on the structure, active-site conformational dynamics, function and relative domain movement of multi-domain human serum albumin (HSA). All the crowders used in this study have zero charges and similar sizes (at least in the dilute region) but different shapes and compositions. Some observations follow the traditional crowding theory. For example, all the crowders increased the α-helicity of HSA and hindered the conformational fluctuation dynamics. However, some observations are not in line with the expectations, as an increase in the size of HSA with PEG-35 and uncorrelated domain movement of HSA with ficoll-70 and PEG-35. The relative domain movement is correlated with the activity, suggesting that such moves are essential for protein function. The interaction between HSA and ficoll-70 is proposed to be hydrophobic in nature. Overall, our results provide a somewhat systematic study of the shape-dependent macromolecular crowding effect on various protein properties and present a possible new insight into the mechanism of macromolecular crowding

Recent studies have led to increased appreciation of the influence of excluded volume in solutions of high total macromolecular content ('macromolecular crowding') upon the various classes of reaction that lead to the assembly of proteins and protein complexes. In general, crowding is expected to stabilize native protein structure relative to less compact non-native structures and to favor the formation of functional complexes of native proteins. Under certain pathological conditions, 'overcrowding' may enhance the formation of nonfunctional aggregates of non-native protein (e.g. amyloid and inclusion bodies).

Biological reactions in the cellular environment differ physicochemically from those performed in dilute buffer solutions due to, in part, slower diffusion of various components in the cellular milieu, increase in their chemical activities, and modulation of their binding affinities and conformational stabilities. In vivo transcription is therefore expected to be strongly influenced by the 'crowdedness' of the cell. Previous studies of transcription under macromolecular crowding conditions have focused mainly on multiple cycles of RNAP-Promoter associations, assuming that the association is the rate-determining step of the entire transcription process. However, recent reports demonstrated that late initiation and promoter escape could be the rate-determining steps for some promoter DNA sequences. The investigation of crowding effects on these steps under single-round conditions is therefore crucial for better understanding of transcription initiation in vivo. Here, we have i...

Protein p6 from Bacillus subtilis phage ϕ29 binds double-stranded DNA, forming a large nucleoprotein complex all along the viral genome, and has been proposed to be an architectural protein with a global role in genome organization. Here, we have characterized quantitatively the DNA binding properties of protein p6 by means of sedimentation velocity and sedimentation equilibrium experiments permitting determination of the strength and stoichiometry of complex formation. The composition dependence of protein binding to DNA is quantitatively consistent with a model in which the protein undergoes a reversible monomer-dimer self-association, and the dimeric species binds noncooperatively to the DNA. We also have found that when the anisotropic bendability periodicity of the nucleotide sequence preferred by p6 is modified, nucleocomplex formation is impaired. In addition, suppression of complex formation at high ionic strength is reversed by the addition of high concentrations of an inert polymer, mimicking the crowded intracellular environment. The results obtained in this work illustrate how macromolecular crowding could act as a metabolic buffer that can significantly extend the range of intracellular conditions under which a specific reaction may occur.

Current technologies and available scaffold materials do not support long-term cell viability, differentiation, and maintenance of podocytes, the ultra-specialized kidney resident cells that are responsible for the filtration of the blood. A new platform which imitates the native kidney microenvironment by decellularizing fibroblasts grown on surfaces with macromolecular crowding is developed. Human immortalized podocytes cultured on this platform display superior viability and metabolic activity up to 28 days compared to podocytes cultured on tissue culture plastic surfaces. The new platform displays a softer surface and an abundance of growth factors and associated molecules. More importantly, it enables podocytes to display molecules responsible for their structure and function and a superior development of intercellular connections/interdigitations, consistent with maturation. The new platform can be used to study podocyte biology, test drug toxicity, and determine whether sera from patients with podocytopathies are involved in the expression of glomerular pathology.

The aim of this publication is to present a general strategy to engineer more efficient photoluminescent dendritic molecules based on polyhedral oligomeric silsesquioxane (POSS) cores. A series of chromophores were grafted on POSS cores to form dendritic molecules for which steric hindrance was used as a trigger to tune their photophysical properties. For fluorescence in the blue/near-UV spectral ranges, 4-vinylbiphenyl molecules were chosen as model chromophores to present a general approach based on stable chemistry and bulky groups grafted to the chromophores to enhance photoluminescence efficiency of the dendritic molecules. Photoluminescence quantum yields as well as steady-state and time-resolved solution spectroscopy along with molecular dynamics investigation and electronic structure calculations on a family of new materials are reported. We highlight an apparent contrast between free chromophore and dendritic molecules photophysical properties and show that chromophores' engineering and confinement around an inorganic core allows the design of more efficient photoluminescent dendritic molecules relevant to sensors and hybrid light-emitting diodes.