Mechanochemical consequences of myopathy-linked mutations in Tpm2.2 on striated muscle contractility.

Tropomyosin (Tpm) is an actin-binding protein central to muscle contraction regulation. The Tpm sequence consists of periodic repeats corresponding to seven actin-binding sites, further divided in two functionally distinct halves. To clarify the importance of the first and second halves of the actin-binding periods in regulating the interaction of myosin with actin, we introduced hypercontractile mutations D20H, E181K located in the N-terminal halves of periods 1 and 5 and hypocontractile mutations E41K, N202K located in the C-terminal halves of periods 1 and 5 of the skeletal muscle Tpm isoform Tpm2.2. Wild-type and mutant Tpms displayed similar actin-binding properties, however, as revealed by FRET experiments, the hypercontractile mutations affected the binding geometry and orientation of Tpm2.2 on actin, causing a stimulation of myosin motor performance. Contrary, the hypocontractile mutations led to an inhibition of both, actin activation of the myosin ATPase and motor activity, that was more pronounced than with wild-type Tpm2.2. Single ATP turnover kinetic experiments indicate that the introduced mutations have opposite effects on product release kinetics. While the hypercontractile Tpm2.2 mutants accelerated product release, the hypocontractile mutants decelerated product release from myosin, thus having either an activating or inhibitory influence on myosin motor performance, which agrees with the muscle disease phenotypes caused by these mutations.

Transient Excursions to Membrane Core as Determinants of Influenza Virus Fusion Peptide Activity.

Fusion of viral and host cell membranes is a critical step in the life cycle of enveloped viruses. In the case of influenza virus, it is mediated by subunit 2 of hemagglutinin (HA) glycoprotein whose N-terminal fragments insert into the target membrane and initiate lipid exchange. These isolated fragments, known as fusion peptides (HAfp), already possess own fusogenic activity towards liposomes. Although they have long been studied with the hope to uncover the details of HA-mediated fusion, their actual mechanism of action remains elusive. Here, we use extensive molecular dynamics simulations combined with experimental studies of three HAfp variants to fully characterize their free energy landscape and interaction with lipid bilayer. In addition to customary assumed peptides localization at lipid-water interface, we characterize membrane-spanning configurations, which turn out to be metastable for active HAfps and unstable for the fusion inactive W14A mutant. We show that, while the degree of membrane perturbation by surface peptide configurations is relatively low and does not show any mutation-related differences, the effect of deeply inserted configurations is significant and correlates with insertion depth of the N-terminal amino group which is the highest for the wild type HAfp. Finally, we demonstrate the feasibility of spontaneous peptide transition to intramembrane location and the critical role of strictly conserved tryptofan residue 14 in this process.

Effect of HIV-1 TAT Peptide Fusion on 5' mRNA Cap Analogs Cell Membrane Permeability and Translation Inhibition.

The development of targeted anticancer drugs has been one of the most challenging goals of current research. Eukaryotic translation initiation factor 4E (eIF4E) is an oncogene that stimulates mRNA translation via binding to the 5' endcap structure. It is well documented that eIF4E is overexpressed in many cancers including breast, prostate, head and neck, and stomach malignancies and leads to oncogenic transformation and metastasis. One approach to block eIF4E function in cancer cells is based on the disruption of the interaction between eIF4E and the 5' mRNA cap structure using cap analog inhibitors. Since analogs are cell-impermeable due to their anionic nature, we used a cell penetrating peptide (CPP) for delivery of model cap analogs into cancer cells. The human immunodeficiency virus I (HIV-1) transactivator of transcription derived peptide (TAT) was conjugated with the analogs m7GMP and m7GpppG using click chemistry methodology. We observed that both conjugates (m7GMP-TAT and m7GpppG-TAT), contrary to TAT alone, did not translocate through the artificial phospholipid membrane of giant unilamellar vesicles. This suggests that passive transport is not the mechanism by which translocation of cap analogs occurs. In contrast, synthesized fluorescently labeled m7GpppG-TAT translocated into the human breast adenocarcinoma cancer cell line MCF-7. Furthermore, we demonstrated that m7GMP-TAT and m7GpppG-TAT inhibited cap-dependent translation up to 30% both in vivo and in vitro while simultaneously not affecting cell growth and viability. These results demonstrate the usefulness of cell penetration peptides as carriers for the internalization of cap analogs.

Charged N-terminus of Influenza Fusion Peptide Facilitates Membrane Fusion.

Cleavage of hemagglutinin precursor (HA0) by cellular proteases results in the formation of two subunits, HA1 and HA2. The N-terminal fragment of HA2, named a fusion peptide (HAfp), possess a charged, amine N-terminus. It has been shown that the N-terminus of HAfp stabilizes the structure of a helical hairpin observed for a 23-amino acid long peptide (HAfp1-23), whose larger activity than HAfp1-20 has been demonstrated recently. In this paper, we analyze the effect of N-terminal charge on peptide-mediated fusion efficiency and conformation changes at the membrane interface by comparison with the corresponding N-acetylated peptides of 20- and 23-amino acid lengths. We found that higher fusogenic activities of peptides with unmodified amino termini correlates with their ability to form helical hairpin structures oriented perpendicularly to the membrane plane. Molecular dynamics simulations showed that acetylated peptides adopt open and surface-bound conformation more often, which induced less disorder of the phospholipid chains, as compared to species with unmodified amino termini.

Translocation of 5' mRNA cap analogue--peptide conjugates across the membranes of giant unilamellar vesicles.

Cell-penetrating peptides (CPPs) have been extensively studied because of their ability to deliver various cargo molecules, which are often potential therapeutic agents. However, in most cases, the exact entry mechanism of CPPs is still unknown. In this study, we focused our attention on the membrane permeability sequence (MPS) peptide (AAVALLPAVLLALLAK) conjugated to analogues of a 5' mRNA cap. This unique RNA structure plays a pivotal role in eukaryotic gene expression and has a large therapeutic application potential. We validated the translocation abilities of conjugates across the membranes of giant unilamellar vesicles (GUVs) composed of POPC lipids by application of fluorescence microscopy. Translocation of the MPS peptide itself was observed in contrast to peptide conjugates containing mono- and dinucleotide cap analogues, indicating that even for such small cargos, passive translocation does not occur. However, membrane permeability was observed in the case of conjugated mononucleotides. Fluorescence lifetime microscopy (FLIM) of the C6-NBD-phospholipid revealed changes in lipid packing induced by a penetrating peptide. Our results support the usefulness of artificial membrane systems applied to elucidate membrane crossing mechanisms.

Molecular recognition of mRNA 5' cap by 3' poly(A)-specific ribonuclease (PARN) differs from interactions known for other cap-binding proteins.

The mRNA 5' cap structure plays a pivotal role in coordination of eukaryotic translation and mRNA degradation. Poly(A)-specific ribonuclease (PARN) is a dimeric exoribonuclease that efficiently degrades mRNA 3' poly(A) tails while also simultaneously interacting with the mRNA 5' cap. The cap binding amplifies the processivity of PARN action. We used surface plasmon resonance kinetic analysis, quantitative equilibrium fluorescence titrations and circular dichroism to study the cap binding properties of PARN. The molecular mechanism of 5' cap recognition by PARN has been demonstrated to differ from interactions seen for other known cap-binding proteins in that: i) the auxiliary biological function of 5' cap binding by the 3' degrading enzyme is accomplished by negative cooperativity of PARN dimer subunits; ii) non-coulombic interactions are major factors in the complex formation; and iii) PARN has versatile activity toward alternative forms of the cap. These characteristics contribute to stabilization of the PARN-cap complex needed for the deadenylation processivity. Our studies provide a consistent biophysical basis for elucidation of the processive mechanism of PARN-mediated 3' mRNA deadenylation and provide a new framework to interpret the role of the 5' cap in mRNA degradation.

Diffusion of Single-Pass Transmembrane Receptors: From the Plasma Membrane into Giant Liposomes.

To quantitatively examine the effect of membrane organization on lateral diffusion, we studied fluorescent carbocyanine lipid analogues and EGFP-tagged, single-pass transmembrane proteins in systems of decreasing complexity: (i) the plasma membrane (PM) of living cells, (ii) paraformaldehyde/dithiothreitol-induced giant plasma membrane vesicles (GPMVs), and (iii) giant unilamellar vesicles (GUVs) under physiological buffer conditions. A truncated, signaling-deficient interleukin-4 receptor subunit, showing efficient accumulation in the plasma membrane, served as a model transmembrane protein. Two-dimensional diffusion coefficients (D) were determined by fluorescence correlation spectroscopy (FCS) either at fixed positions (single-point, spFCS) or while scanning a circular orbit (circular scanning, csFCS). Consistent with a different inclusion sizes in the membrane, lipids diffuse slightly faster than the single-spanning membrane proteins in both membrane systems, GUVs and GPMVs. In GPMVs lipids and proteins consistently experienced a fivefold larger viscosity than in GUVs, reflecting the significant fraction of plasma membrane-derived proteins partitioning into GPMVs. Lipid and protein diffusion in the PM was, respectively, 2 times and 4-5 times slower in comparison to GPMVs. This discrepancy was quantitatively confirmed by csFCS. The similarity of diffusion of receptors and lipids in GPMVs and GUVs and its significant difference in the plasma membrane suggest that protein domains as small as EGFP convey sensitivity to the actin cortex on various length scales.

Three conserved C-terminal residues of influenza fusion peptide alter its behavior at the membrane interface.

The N-terminal fragment of the viral hemagglutinin HA2 subunit is termed a fusion peptide (HAfp). The 23-amino acid peptide (HAfp1-23) contains three C-terminal W21-Y22-G23 residues which are highly conserved among serotypes of influenza A and has been shown to form a tight helical hairpin very distinct from the boomerang structure of HAfp1-20. We studied the effect of peptide length on fusion properties, structural dynamics, and binding to the membrane interface. We developed a novel fusion visualization assay based on FLIM microscopy on giant unilamellar vesicles (GUV). By means of molecular dynamics simulations and spectroscopic measurements, we show that the presence of the three C-terminal W21-Y22-G23 residues promotes the hairpin formation, which orients perpendicularly to the membrane plane and induces more disorder in the surrounding lipids than the less structured HAfp1-20. Moreover, we report cholesterol-enriched domain formation induced exclusively by the longer fusion peptide.

Dynamics and interaction of interleukin-4 receptor subunits in living cells.

It has long been established that dimerization of Interleukin-4 receptor (IL-4R) subunits is a pivotal step for JAK/STAT signal transduction. However, ligand-induced complex formation at the surface of living cells has been challenging to observe. Here we report an experimental assay employing trisNTA dyes for orthogonal, external labeling of eGFP-tagged receptor constructs that allows the quantification of receptor heterodimerization by dual-color fluorescence cross-correlation spectroscopy. Fluorescence cross-correlation spectroscopy analysis at the plasma membrane shows that IL-4R subunit dimerization is indeed a strictly ligand-induced process. Under conditions of saturating cytokine occupancy, we determined intramembrane dissociation constants (K(d,2D)) of 180 and 480 receptors per µm(2) for the type-2 complexes IL-4:IL-4Rα/IL-13Rα1 and IL-13:IL-13Rα1/IL-4Rα, respectively. For the lower affinity type-1 complex IL-4:IL-4Rα/IL-2Rγ, we estimated a K(d,2D) of ∼1000 receptors per µm(2). The receptor densities required for effective dimerization thus exceed the typical, average expression levels by several orders of magnitude. In addition, we find that all three receptor subunits accumulate rapidly within a subpopulation of early sorting and recycling endosomes stably anchored just beneath the plasma membrane (cortical endosomes, CEs). The receptors, as well as labeled IL-4 and trisNTA ligands are specifically trafficked into CEs by a constitutive internalization mechanism. This may compensate for the inherent weak affinities that govern ligand-induced receptor dimerization at the plasma membrane. Consistently, activated receptors are also concentrated at the CEs. Our observations thus suggest that receptor trafficking may play an important role for the regulation of IL-4R-mediated JAK/STAT signaling.

Cholesterol and sphingomyelin drive ligand-independent T-cell antigen receptor nanoclustering.

The T-cell antigen receptor (TCR) exists in monomeric and nanoclustered forms independently of antigen binding. Although the clustering is involved in the regulation of T-cell sensitivity, it is unknown how the TCR nanoclusters form. We show that cholesterol is required for TCR nanoclustering in T cells and that this clustering enhances the avidity but not the affinity of the TCR-antigen interaction. Investigating the mechanism of the nanoclustering, we found that radioactive photocholesterol specifically binds to the TCRß chain in vivo. In order to reduce the complexity of cellular membranes, we used a synthetic biology approach and reconstituted the TCR in liposomes of defined lipid composition. Both cholesterol and sphingomyelin were required for the formation of TCR dimers in phosphatidylcholine-containing large unilamellar vesicles. Further, the TCR was localized in the liquid disordered phase in giant unilamellar vesicles. We propose a model in which cholesterol and sphingomyelin binding to the TCRß chain causes TCR dimerization. The lipid-induced TCR nanoclustering enhances the avidity to antigen and thus might be involved in enhanced sensitivity of memory compared with naive T cells. Our work contributes to the understanding of the function of specific nonannular lipid-membrane protein interactions.

Application of Phosphoramidate ProTide Technology for the Synthesis of 5'-mRNA Cap Analogs Modified on the Exocyclic Amine Group.

Aryloxy triester phosphoramidate methodology, commonly known as ProTide technology, is one of the most widely used prodrug approaches applied to therapeutic nucleosides. This approach has been used extensively by the pharmaceutical industry and researchers in medicinal chemistry. Herein we report our adaptation of this effective method for the synthesis of bioactive 5'-mRNA cap analogues as inhibitors for targeting cap-dependent translation. The synthesis was performed in two main stages: preparation of N2-modified guanosine analogues and their subsequent transformation into prodrugs using phenylethoxy-l-alaninyl phosphorochloridate. The prepared pro-nucleotide cap analogues were tested for their capacity in enzymatic activation, inhibitory properties in a rabbit reticulocyte lysate system, and passive membrane translocation properties.

Astrocytic CD44 Deficiency Reduces the Severity of Kainate-Induced Epilepsy.

BACKGROUND: Epilepsy affects millions of people worldwide, yet we still lack a successful treatment for all epileptic patients. Most of the available drugs modulate neuronal activity. Astrocytes, the most abundant cells in the brain, may constitute alternative drug targets. A robust expansion of astrocytic cell bodies and processes occurs after seizures. Highly expressed in astrocytes, CD44 adhesion protein is upregulated during injury and is suggested to be one of the most important proteins associated with epilepsy. It connects the astrocytic cytoskeleton to hyaluronan in the extracellular matrix, influencing both structural and functional aspects of brain plasticity. METHODS: Herein, we used transgenic mice with an astrocyte CD44 knockout to evaluate the impact of the hippocampal CD44 absence on the development of epileptogenesis and ultrastructural changes at the tripartite synapse. RESULTS: We demonstrated that local, virally-induced CD44 deficiency in hippocampal astrocytes reduces reactive astrogliosis and decreases the progression of kainic acid-induced epileptogenesis. We also observed that CD44 deficiency resulted in structural changes evident in a higher dendritic spine number along with a lower percentage of astrocyte-synapse contacts, and decreased post-synaptic density size in the hippocampal molecular layer of the dentate gyrus. CONCLUSIONS: Overall, our study indicates that CD44 signaling may be important for astrocytic coverage of synapses in the hippocampus and that alterations of astrocytes translate to functional changes in the pathology of epilepsy.

Restorative effect of NitroSynapsin on synaptic plasticity in an animal model of depression.

In the search for new options for the pharmacological treatment of major depressive disorder, compounds with a rapid onset of action and high efficacy but lacking a psychotomimetic effect are of particular interest. In the present study, we evaluated the antidepressant potential of NitroSynapsin (NS) at behavioural, structural, and functional levels. NS is a memantine derivative and a dual allosteric N-methyl-d-aspartate receptors (NMDAR) antagonist using targeted delivery by the aminoadamantane of a warhead nitro group to inhibitory redox sites on the NMDAR. In a chronic restraint stress (CRS) mouse model of depression, five doses of NS administered on three consecutive days evoked antidepressant-like activity in the chronically stressed male C57BL/6J mice, reversing CRS-induced behavioural disturbances in sucrose preference and tail suspension tests. CRS-induced changes in morphology and density of dendritic spines in cerebrocortical neurons in the medial prefrontal cortex (mPFC) were also reversed by NS. Moreover, CRS-induced reduction in long-term potentiation (LTP) in the mPFC was found to be prevented by NS based on the electrophysiological recordings. Our study showed that NS restores structural and functional synaptic plasticity and reduces depressive behaviour to the level found in naïve animals. These results preliminarily revealed an antidepressant-like potency of NS.

Unique properties of Coronaviridae single-pass transmembrane domain regions as an adaptation to diverse membrane systems.

Enveloped viruses such as Coronaviridae (CoV) enter the host cell by fusing the viral envelope directly with the plasma membrane (PM) or with the membrane of the endosome. Replication of the CoV genome takes place in membrane compartments formed by rearrangement of the endoplasmic reticulum (ER) membrane network. Budding of these viruses occurs from the ER-Golgi intermediate compartment (ERGIC). The relationship between proteins and various membranes is crucial for the replication cycle of CoVs. The role of transmembrane domains (TMDs) and pre-transmembrane domains (pre-TMD) of viral proteins in this process is gaining more recognition. Here we present a thorough analysis of physico-chemical parameters, such as accessible surface area (ASA), average hydrophobicity (Hav), and contribution of specific amino acids in TMDs and pre-TMDs of single-span membrane proteins of human viruses. We focus on unique properties of these elements in CoV and postulate their role in adaptation to diverse host membranes and regulation of retention of membrane proteins during replication.

Single cell analysis of ligand binding and complex formation of interleukin-4 receptor subunits.

Interleukin-4 (IL-4) is an important class I cytokine involved in adaptive immunity. IL-4 binds with high affinity to the single-pass transmembrane receptor IL-4Rα. Subsequently, IL-4Rα/IL-4 is believed to engage a second receptor chain, either IL-2Rγ or IL-13Rα1, to form type I or II receptor complexes, respectively. This ternary complex formation then triggers downstream signaling via intracellular Janus kinases bound to the cytoplasmic receptor tails. Here, we study the successive steps of complex formation at the single cell level with confocal fluorescence imaging and correlation spectroscopy. We characterize binding and signaling of fluorescently labeled IL-4 by flow cytometry of IL-4-dependent BaF3 cells. The affinity to ectopically expressed IL-4Rα was then measured by single-color fluorescence correlation spectroscopy in adherent HEK293T cells that express the components of the type II IL-4R but not type I. Finally, IL-4-induced complex formation was tested by dual-color fluorescence cross-correlation spectroscopy. The data provide evidence for codiffusion of IL-4-A647 bound IL-4Rα and the type II subunit IL-13Rα1 fused to enhanced green fluorescent protein, whereas type I complexes containing IL-2Rγ and JAK3 were not detected at the cell surface. This behavior may reflect hitherto undefined differences in the mode of receptor activation between type I (lymphoid) and type II (epithelial) receptor expressing cells.

Competition between Photoinduced Electron Transfer and Resonance Energy Transfer in an Example of Substituted Cytochrome c-Quantum Dot Systems.

Colloidal quantum dots (QDs) are nanoparticles that are able to photoreduce redox proteins by electron transfer (ET). QDs are also able to transfer energy by resonance energy transfer (RET). Here, we address the question of the competition between these two routes of QDs' excitation quenching, using cadmium telluride QDs and cytochrome c (CytC) or its metal-substituted derivatives. We used both oxidized and reduced versions of native CytC, as well as fluorescent, nonreducible Zn(II)CytC, Sn(II)CytC, and metal-free porphyrin CytC. We found that all of the CytC versions quench QD fluorescence, although the interaction may be described differently in terms of static and dynamic quenching. QDs may be quenchers of fluorescent CytC derivatives, with significant differences in effectiveness depending on QD size. SnCytC and porphyrin CytC increased the rate of Fe(III)CytC photoreduction, and Fe(II)CytC slightly decreased the rate and ZnCytC presence significantly decreased the rate and final level of reduced FeCytC. These might be partially explained by the tendency to form a stable complex between protein and QDs, which promoted RET and collisional quenching. Our findings show that there is a net preference for photoinduced ET over other ways of energy transfer, at least partially, due to a lack of donors, regenerating a hole at QDs and leading to irreversibility of ET events. There may also be a common part of pathways leading to photoinduced ET and RET. The nature of synergistic action observed in some cases allows the hypothesis that RET may be an additional way to power up the ET.

Focus on composition and interaction potential of single-pass transmembrane domains.

Transmembrane domains (TMD) connect the inner with the outer world of a living cell. Single TMD containing (bitopic) receptors are of particular interest, because their oligomerization seems to be a common activation mechanism in cell signaling. We analyzed the composition of TMDs in bitopic proteins within the proteomes of 12 model organisms. The average number of strongly polar and charged residues decreases during evolution, while the occurrence of a dimerization motif, GxxxG, remains unchanged. This may reflect the avoidance of unspecific binding within a growing receptor interaction network. In addition, we propose a new experimental approach for studying helix-helix interactions in giant plasma membrane vesicles using scanning fluorescence cross-correlation spectroscopy. Measuring eGFP/mRFP tagged versions of cytokine receptors confirms the homotypic interactions of the erythropoietin receptor in contrast to the Interleukin-4 receptor chains. As a proof of principle, by swapping the TMDs, the interaction potential of erythropoietin receptor was partially transferred to Interleukin-4 receptor α and vice versa. Non-interacting receptors can therefore serve as host molecules for TMDs whose oligomerization capability must be assessed. Computational analysis of the free energy gain resulting from TMD dimer formation strongly corroborates the experimental findings, potentially allowing in silico pre-screening of interacting pairs.

Influenza A H1 and H3 Transmembrane Domains Interact Differently with Each Other and with Surrounding Membrane Lipids.

Hemagglutinin (HA) is a class I viral membrane fusion protein, which is the most abundant transmembrane protein on the surface of influenza A virus (IAV) particles. HA plays a crucial role in the recognition of the host cell, fusion of the viral envelope and the host cell membrane, and is the major antigen in the immune response during the infection. Mature HA organizes in homotrimers consisting of a sequentially highly variable globular head and a relatively conserved stalk region. Every HA monomer comprises a hydrophilic ectodomain, a pre-transmembrane domain (pre-TMD), a hydrophobic transmembrane domain (TMD), and a cytoplasmic tail (CT). In recent years the effect of the pre-TMD and TMD on the structure and function of HA has drawn some attention. Using bioinformatic tools we analyzed all available full-length amino acid sequences of HA from 16 subtypes across various host species. We calculated several physico-chemical parameters of HA pre-TMDs and TMDs including accessible surface area (ASA), average hydrophobicity (Hav), and the hydrophobic moment (µH). Our data suggests that distinct differences in these parameters between the two major phylogenetic groups, represented by H1 and H3 subtypes, could have profound effects on protein-lipid interactions, trimer formation, and the overall HA ectodomain orientation and antigen exposure.

Heterodimerizing helices as tools for nanoscale control of the organization of protein-protein and protein-quantum dots.

In this study, we tested the possibility of creating complexes of two proteins by fusing them with heterodimerizing helices. We used the fluorescent proteins GFP and mCHERRY expressed with a His-tag as our model system. We added heterodimer-forming sequences at the C- or N- termini of the proteins, opposite to the His-tag position. Heterodimerization was tested for both helices at the C-terminus or at the N- terminus and C-terminus. We observed complex formation with a nanomolar dissociation constant in both cases that was higher by one order of magnitude than the Kds measured for helices alone. The binding of two C-terminal helices was accompanied by an increased enthalpy change. The binding between helices could be stabilized by introducing an additional turn of the helix with cysteine, which was capable of forming disulphide bridges. Covalently linked proteins were obtained using this strategy and observed using fluorescence cross-correlation spectroscopy. Finally, we demonstrated the formation of complexes of protein dimers and quantum dots.

Comparison of α-Helix and ß-Sheet Structure Adaptation to a Quantum Dot Geometry: Toward the Identification of an Optimal Motif for a Protein Nanoparticle Cover.

While quantum dots (QDs) are useful as fluorescent labels, their application in biosciences is limited due to the stability and hydrophobicity of their surface. In this study, we tested two types of proteins for use as a cover for spherical QDs, composed of cadmium selenide. Pumilio homology domain (Puf), which is mostly α-helical, and leucine-rich repeat (LRR) domain, which is rich in ß-sheets, were selected to determine if there is a preference for one of these secondary structure types for nanoparticle covers. The protein sequences were optimized to improve their interaction with the surface of QDs. The solubilization of the apoproteins and their assembly with nanoparticles required the application of a detergent, which was removed in subsequent steps. Finally, only the Puf-based cover was successful enough as a QD hydrophilic cover. We showed that a single polypeptide dimer of Puf, PufPuf, can form a cover. We characterized the size and fluorescent properties of the obtained QD:protein assemblies. We showed that the secondary structure of the Puf proteins was not destroyed upon contact with the QDs. We demonstrated that these assemblies do not promote the formation of reactive oxygen species during illumination of the nanoparticles. The data represent advances in the effort to obtain a stable biocompatible cover for QDs.