Structural Determinants for Protein adsorption/non-adsorption to Silica Surface

The understanding of the mechanisms involved in the interaction of proteins with inorganic surfaces is of major interest in both fundamental research and applications such as nanotechnology. However, despite intense research, the mechanisms and the structural determinants of protein/surface interactions are still unclear. We developed a strategy consisting in identifying, in a mixture of hundreds of soluble proteins, those proteins that are adsorbed on the surface and those that are not. If the two protein subsets are large enough, their statistical comparative analysis must reveal the physicochemical determinants relevant for adsorption versus non-adsorption. This methodology was tested with silica nanoparticles. We found that the adsorbed proteins contain a higher number of charged amino acids, particularly arginine, which is consistent with involvement of this basic amino acid in electrostatic interactions with silica. The analysis also identified a marked bias toward low aromatic amino acid content (phenylalanine, tryptophan, tyrosine and histidine) in adsorbed proteins. Structural analyses and molecular dynamics simulations of proteins from the two groups indicate that non-adsorbed proteins have twice as many π-π interactions and higher structural rigidity. The data are consistent with the notion that adsorption is correlated with the flexibility of the protein and with its ability to spread on the surface. Our findings led us to propose a refined model of protein adsorption.     

Discovery of similar regions on protein surfaces.

Discovery of a similar region on two protein surfaces can lead to important inference about the functional role or molecular interaction of this region for one of the proteins if such information is available for the other. We propose a new characterization of protein surfaces based on a spin-image representation of the surfaces that facilitates the simultaneous search of the entire surface of each of two proteins for a matching region. For a surface point, we introduce spin-image profiles that are related to the degree of exposure of the point to identify structurally equivalent surface regions in two proteins. Unlike some related methods, we do not assume that a known fixed region of one of the protein surfaces is to be matched on the other protein surface. Rather, we search for the largest similar regions on each of the two surfaces. In spite of the fact that this approach is entirely geometric and no use is made of physicochemical properties of the protein surfaces or fold information, it is effective in identifying similar regions on both surfaces even when the region corresponds to a binding site on one of the proteins. The discovery of similar regions on two or more proteins also has implications for drug design and pharmacophore identification. We present experimental results from datasets of more than 50 protein surfaces.     

UNDERSTANDING INTERCELLULAR INTERACTIONS AND CELL ADHESION: LESSONS FROM STUDIES ON PROTEIN—METAL INTERACTIONS

To understand cell—cell interactions and the interactions of cells to non-biological materials, studies on binding forces between cellular proteins and between proteins and non-biological material such as metal surfaces are essential. The adsorption of proteins to solid—water interfaces is a multifactorial and a multistep process. First steps are determined by long-range interactions where surface properties such as hydrophobicity, distribution of charged groups, ion concentrations and pH play important roles. In later steps structural rearrangements in the protein molecule and dehydration effects become more important making the adsorption process often irreversible. In the following we demonstrate that protein A and tubulin have a specific type of interaction to metal surfaces probably as an intermediate step in the adsorption process. The proteins were attached to the tip of a microfabricated cantilever in such a way that only one molecule interacts with the surface. By recording force—distance curves with an atomic force microscope the adhesion forces of single molecules binding to gold, titanium and indium—tinoxid surfaces were measured.     

The Selective Interaction between Silica Nanoparticles and Enzymes from Molecular Dynamics Simulations

Nanoscale particles have become promising materials in many fields, such as cancer therapeutics, diagnosis, imaging, drug delivery, catalysis, as well as biosensors. In order to stimulate and facilitate these applications, there is an urgent need for the understanding of the interaction mode between the nano-particles and proteins. In this study, we investigate the orientation and adsorption between several enzymes (cytochrome c, RNase A, lysozyme) and 4 nm/11 nm silica nanoparticles (SNPs) by using molecular dynamics (MD) simulation. Our results show that three enzymes are adsorbed onto the surfaces of both 4 nm and 11 nm SNPs during our MD simulations and the small SNPs induce greater structural stabilization. The active site of cytochrome c is far away from the surface of 4 nm SNPs, while it is adsorbed onto the surface of 11 nm SNPs. We also explore the influences of different groups (-OH, -COOH, -NH₂ and CH₃) coated onto silica nanoparticles, which show significantly different impacts. Our molecular dynamics results indicate the selective interaction between silicon nanoparticles and enzymes, which is consistent with experimental results. Our study provides useful guides for designing/modifying nanomaterials to interact with proteins for their bio-applications.     

Protein interactions with surfaces: Computational approaches and repellency

Study of protein adsorption to solid surfaces continues to be substantial because of its role in cellular responses to biomaterials, interest in molecular aspects such as conformation and orientation, new methods for making protein repellent surfaces, and new application areas such as nanoparticles and microfluidics. This brief review is based only on very recent articles of particular interest to the authors, who each have worked in this area for some time. Simulations of protein interactions with surfaces and protein repellent surfaces are the only subtopics reviewed here.     

RosettaSurf—A surface-centric computational design approach

Proteins are typically represented by discrete atomic coordinates providing an accessible framework to describe different conformations. However, in some fields proteins are more accurately represented as near-continuous surfaces, as these are imprinted with geometric (shape) and chemical (electrostatics) features of the underlying protein structure. Protein surfaces are dependent on their chemical composition and, ultimately determine protein function, acting as the interface that engages in interactions with other molecules. In the past, such representations were utilized to compare protein structures on global and local scales and have shed light on functional properties of proteins. Here we describe RosettaSurf, a surface-centric computational design protocol, that focuses on the molecular surface shape and electrostatic properties as means for protein engineering, offering a unique approach for the design of proteins and their functions. The RosettaSurf protocol combines the explicit optimization of molecular surface features with a global scoring function during the sequence design process, diverging from the typical design approaches that rely solely on an energy scoring function. With this computational approach, we attempt to address a fundamental problem in protein design related to the design of functional sites in proteins, even when structurally similar templates are absent in the characterized structural repertoire. Surface-centric design exploits the premise that molecular surfaces are, to a certain extent, independent of the underlying sequence and backbone configuration, meaning that different sequences in different proteins may present similar surfaces. We benchmarked RosettaSurf on various sequence recovery datasets and showcased its design capabilities by generating epitope mimics that were biochemically validated. Overall, our results indicate that the explicit optimization of surface features may lead to new routes for the design of functional proteins.     

Denaturing and refolding of protein molecules on surfaces

Keeping protein molecules in the active state on a solid surface is essential to protein microarrays and other protein-based biosensors. Here, we show that the 2-D chemical environment controls the refolding of the denatured green fluorescent proteins tethered to solid surfaces. Refolding occurs readily on the repulsive PEG functionalized surface but is inhibited on the attractive--NH(2) functionalized surface. This result shows the critical importance of the 2-D chemical environment in the maintenance and revival of protein activity on surfaces and opens the door to designing 2-D molecular chaperones for protein folding.     

Protein nanoarray on Prolinker surface constructed by atomic force microscopy dip-pen nanolithography for analysis of protein interaction

Protein nanoarrays are addressable ensembles of nano-scale protein domain on solid surfaces. This method can serve as a useful platform for ultraminiaturized bioanalysis. In this study, we investigated single molecular nanopatterning and molecular interaction of proteins that were immobilized on Prolinker surface of gold-coated silicon wafer by using dip-pen nanolithography (DPN) method. Contact force and humidity were optimized at 0.01 nN and 80%, respectively. The domain features of protein nanoarrays were developed at the contact time of 5 s. The optimized conditions for the nanoarray process were applied to create protein nanoarray using integrin alpha(v)beta3 and angiogenin. Constructed protein nanoarrays using integrin alpha(v)beta3 have single molecular monolayer with regular domain shape (height 15 +/- 5 nm). The changed height value due to the single molecular interaction between integrin alpha(v)beta3 and vitronectin was approximately 30 +/- 5 nm on Prolinker surface as measured with atomic force microscopy tip. Taken together, these results suggest that protein nanoarray on Prolinker surface fabricated by well-controlled DPN process can be used to analyze single molecular interaction of protein.     

A novel method for protein-protein interaction site prediction using phylogenetic substitution models

Protein-protein binding events mediate many critical biological functions in the cell. Typically, functionally important sites in proteins can be well identified by considering sequence conservation. However, protein-protein interaction sites exhibit higher sequence variation than other functional regions, such as catalytic sites of enzymes. Consequently, the mutational behavior leading to weak sequence conservation poses significant challenges to the protein-protein interaction site prediction. Here, we present a phylogenetic framework to capture critical sequence variations that favor the selection of residues essential for protein-protein binding. Through the comprehensive analysis of diverse protein families, we show that protein binding interfaces exhibit distinct amino acid substitution as compared with other surface residues. On the basis of this analysis, we have developed a novel method, BindML, which utilizes the substitution models to predict protein-protein binding sites of protein with unknown interacting partners. BindML estimates the likelihood that a phylogenetic tree of a local surface region in a query protein structure follows the substitution patterns of protein binding interface and nonbinding surfaces. BindML is shown to perform well compared to alternative methods for protein binding interface prediction. The methodology developed in this study is very versatile in the sense that it can be generally applied for predicting other types of functional sites, such as DNA, RNA, and membrane binding sites in proteins.     

Molecular and cellular approaches for the detection of protein-protein interactions: latest techniques and current limitations

Homotypic and heterotypic protein interactions are crucial for all levels of cellular function, including architecture, regulation, metabolism, and signaling. Therefore, protein interaction maps represent essential components of post-genomic toolkits needed for understanding biological processes at a systems level. Over the past decade, a wide variety of methods have been developed to detect, analyze, and quantify protein interactions, including surface plasmon resonance spectroscopy, NMR, yeast two-hybrid screens, peptide tagging combined with mass spectrometry and fluorescence-based technologies. Fluorescence techniques range from co-localization of tags, which may be limited by the optical resolution of the microscope, to fluorescence resonance energy transfer-based methods that have molecular resolution and can also report on the dynamics and localization of the interactions within a cell. Proteins interact via highly evolved complementary surfaces with affinities that can vary over many orders of magnitude. Some of the techniques described in this review, such as surface plasmon resonance, provide detailed information on physical properties of these interactions, while others, such as two-hybrid techniques and mass spectrometry, are amenable to high-throughput analysis using robotics. In addition to providing an overview of these methods, this review emphasizes techniques that can be applied to determine interactions involving membrane proteins, including the split ubiquitin system and fluorescence-based technologies for characterizing hits obtained with high-throughput approaches. Mass spectrometry-based methods are covered by a review by Miernyk and Thelen (2008; this issue, pp. 597–609 ). In addition, we discuss the use of interaction data to construct interaction networks and as the basis for the exciting possibility of using to predict interaction surfaces.     

Restricted mobility of side chains on concave surfaces of solenoid proteins may impart heightened potential for intermolecular interactions

Significant progress has been made in the determination of the protein structures with their number today passing over a hundred thousand structures. The next challenge is the understanding and prediction of protein–protein and protein–ligand interactions. In this work we address this problem by analyzing curved solenoid proteins. Many of these proteins are considered as “hub molecules” for their high potential to interact with many different molecules and to be a scaffold for multisubunit protein machineries. Our analysis of these structures through molecular dynamics simulations reveals that the mobility of the side‐chains on the concave surfaces of the solenoids is lower than on the convex ones. This result provides an explanation to the observed preferential binding of the ligands, including small and flexible ligands, to the concave surface of the curved solenoid proteins. The relationship between the landscapes and dynamic properties of the protein surfaces can be further generalized to the other types of protein structures and eventually used in the computer algorithms, allowing prediction of protein–ligand interactions by analysis of protein surfaces . Proteins 2015; 83:1654–1664. © 2015 Wiley Periodicals, Inc.     

Transferrin coated nanoparticles: study of the bionano interface in human plasma

It is now well established that the surface of nanoparticles (NPs) in a biological environment is immediately modified by the adsorption of biomolecules with the formation of a protein corona and it is also accepted that the protein corona, rather than the original nanoparticle surface, defines a new biological identity. Consequently, a methodology to effectively study the interaction between nanomaterials and the biological corona encountered within an organism is a key objective in nanoscience for understanding the impact of the nanoparticle-protein interactions on the biological response in vitro and in vivo. Here, we outline an integrated methodology to address the different aspects governing the formation and the function of the protein corona of polystyrene nanoparticles coated with Transferrin by different strategies. Protein-NP complexes are studied both in situ (in human plasma, full corona FC) and after washing (hard corona, HC) in terms of structural properties, composition and second-order interactions with protein microarrays. Human protein microarrays are used to effectively study NP-corona/proteins interactions addressing the growing demand to advance investigations of the extrinsic function of corona complexes. Our data highlight the importance of this methodology as an analysis to be used in advance of the application of engineered NPs in biological environments.     

Photonic immobilization of BSA for nanobiomedical applications: creation of high density microarrays and superparamagnetic bioconjugates

Light assisted molecular immobilization has been used for the first time to engineer covalent bioconjugates of superparamagnetic nanoparticles and proteins. The technology involves disulfide bridge disruption upon UV excitation of nearby aromatic residues. The close spatial proximity of aromatic residues and disulfide bridges is a conserved structural feature in proteins. The created thiol groups bind thiol reactive surfaces leading to oriented covalent protein immobilization. We have immobilized a model carrier protein, bovine serum albumin, onto Fe(3)O(4)@Au core-shell nanoparticles as well as arrayed it onto optically flat thiol reactive surfaces. This new immobilization technology allows for ultra high dense packing of different bio-molecules on a surface, allowing the creation of multi-potent functionalized active new biosensor materials, biomarkers identification and the development of nanoparticles based novel drug delivery system.     

In silico Structure Modeling and Comparative Analysis of Characterization Properties of Protein Polymers Useful for Protein-Based Nano Particulate Drug Delivery Systems (NPDDS): A Bioinformatics Approach

With an increasing interest in nanoparticulate delivery systems, there is a greater need to identify biomaterials that are biocompatible and safe for human applications. Protein polymers from animal and plant sources are promising materials for designing nanocarriers. Composition of the protein plays an important role for specific drug delivery applications such as drug release, targeting, and stimuli responsive drug release. An important issue in protein polymers is characteristics such as size, charge, and hydrophobicity may play a significant role in phagocytic uptake and initiating a subsequent immune response. This remains to be investigated systematically by analyzing factors that influence nanoparticle characteristics of protein and reduce phagocytic uptake and does not initiate immune response too. Although protein polymers are biodegradable, it is essential to ensure that there must not be premature enzymatic breakdown of the protein nanoparticles in the systemic circulation. Surface modification of the protein nanoparticles can be used to address this issue to propose the necessary modification in the surface of the protein would be great contribution in the nano particulate drug delivery systems (NPPDS). Of the various proteins, gelatin and albumin have been widely studied for drug delivery applications. Plant proteins are yet to be investigated widely for drug delivery applications so there is need to find out the plant proteins capable to act as nanoparticles. The commercial success of albumin-based nanoparticles has created an interest in other proteins. An increased understanding of the physicochemical properties coupled with the developments in rDNA technology will open up new opportunities for protein-based nanoparticulate systems. In the present studies several proteins currently useful for drug delivery system were structurally modeled and has been analyzed to propose the essential characteristics of protein for protein-based NPDDS.     

A Hydrophobic Gold Surface Triggers Misfolding and Aggregation of the Amyloidogenic Josephin Domain in Monomeric Form,While Leaving the Oligomers Unaffected

Protein misfolding and aggregation in intracellular and extracellular spaces is regarded as a main marker of the presence of degenerative disorders such as amyloidoses. To elucidate the mechanisms of protein misfolding, the interaction of proteins with inorganic surfaces is of particular relevance, since surfaces displaying different wettability properties may represent model systems of the cell membrane. Here, we unveil the role of surface hydrophobicity/hydrophilicity in the misfolding of the Josephin domain (JD), a globular-shaped domain of ataxin-3, the protein responsible for the spinocerebellar ataxia type 3. By means of a combined experimental and theoretical approach based on atomic force microscopy, Fourier transform infrared spectroscopy and molecular dynamics simulations, we reveal changes in JD morphology and secondary structure elicited by the interaction with the hydrophobic gold substrate, but not by the hydrophilic mica. Our results demonstrate that the interaction with the gold surface triggers misfolding of the JD when it is in native-like configuration, while no structural modification is observed after the protein has undergone oligomerization. This raises the possibility that biological membranes would be unable to affect amyloid oligomeric structures and toxicity.     

The interaction of proteins with solid surfaces

The interaction of proteins with solid surfaces is a fundamental phenomenon with implications for nanotechnology, biomaterials and biotechnological processes. Kinetic and thermodynamic studies have long indicated that significant conformational changes may occur as a protein encounters a surface; new techniques are measuring and modeling these changes. Combinatorial and directed evolution techniques have created new peptide sequences that bind specifically to solid surfaces, similar to the natural proteins that regulate crystal growth. Modeling efforts capture kinetics and thermodynamics on the colloidal scale, but detailed treatments of atomic structure are still in development and face the usual challenges of protein modeling. Opportunities abound for fundamental discovery, as well as breakthroughs in biomaterials, biotechnology and nanotechnology.     

Importance of Metal–Adsorbate Interactions for the Surface-enhanced Raman Scattering of Molecules Adsorbed on Plasmonic Nanoparticles

The interaction between adsorbates of different nature and plasmonic nanoparticles is reviewed here on the basis of the work done in our laboratory in the past few years. The paper is structured for analyzing the interaction of adsorbates with metal nanoparticles as function of the interacting atom (O, N, or S) and the adsorbate conformation. In the study of the adsorption of molecular species on metals, it is necessary to take into account that different interaction mechanisms are possible, leading to the existence of different molecular forms (isomers or conformers). These forms can be evidenced by changing the excitation wavelength, due to a resonant selection of these wavelengths. Charge-transfer complexes and electrostatic interactions are the usual driving forces involved in the interaction of adsorbates on metal surfaces when these metallic systems are used in wet conditions. The understanding of the metal–adsorbate interaction is crucial in the surface functionalization of metal surfaces, which has a growing importance in the development of sensing systems or optoelectronic devices. In relation to this, special attention is paid in this work to the study of the adsorption of calixarene host molecules on plasmonic nanoparticles.     

Toward the understanding of MNEI sweetness from hydration map surfaces

The binding mechanism of sweet proteins to their receptor, a G-protein-coupled receptor, is not supported by direct structural information. In principle, the key groups responsible for biological activity (glucophores) can be localized on a small structural unit (sweet finger) or spread on a larger surface area. A recently proposed model, called "wedge model", implies a large surface of interaction with the receptor. To explore this model in greater detail, it is necessary to examine the physicochemical features of the surfaces of sweet proteins, since their interaction with the receptor, with respect to that of small sweeteners, is more dependent on general physicochemical properties of the interface, such as electrostatic potential and hydration. In this study, we performed exhaustive molecular dynamics simulations in explicit water of the sweet protein MNEI and of its structural mutant G-16A, whose sweetness is one order of magnitude lower than that of MNEI. Solvent density and self-diffusion calculated from molecular dynamics simulations suggest a likely area of interaction delimited by four stretches arranged as a tetrahedron whose shape is complementary to that of a cavity on the surface of the receptor, in agreement with the wedge model. The suggested area of interaction is amazingly consistent with known mutagenesis data. In addition, the asymmetric hydration of the only helix in both proteins hints at a specific role for this secondary structure element in orienting the protein during the binding process.     

Role of Nonspecific Interactions in Molecular Chaperones through Model-Based Bioinformatics

Molecular chaperones are large proteins or protein complexes from which many proteins require assistance in order to fold. One unique property of molecular chaperones is the cavity they provide in which proteins fold. The interior surface residues which make up the cavities of molecular chaperone complexes from different organisms has recently been identified, including the well-studied GroEL-GroES chaperonin complex found in Escherichia coli. It was found that the interior of these protein complexes is significantly different than other protein surfaces and that the residues found on the protein surface are able to resist protein adsorption when immobilized on a surface. Yet it remains unknown if these residues passively resist protein binding inside GroEL-GroEs (as demonstrated by experiments that created synthetic mimics of the interior cavity) or if the interior also actively stabilizes protein folding. To answer this question, we have extended entropic models of substrate protein folding inside GroEL-GroES to include interaction energies between substrate proteins and the GroEL-GroES chaperone complex. This model was tested on a set of 528 proteins and the results qualitatively match experimental observations. The interior residues were found to strongly discourage the exposure of any hydrophobic residues, providing an enhanced hydrophobic effect inside the cavity that actively influences protein folding. This work provides both a mechanism for active protein stabilization in GroEL-GroES and a model that matches contemporary understanding of the chaperone protein.