Influence of the aggregation state in solution on the supramolecular organization of adsorbed type I collagen layers

In the last years, adsorbed collagen was shown to form layers with a supramolecular organization depending on the substrate surface properties and on the preparation procedure. If the concentration of collagen and the duration of adsorption are sufficient, fibrillar collagen structures are formed, corresponding to assemblies of a few molecules. This occurs more readily on hydrophobic compared to hydrophilic surfaces. This study aims at understanding the origin of such fibrillar structures and in particular at determining whether they result from the deposition of fibrils formed in solution or from the building of assemblies at the interface. Therefore, type I collagen solutions with an increasing degree of aggregation were prepared, using the “neutral-start” approach, by ageing pH 5.8 solutions at 37 °C for 15 min, 2 or 7 days. The obtained solutions were used to investigate the influence of collagen aggregation in solution on the supramolecular organization of adsorbed collagen layers, which was characterized by X-ray photoelectron spectroscopy and atomic force microscopy. Polystyrene and plasma-oxidized polystyrene were chosen as substrates for the adsorption. The size and the density of collagen fibrils at the interface decreased upon increasing the degree of aggregation of collagen in solution. This is explained by a competitive adsorption process between monomers and aggregates of the solution, turning at the advantage of the monomers. More aggregated solutions, which are thus depleted in free monomers, behave like less concentrated solutions, i.e. lead to a lower adsorbed amount and less fibril formation at the interface. This study shows that the supramolecular fibrils observed in adsorbed collagen layers, especially on hydrophobic substrates, are not formed in the solution, prior to adsorption, but are built at the interface, through the assembly of free segments of adsorbed molecules.     

Formation of a protein corona around nanoparticles

In biofluids, nanoparticles rapidly become surrounded by a protein corona. This phenomenon started to attract attention 10 years ago. Since then, this subfield of colloid and interface science was among the most rapidly expanding and progressing. Owing to its strong relation to biology and applications, this area is rich in various questions to explore. The reviews of the corresponding experiments are already numerous. Herein, I focus on the related theory including conventional mean-field kinetic models, dynamic density functional theory, Monte Carlo simulations, and molecular dynamics simulations. The key concept here is that the formation of a protein corona depends on the interplay of competition of different proteins for the location near the nanoparticle–solution interface (Vroman effect) and denaturation at this interface. Although this concept is not new, many details of this interplay are still open for debate.     

Monitoring folding transitions of synthetic,branched-chain polypeptides by capillary zone electrophoresis

The coil/helix transition of a synthetic, branched-chain polymeric polypeptide (poly (Lys(Glu(1)-DL-Ala(3))EAK), 50-Lys residues long in the backbone, as a function of increasing molarities of methanol in solution, is here studied by both, circular dichroism (CD) and capillary zone electrophoresis. CD spectra showed that, at 75% v/v methanol, the transition from random coil to fully helical structure was obtained, in a pH 1.1 HCI solution in the presence of 20 mM NaCI. CZE studies, run in parallel, exhibited the classical unfolding to folding sigmoidal transition, with mid-point at 60% v/v methanol concentration, plateauing at ca. 80% v/v organic solvent. Surprisingly, though, such unfolding to folding transition was accompanied by an expansion, rather than a contraction, of the resulting ordered polypeptide. As the charge of the polypeptide (a pure polycation at a pH of 2.1 in CZE) was kept rigorously constant, a plot of the radius of the polymer along the sigmoidal transition clearly showed that the radius of gyration of the helical, structured polypeptide was in fact larger than that of the random coil. Such results were confirmed by molecular dynamics simulations, which indicated that the dimensions of such polypeptide, in alpha-helix configuration, were 8.5 nm (in length) and 3.2 nm (in diameter), whereas those of the corresponding random coil were 7.2 nm (in length) and 5.1 nm (length of shorter axis). It would thus appear that the randomized structure assumes the shape of a more compact object, roughly resembling a "rugby ball".     

Multiscale modeling and simulations of protein adsorption: progresses and perspectives

Protein adsorption, which shows wide prospects in many practical applications such as biosensors, biofuel cells, and biomaterials, has long been identified as a very complex problem in interface science. Here, we present a review on the multiscale modeling and simulation methods of protein adsorption on surfaces with different properties. First, various simulation algorithms (replica exchange, metadynamics, TIGER2A, and PSOVina) and protein models (colloidal, coarse-grained, and all-atom models) are introduced. Then, recent molecular simulation progresses about protein adsorption on different material surfaces (such as charged, hydrophobic, hydrophilic, and responsive surfaces) are retrospected. It has been demonstrated that the adsorption orientation of proteins on charged surfaces and hydrophobic surfaces can be controlled by the electrical dipole and the hydrophobic dipole of proteins, respectively. Superhydrophilic zwitterionic surfaces can resist protein adsorption because of the strong hydration. Under the stimuli of external conditions, the surface properties of materials can be modulated, and thus, the adsorption/desorption of proteins on responsive surfaces can be controlled. Finally, the future directions of molecular simulation study of protein adsorption are discussed.     

Changes in solvent exposure reveal the kinetics and equilibria of adsorbed protein unfolding in hydrophobic interaction chromatography

Hydrogen exchange has been a useful technique for studying the conformational state of proteins, both in bulk solution and at interfaces, for several decades. Here, we propose a physically based model of simultaneous protein adsorption, unfolding and hydrogen exchange in HIC. An accompanying experimental protocol, utilizing mass spectrometry to quantify deuterium labeling, enables the determination of both the equilibrium partitioning between conformational states and pseudo-first order rate constants for folding and unfolding of adsorbed protein. Unlike chromatographic techniques, which rely on the interpretation of bulk phase behavior, this methodology utilizes the measurement of a molecular property (solvent exposure) and provides insight into the nature of the unfolded conformation in the adsorbed phase. Three model proteins of varying conformational stability, α-chymotrypsinogen A, β-lactoglobulin B, and holo α-lactalbumin, are studied on Sepharose™ HIC resins possessing assorted ligand chemistries and densities. α-Chymotrypsinogen, conformationally the most stable protein in the set, exhibits no change in solvent exposure at all the conditions studied, even when isocratic pulse-response chromatography suggests nearly irreversible adsorption. Apparent unfolding energies of adsorbed β-lactoglobulin B and holo α-lactalbumin range from −4 to 3 kJ/mol and are dependent on resin properties and salt concentration. Characteristic pseudo-first order rate constants for surface-induced unfolding are 0.2–0.9 min⁻¹. While poor protein recovery in HIC is often associated with irreversible unfolding, this study documents that non-eluting behavior can occur when surface unfolding is reversible or does not occur at all. Further, this hydrogen exchange technique can be used to assess the conformation of adsorbed protein under conditions where the protein is non-eluting and chromatographic methods are not applicable.     

Design and characterization of an engineered gp41 protein from human immunodeficiency virus-1 as a tool for drug discovery

Two new proteins of approximately 70 amino acids in length, corresponding to an unnaturally-linked N- and C-helix of the ectodomain of the gp41 protein from the human immunodeficiency virus (HIV) type 1, were designed and characterized. A designed tripeptide links the C-terminus of the C-helix with the N-terminus of the N-helix in a circular permutation so that the C-helix precedes the N-helix in sequence. In addition to the artificial peptide linkage, the C-helix is truncated at its N-terminus to expose a region of the N-helix known as the “Trp-Trp-Ile” binding pocket. Sedimentation, crystallographic, and nuclear magnetic resonance studies confirmed that the protein had the desired trimeric structure with an unoccupied binding site. Spectroscopic and centrifugation studies demonstrated that the engineered protein had ligand binding characteristics similar to previously reported constructs. Unlike previous constructs which expose additional, shallow, non-conserved, and undesired binding pockets, only the single deep and conserved Trp-Trp-Ile pocket is exposed in the proteins of this study. This engineered version of gp41 protein will be potentially useful in research programs aimed at discovery of new drugs for therapy of HIV-infection in humans.     

157-nm Laser ablation of polymeric layers for fabrication of biomolecule microarrays

A new methodology for protein microarray fabrication is proposed based on the ablation of polymer film using laser at 157 nm (F₂). The polymer has been selected among others with the criterion of negligible protein adsorption. Improved results have been obtained by pretreatment of the polymer surface with an inert protein. The use of 157-nm laser radiation allowed very good depth control during the polymeric layer ablation process. In addition the importance of laser ablation at 157 nm is based on the fact that irradiated surfaces indicate limited chemical change due to the fact that laser ablation at 157 nm is only photochemical, thus avoiding excessive surface heating and damage. Results of protein microarray fabrication are presented to illustrate the viability of the proposed method.     

Wavelets and molecular structure

Summary The wavelet method offers possibilities for display, editing, and topological comparison of proteins at a user-specified level of detail. Wavelets are a mathematical tool that first found application in signal processing. The multiresolution analysis of a signal via wavelets provides a hierarchical series of best lower-resolution approximations. B-spline ribbons model the protein fold, with one control point per residue. Wavelet analysis sets limits on the information required to define the winding of the backbone through space, suggesting a recognizable fold is generated from a number of points equal to 1/4 or less the number of residues. Wavelets applied to surfaces and volumes show promise in structure-based drug design.This paper is based on a presentation given at the 14th Molecular Graphics and Modelling Society Conference, held in Cairns, Australia, August 27–September 1, 1995.     

Effect of protein aggregates on foaming properties of β-lactoglobulin

Our paper aims at determining the respective part of protein aggregates and non-aggregated proteins in the foam formation and stability of β-lactoglobulin. We report results on fractal aggregates formed at neutral pH and strong ionic strength (aggregates size from 30 to 190 nm). Pure aggregates and mixtures of non-aggregated/aggregated proteins at varying ratios were used. The capacity of aggregates to form and stabilize foams has been studied in relation with their ability to absorb at air/water interfaces. Our results show that protein aggregates are not able by themselves to improve the foaming properties but participate to a better foam stabilization in the presence of non-aggregated proteins. Non-aggregated proteins appear to be necessary to produce stable foams. We have shown that the amount and the size of aggregates had an influence on the drainage rate.     

High-throughput isotherm determination and thermodynamic modeling of protein adsorption on mixed mode adsorbents

The thermodynamic modeling of protein adsorption on mixed-mode adsorbents functionalized with ligands carrying both hydrophobic and electrostatic groups was undertaken. The developed mixed mode isotherm was fitted with protein adsorption data obtained for five different proteins on four different mixed mode adsorbents by 96-well microtitre plate high throughput batch experiments on a robotic workstation. The developed mixed mode isotherm was capable of describing the adsorption isotherms of all five proteins (having widely different molecular masses and iso-electric points) on the four mixed mode adsorbents and over a wide range of salt concentrations and solution pH, and provided a unique set of physically meaningful parameters for each resin-protein-pH combination. The model could capture the typically observed minimum in mixed mode protein adsorption and predict the precise salt concentration at which this minimum occurs. The possibility of predicting the salt concentration at which minimum protein binding occurs presents new opportunities for designing better elution strategies in mixed mode protein chromatography. Salt-protein interactions were shown to have important consequences on mixed mode protein adsorption when they occur. Finally, the mixed mode isotherm also gave very good fit with literature data of BSA adsorption on a different mixed mode adsorbent not examined in this study. Hence, the mixed mode isotherm formalism presented in this study can be used with any mixed mode adsorbent having the hydrophobic and electrostatic functional groups. It also provides the basis for detailed modeling and optimization of mixed mode chromatographic separation of proteins.     

Protein Oligomer Engineering: A New Frontier for Studying Protein Structure,Function, and Toxicity

Prevalent in nature, protein oligomers play critical roles both physiologically and pathologically. The multimeric nature and conformational transiency of protein oligomers greatly complicate a more detailed glimpse into the molecular structure as well as function. In this minireview, the oligomers are classified and described on the basis of biological function, toxicity, and application. We also define the bottlenecks in recent oligomer studies and further review numerous frontier methods for engineering protein oligomers. Progress is being made on many fronts for a wide variety of applications, and protein grafting is highlighted as a promising and robust method for oligomer engineering. These advances collectively allow the engineering and design of stabilized oligomers that bring us one step closer to understanding their biological functions, toxicity, and a wide range of applications.     

FTIR investigation of the conformational properties of the cyanide bound human hemoglobin

Infrared spectra at 4 cm⁻¹ resolution of the cyanide ligated human methemoglobin (Hb-CN) were examined in the C---N stretching region. The FTIR spectra of hemoglobin ligated with the various isotopomeric forms of the cyanide ion support the existence of three conformational states for Hb-CN. In potassium phosphate buffer at pH of 7.5, the three bands were observed at 2116, 2122 and 2127 cm⁻¹ for natural abundance Hb-CN. These bands shift to 2086, 2091 and 2095 cm⁻¹ for Hb-¹²C¹⁵N and 2073, 2077 and 2081 cm⁻¹ for Hb-¹³C¹⁴N. Two extra bands have been identified in the IR spectra of solid Hb-CN in KBr pellets. The peaks persist in the pH range between 3.5 and 10.5 with small changes in frequency and intensity. The appearance of several C---N stretching bands is consistent with C---N vibrators residing in different environment and support the hypothesis that Hb-CN assumes multiple conformers under the conditions studied.     

Boltzmann's principle,knowledge-based mean fields and protein folding. An approach to the computational determination of protein structures

Summary The data base of known protein structures contains a tremendous amount of information on protein-solvent systems. Boltzmann's principle enables the extraction of this information in the form of potentials of mean force. The resulting force field constitutes an energetic model for protein-solvent systems. We outline the basic physical principles of this approach to protein folding and summarize several techniques which are useful in the development of knowledge-based force fields. Among the applications presented are the validation of experimentally determined protein structures, data base searches which aim at the identification of native-like sequence structure pairs, sequence structure alignments and the calculation of protein conformations from amino acid sequences.     

Discovery of a Potent Allosteric Kinase Modulator by Combining Computational and Synthetic Methods

The rational design of allosteric kinase modulators is challenging but rewarding. The protein kinase PDK1, which lies at the center of the growth‐factor signaling pathway, possesses an allosteric regulatory site previously validated both in vitro and in cells. ANCHOR.QUERY software was used to discover a potent allosteric PDK1 kinase modulator. Using a recently published PDK1 compound as a template, several new scaffolds that bind to the allosteric target site were generated and one example was validated. The inhibitor can be synthesized in one step by multicomponent reaction (MCR) chemistry when using the ANCHOR.QUERY approach. Our results are significant because the outlined approach allows rapid and efficient scaffold hopping from known molecules into new easily accessible and biologically active ones. Based on increasing interest in allosteric‐site drug discovery, we foresee many potential applications for this approach.     

Protein adsorption and transport in dextran-modified ion-exchange media. I: Adsorption

Adsorption behavior is compared on a traditional agarose-based ion-exchange resin and on two dextran-modified resins, using three proteins to examine the effect of protein size. The latter resins typically exhibit higher static capacities at low ionic strengths and electron microscopy provides direct visual evidence supporting the view that the higher static capacities are due to the larger available binding volume afforded by the dextran. However, isocratic retention experiments reveal that the larger proteins can be almost completely excluded from the dextran layer at high ionic strengths, potentially leading to significant losses in static capacity at relevant column loading conditions. Knowledge of resin and protein properties is used to estimate physical limits on the static capacities of the resins in order to provide a meaningful interpretation of the observed static capacities. Results of such estimates are consistent with the expectation that available surface area is limiting for traditional resins. In dextran-modified media, however, the volume of the dextran layer appears to limit adsorption when the protein charge is low relative to the resin charge, but the protein–resin electroneutrality may be limiting when the protein charge is relatively high. Such analyses may prove useful for semiquantitative prediction of maximum static capacities and selection of operating conditions when combined with protein transport information.     

反相高效液相色谱对DABTH—氨基酸的分离与鉴定

应用反相高效液相色谱(RP-HPLC)技术,采用等度洗脱。对20种标准DABTH-氨基酸进行了分离与鉴定,灵敏度达到pmol水平;并以溶菌酶为模式蛋白,经手工DABITC/PITC双偶联方法对其N端部分序列进行Edman降解,其降解产物DABTH-氨基酸用RP-HPLC技术进行了鉴定,蛋白质微量序列分析的灵敏度达到nmol水平.     

Site-selective protein glycation and PEGylation

A two step protocol has been set up to selectively conjugate PEG to buried amino acids of proteins. The process involves site-specific glycation followed by PEGylation of the oxidized glycosides. Aimed at glycating the cysteine groups of proteins, two maleimide-glycosylic linkers have been synthesised: galactosyl-glucono-CO–NH–(CH₂)₁₂–NH–CO–(CH₂)₂-maleimide and maltosyl-glucono-CO–NH–(CH₂)₁₂–NH–CO–(CH₂)₂-maleimide. The linkers were extensively characterized by ¹H NMR, FT-IR, ESI–TOF mass spectrometry and colorimetric assays. Complete conjugation of the activated linkers to Cys³⁴ of human serum albumin was obtained in about 2 h. The selective oxidation of the galactosyl and maltosyl moieties by periodate treatment yielded two and three available aldehyde groups, respectively. The PEG-hydrazide conjugation to the aldehyde groups was found to be 100% in about 40 h, whereas less than 30% protein modification was obtained by direct conjugation of commercial PEG-maleimide to Cys³⁴. The pH dependent PEG-glycosyl hydrazone bond hydrolysis at various pH values was verified. PEG release was faster under mild acidic and basic conditions than at neutral pH. Furthermore, the maltosyl derivatives, by virtue of the higher number of coupled PEG chains, showed a slower protein release as compared to the galactosyl counterpart, indicating that the choice of the glycosylic linker allows for control of protein release kinetics.     

Molecular Machines: How Motion and Other Functions of Living Organisms Can Result from Reversible Chemical Changes

Certain model proteins dramatically fold and become more ordered on raising the temperature. When the temperature is raised to drive folding and assembly, these model proteins can lift weights and perform work; they can produce motion. The temperature of warm-blooded animals, however, is kept constant. Therefore, motion cannot result from a change in temperature. In this case, a free energy change, caused, for example, by an increase in the concentration of a chemical, can lower the temperature at which the protein folding and assembly transition occurs from above to below physiological temperature. Raising the concentration of a chemical isothermally has indeed been shown to result in motion and the efficient performance of work. These model proteins and the mechanism they reveal provide insight into the molecular basis for diverse biological functions; they are models for the molecular machines that comprise the living organism, and they provide a new class of materials for both medical and nonmedical applications.