Surface Fractality of Proteins From Theory and NMR Data

Abstract

Different approaches to study protein surface fractality are considered. An approach based on analysis of surface versus molecular weight dependence is shown to be an informative tool for investigation of protein surface behaviour. An evidence for protein surface fractality, obtained with the use of this analysis from the data of both NMR measurements in protein solutions and computer analysis of protein structures, is presented. Obtained value of fractal dimension of protein surface (d _s ? 2.2) is in a good agreement with the results of conventional approach (with variation of yardstick length) to protein surface fractality. A conclusion is made that surface enlargement due to the rise of protein molecular weight is accompanied by the increase of maximum scale of irregularities on protein surface. Possible effect of surface fractality on hydrodynamic characteristics of protein molecules in solution is discussed.     

Surface fractality as a guide for studying protein-protein interactions

The concept of fractal dimension is applied to protein surfaces. Satellite tobacco necrosis virus, prealhumin, retinol binding protein and lysozyme have been studied. A residue fractal index has been defined, which provides a suitable colour code when using computer graphics for visualizing surfaces. Some provisions are made that render the MS algorithm useful to calculate protein surface fractal dimensions. It has been found that a correlation exists between regions of high fractal dimension and those involved in protein-protein interactions. The usefulness of surface fractality in this context is demonstrated by a molecular docking experiment.     

Many length scales surface fractality in monomolecular films of whole myelin lipids and proteins

Monomolecular films prepared with all the lipid and protein components of myelin were spread at the air/aqueous buffer interface from isolated bovine spinal cord myelin fully dissolved in chloroform:methanol (2:1) or by surface free energy shock of myelin membrane microvesicles. These monolayers show indistinguishable surface behavior, with similar compositional phase coexistence through all the compression isotherm on several subphase conditions. The domains were observed through epifluorescence and Brewster angle microscopy on the air/water interface and on Langmuir-Blodgett films. Their thickness was measured ellipsometrically. Under molecular packing conditions resembling those found in the natural membrane, the morphology and size of the domains are highly self-similar, displaying no characteristic length scale. These properties are the hallmark of fractal objects. The fractality extends at least three orders of magnitudes, from the micrometer to the millimeter range, the fractal dimension being about 1.7. A possible implication of fractality in membrane structure and/or function is demonstrated through the high fluctuation of the propagation of signals through constrained diffusion in corrals formed by domains in the plane of the monolayer, which restricts the diffusion of a fluorescent probe over many length scale domains.     

Human fibroblast-conditioned medium contains a 100K dalton glucose-regulated cell surface protein

This report describes the identification and partial characterization of a 100K dalton “glucose-regulated” cell surface protein of human diploid fibroblasts (HDF). This protein is released into and can be recovered virtually intact from the surrounding culture medium. At the present level of analysis, the protein recovered from the culture medium (“conditioned medium”) is indistinguishable from the protein extracted directly from the cell surface by 1 M urea treatment. Both proteins have molecular weights of 100K daltons when analyzed by gel electrophoresis. The protein is readily labeled at the cell surface via lactoperoxidase-catalyzed iodination, and the label can be chased into the released form of this protein in conditioned medium. Antiserum raised against the medium form of the protein reacts with the surface form of the protein but does not react with fibronectin, the major cell surface protein of HDF. Conditioned medium from SV40-transformed human fibroblasts does not contain the 100K protein, but instead contains a component that has a slightly lower molecular weight (97K daltons). The lower molecular weight band does not iodinate at the cell surface and is apparently an underglycosylated form of the 100K protein. Its molecular weight is shifted back to 100K by growing transformed cells in medium containing excess glucose. After the shift, the component becomes accessible to the radioiodine label. We suggest that the 100K protein is a glucose-regulated protein (Shiu, Pouyssegur and Pastan, 1977; Pouyssegur and Yamada, 1978) that is released into the culture medium. An underglycosylated form of the same glycoprotein is released from transformed cells.     

Synthesis and expression of the DNA fragment coding the antigenic determinant of the surface antigen protein of hepatitis B virus

With the use of chemical-enzymatic synthesis, a polynucleotide (I) has been obtained which codes the amino acid sequence 93-109 of the hepatitis B surface antigen (HBsAg). The plasmid pHS12, constructed by insertion of (I) into pBR325 between EcoRI and BamHI sites, transformed E. coli HB101 cells. The plasmid expression in cell-free system produced a chimeric protein, its molecular weight corresponding to that a polypeptide containing the heptadecapeptide from HBsAg. An approach is discussed for creating a new type of vaccines on the basis of chimeric proteins containing the antigenic determinants of virus proteins.     

Isolation and characterization of autoagglutination factor of Yersinia pestis Hms− cells

An approach for isolation of an autoagglutination factor (AF) from Hms(-) cells of the plague agent has been developed. Purified AF has been obtained and characterized in physicochemical properties. The AF is found to be a complex of a 17.5-kD protein with a low molecular weight peptide component, which binds iron ions and shows siderophore activity. This low molecular weight component is responsible for hydrophobic properties and immunochemical activity of the AF, as well as for its ability to interact with the plague diagnosticum L-413c bacteriophage.     

Kinetics of protein adsorption and desorption on surfaces with grafted polymers

The kinetics of protein adsorption are studied using a generalized diffusion approach which shows that the time-determining step in the adsorption is the crossing of the kinetic barrier presented by the polymers and already adsorbed proteins. The potential of mean-force between the adsorbing protein and the polymer-protein surface changes as a function of time due to the deformation of the polymer layers as the proteins adsorb. Furthermore, the range and strength of the repulsive interaction felt by the approaching proteins increases with grafted polymer molecular weight and surface coverage. The effect of molecular weight on the kinetics is very complex and different than its role on the equilibrium adsorption isotherms. The very large kinetic barriers make the timescale for the adsorption process very long and the computational effort increases with time, thus, an approximate kinetic approach is developed. The kinetic theory is based on the knowledge that the time-determining step is crossing the potential-of-mean-force barrier. Kinetic equations for two states (adsorbed and bulk) are written where the kinetic coefficients are the product of the Boltzmann factor for the free energy of adsorption (desorption) multiplied by a preexponential factor determined from a Kramers-like theory. The predictions from the kinetic approach are in excellent quantitative agreement with the full diffusion equation solutions demonstrating that the two most important physical processes are the crossing of the barrier and the changes in the barrier with time due to the deformation of the polymer layer as the proteins adsorb/desorb. The kinetic coefficients can be calculated a priori allowing for systematic calculations over very long timescales. It is found that, in many cases where the equilibrium adsorption shows a finite value, the kinetics of the process is so slow that the experimental system will show no adsorption. This effect is particularly important at high grafted polymer surface coverage. The construction of guidelines for molecular weight/surface coverage necessary for kinetic prevention of protein adsorption in a desired timescale is shown. The time-dependent desorption is also studied by modeling how adsorbed proteins leave the surface when in contact with a pure water solution. It is found that the kinetics of desorption are very slow and depend in a nonmonotonic way in the polymer chain length. When the polymer layer thickness is shorter than the size of the protein, increasing polymer chain length, at fixed surface coverage, makes the desorption process faster. For polymer layers with thickness larger than the protein size, increases in molecular weight results in a longer time for desorption. This is due to the grafted polymers trapping the adsorbed proteins and slowing down the desorption process. These results offer a possible explanation to some experimental data on adsorption. Limitations and extension of the developed approaches for practical applications are discussed.     

Modeling microdomains: the surface area of globin helices.

The accessible surface areas of 53 high-resolution globin helices are correlated with molecular weight. The linear fit is assessed for statistical accuracy using a boot-strap analysis, and by comparison to the areas of 13 ideal polyalanine alpha-helices. The accessible area of the unfolded helices is compared with the folded values before helix-helix packing. An analytical physical model is presented to explain the correlation, and to provide an analytical value for the surface area parameter in the diffusion-collision model of protein folding.     

Optical surface plasmon resonance biosensors in molecular fishing

An optical biosensor employing surface plasmon resonance (SPR; SPR-biosensor) is a highly efficient instrument applicable for direct real time registration of molecular interactions without additional use of any labels or coupled processes. As an independent approach it is especially effective in analysis of various ligand receptor interactions. SPR-biosensors are used for validation of studies on intermolecular interactions in complex biological systems (affinity profiling of various groups of proteins, etc.). Recently, potential application of the SPR-biosensor for molecular fishing (direct affinity binding of target molecules from complex biological mixtures on the optical biosensor surface followed by their elution for identification by LCMS/MS) has been demonstrated. Using SPR-biosensors in such studies it is possible to solve the following tasks: (a) SPR-based selection of immobilization conditions required for the most effective affinity separation of a particular biological sample; (b) SPR-based molecular fishing for subsequent protein identification by mass spectrometry; (c) SPR-based validation of the interaction of identified proteins with immobilized ligand. This review considers practical application of the SPR technology in the context of recent studies performed in the Institute of Biomedical Chemistry on molecular fishing of real biological objects.     

Cell surface protein of Pseudomonas (Hydrogenomonas) facilis

Intact cells of Pseudomonas facilis contain one major molecular weight class of protein that is exposed at the cell surface as revealed by lactoperoxidase-catalyzed iodination with (125)I. All molecular weight classes of protein in derived cell envelope preparations are apparently saturated by iodination by lactoperoxidase after prolonged sonic treatment. The molecular weight of the predominantly exposed protein in intact cells is approximately 16,000, which is the minimal molecular weight of a cell envelope protein that precipitates as a complex with phospholipid from extracts of P. facilis. The isolation of labeled phospholipoprotein (PLP) after labeling intact cells with (125)I corroborates previous experiments which suggested a surface location for the protein portion of the phospholipoprotein (P(PLP)). Solvent extraction of cells and immunological evidence, including studies with ferritin-coupled antibodies, indicate that P(PLP) is located at the cell surface and may also be within the cell envelope. These experiments suggest that P(PLP) is the major cell surface protein in P. facilis.     

Probing the reactivity of nucleophile residues in human 2,3-diphosphoglycerate/deoxy-hemoglobin complex by aspecific chemical modifications.

The use of aspecific methylation reaction in combination with MS procedures has been employed for the characterization of the nucleophilic residues present on the molecular surface of the human 2,3-diphosphoglycerate/deoxy-hemoglobin complex. In particular, direct molecular weight determinations by ESMS allowed to control the reaction conditions, limiting the number of methyl groups introduced in the modified globin chains. A combined LCESMS-Edman degradation approach for the analysis of the tryptic peptide mixtures yielded to the exact identification of methylation sites together with the quantitative estimation of their degree of modification. The reactivities observed were directly correlated with the pKa and the relative surface accessibility of the nucleophilic residues, calculated from the X-ray crystallographic structure of the protein. The results here described indicate that this methodology can be efficiently used in aspecific modification experiments directed to the molecular characterization of the surface topology in proteins and protein complexes.     

Analysis of the arrangement of protein components in the sarcomplasmic reticulum of rat skeletal muscle.

The nature of the protein components and their location in the sarcoplasmic reticulum membrane were studied using sarcoplasmic reticulum vesicles isolated from rat skeletal muscle and purified by a density gradient centrifugation system. On the basis of analysis by means of sodium dodecyl sulfate gel electrophoresis, the protein components appear to be similar if not identical with those reported by others for rabbit sarcoplasmic reticulum, and the relative amount of each component is also similar to that found with rabbit sarcoplasmic reticulum. Evidence is presented that radioiodine-labeled diazotized diiodosulfanilic acid is a nonpermeant labeling agent of the protein components of sarcoplasmic reticulum vesicles; this agent minimally disturbs the functional activities of these membranes. By means of this labeling agent and perturbing agents, it is concluded that the protein components with molecular weights greater than 120,000 and the (Ca2+ + Mg2+)-adenosine triphosphatase partially or totally reside on or at the external surface of the sarcoplasmic reticulum vesicles. In the case of the adenosine triphosphatase, highly controlled trypsin treatment cleaves the molecule into two products, a 65,000 molecular weight fragment and a 56,000 molecular weight fragment. The evidence indicates that the 65,000 molecular weight component of the (Ca2+ + Mg2+)-adenosine triphosphatase is located in a more exposed fashion on the external surface of the vesicles than the 56,000 molecular weight compoenet and that some adenosine triphosphatase molecules have a more exposed position on the external surface of the vesicle than others. The protein components designated by MacLennan (MacLennan, D. H. (1975) Can. J. Biochem. 53, 251-261) as "calsequestrin" and "high affinity Ca2+ binding protein" are shown not to be on the external surface of the rat sarcoplasmic reticulum vesicle but rather to reside either within the core of the membrane or on the inside surface of the vesicle. The results of this study are in agreement with the model for the organization of the protein components of the sarcoplasmic reticulum membrene recently proposed by MacLennan (MacLennan, D. H. (1975) Can. J. Biochem. 53, 251-261).     

Expression of major piroplasm protein (p33) of Theileria sergenti (Korean isolate) and its immunogenicity in guinea pigs

To investigate the development of a subunit vaccine against theileriosis in cattle, the DNA fragments encoding piroplasm surface protein (p33) of Theileria sergenti of a Korean isolate were expressed in baculoviruses. The expressed p33 was characterized by indirect fluorescent antibody (IFA) and western blotting analysis. The expression of p33 was mainly detected on the surface of infected Sf21 cells by IFA. The immunoblotting analysis revealed the presence of a same molecular weight protein band of p33. The antigenicity of expressed polypeptide was further examined through the inoculation of a guinea pig. The sera of guinea pigs immunized with p33 expressed cell lysate showed similar fluorescent antibody patterns and reacted with the same molecular weight protein of T. sergenti in immunoblotting analysis, thus indicating that this protein can be a promising candidate for a subunit vaccine in the future.     

Thermodynamic study of forces involved in bovine serum albumin and ovalbumin partitioning in aqueous two-phase systems

The partitioning of bovine serum albumin and ovalbumin in different two-phase aqueous polymer systems is investigated using a thermodynamic approach. Systems used were polyethylene glycols (PEGs) of molecular weights 1000 to 10,000 Da and Dextran T500 (500,000 Da). Ovalbumin transfer to the top phase is exothermic, which suggests an electrostatic interaction between the hydroxyl groups of PEG and the hydrophilic side chain of the protein, whereas the bovine serum albumin partition is an endothermic process that is entropically driven, which coincides with its high surface hydrophobicity. The effect of PEG molecular weight on enthalpy and heat capacity changes, associated with the partition of both proteins, is examined on the basis of a preferential interaction of low-molecular-weight PEG with the protein surface.     

De facto molecular weight distributions of glucans by size-exclusion chromatography combined with mass/molar-detection of fluorescence labeled terminal hemiacetals

A major capability of polysaccharides in aqueous media is their tendency for aggregation and dynamic formation of supermolecular structures. Even extended dissolution processes will not eliminate these structures which dominate many analytical approaches, in particular absolute molecular weight determinations referring to light scattering data. An alternative approach for determination of de facto molecular weight for glucans with free terminal hemiacetal functionality (reducing end group) has been adjusted from carbohydrates for midrange and high-dp glucans: quantitative and stabilized labeling as aminopyridyl-derivatives (AP-glucans) and subsequent analysis of SEC-separated elution profiles based on simultaneously monitored mass and molar fractions by refractive index and fluorescence detection. SEC-DRI/FL of AP-glucans proved as an appropriate approach for determination of de facto molecular weight of constituting glucan molecules even in the presence of supermolecular structures for non-branched (pullulan), branched (dextran), narrow distributed and broad distributed and for mixes of compact and loose packed polymer coils (starch glucan hydrolizate).     

Dendric nanostructures for SARS-CoV-2 proteins detection based on surface enhanced infrared absorption spectroscopy

Infrared spectroscopy is a non-destructive and rapid characterization tool that can distinguish different viral proteins by spectral details. However, traditional infrared spectroscopy has insufficient absorption signal intensity contrast when measuring low-concentration samples. In this work, surface enhanced infrared absorption (SEIRA) spectroscopy is proposed by deploying a novel nanostructure array as SEIRA substrates. An array of gold dendric nanostructures are designed and fabricated with a precision resonance control to achieve surface enhancement covering a broadband molecular “finger-print” region. The spectral positions of the multiple resonances accurately correspond to the characteristic absorption peaks of the SARS-CoV-2 proteins. An approach for SARS-CoV-2 protein detection based on SEIRA spectroscopy is then proposed. A low concentration detection of 40 μg/ml diluted SARS-CoV-2 nucleocapsid protein is experimentally demonstrated and the enhancement factor (EF) achieved is in good agreement with simulation results. The SEIRA methodology based on broadband resonance nanostructure design provides a systematic approach for sensitive, non-destructive and rapid protein molecular detection, which could be extended to various kind of molecular characterization and biomedical diagnostics.     

黄鳝血清和体表粘液蛋白的比较研究

本文用反相高效液相色谱法测定了黄鳝血清和体表粘液蛋白的氨基酸种类和含量,比较了二者的氨基酸组成变化。结果表明：二者都含有17种氨基酸,血清的氨基酸总量为397．11mg／l00ml,体表粘液蛋白的氨基酸总量为259．29mg／l00m1,血清与粘液蛋白中氨基酸含量差异最大的是蛋氨酸、半胱氨酸。应用SDS—PAGE分析和比较了血清和体表粘液蛋白的分子量大小及特有区带效,黄鳝血清与体表粘液蛋白分子量相同的蛋白区带数为2条,其分子量大小分别为19．5kDa、96．0kDa。其中血清的特有蛋白带为18条,体表粘液的特有蛋白带为8条。此外,对二者的相关性和体表粘液特异性的免疫机制作了探讨。     

Inhibition of cell adhesion by high molecular weight kininogen

An anti-cell adhesion globulin was purified from human plasma by heparin-affinity chromatography. The purified globulin inhibited spreading of osteosarcoma and melanoma cells on vitronectin, and of endothelial cells, platelets, and mononuclear blood cells on vitronectin or fibrinogen. It did not inhibit cell spreading on fibronectin. The protein had the strongest antiadhesive effect when preadsorbed onto the otherwise adhesive surfaces. Amino acid sequence analysis revealed that the globulin is cleaved (kinin-free) high molecular weight kininogen (HKa). Globulin fractions from normal plasma immunodepleted of high molecular weight kininogen (HK) or from an individual deficient of HK lacked adhesive activity. Uncleaved single-chain HK preadsorbed at neutral pH, HKa preadsorbed at pH greater than 8.0, and HKa degraded further to release its histidine-rich domain had little anti-adhesive activity. These results indicate that the cationic histidine-rich domain is critical for anti-adhesive activity and is somehow mobilized upon cleavage. Vitronectin was not displaced from the surface by HKa. Thus, cleavage of HK by kallikrein results in both release of bradykinin, a potent vasoactive and growth-promoting peptide, and formation of a potent anti-adhesive protein.     

Identification of disulfide-containing peptides by performic acid oxidation and mass spectrometry

In addition to reducing the analysis time, the direct examination of proteolytic digests by fast atom bombardment mass spectrometry (FABMS) greatly extends the information that is available from peptide mapping experiments. Mass spectral data are particularly useful for identifying post-translationally modified peptides. For example, the molecular weight of a disulfide-containing peptide may be used to locate the disulfide bond in the protein from which the peptide was derived. This paper describes a new procedure, which is useful for identifying disulfide-bonded peptides. Peptides are treated with performic acid to modify certain residues and thereby cause a characteristic change in the peptide molecular weight. This change in molecular weight is determined by FABMS and used to help identify peptides. Results for a series of small peptides demonstrate that Cys, Met, and Trp are the only residues that undergo a change in molecular weight under the conditions used here. Furthermore, these changes in molecular weight are diagnostic for each of the residues. Cysteinyl-containing peptides are of particular interest, because their identification is essential for locating disulfide bonds. The molecular weight of a peptide increases by 48 mu for each cysteinyl residue present. This approach is used to identify peptides that contain both cysteinyl and cystinyl residues in the peptic digest of bovine insulin. The method is extended to the analysis of a tryptic digest of cyanogen bromide-treated ribonuclease A. A computer-assisted analysis procedure is used to demonstrate the specificity with which peptide molecular weight is related to specific segments of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)