Cryo-EM of kinesin-binding protein: challenges and opportunities from protein-surface interactions

Kinesin-binding protein (KBP) is an important selective inhibitor of specific kinesin family members and its genetic disruption causes Goldberg–Shprintzen syndrome. Cryo-electron microscopy (cryo-EM) has recently been used to reveal the structure of KBP alone (72 kDa) and in complex with the motor domain of the mitotic kinesin-12 KIF15 (110 kDa). KBP is an α-solenoid, tetratricopeptide-repeat protein that interacts with the microtubule-binding region of the kinesin motor domain and blocks microtubule attachment. Numerous challenges arose relating to the behavior of KBP and KBP–kinesin complexes during cryo-EM sample preparation. These included the partial denaturation of KBP by air–water interfaces, protein aggregation resulting from carbon interaction and preferential orientation. Sample preparation with a graphene oxide substrate enabled the eventual structure determination. Here, experiences with preparing these samples are detailed, bringing attention to some of the challenges and opportunities that are likely to arise from protein-surface interactions.

Structural Consequences of the Villin Headpiece Interaction with a Carbon Nitride Polyaniline (C3N) Nanosheet

ABSTRACTCarbon nitride polyaniline (C3N) nanosheets shared a similar structure with graphene and have been utilized in biomedical applications since its recent successful synthesis. However, limited information was known about the interaction of this next-generation nanomaterial with biomolecules, which might hamper its applications in living tissues. Here, by using all-atom molecular dynamics (MD) simulations, we investigated the interaction between a C3N nanosheet and the prototypical protein villin headpiece (HP35), in order to identify the mechanistic determinants of such interaction; this knowledge will provide guidelines about C3N’s biocompatibility. Our MD simulations revealed that the C3N-based nanomaterial caused the partial denaturation of HP35 once the protein was bound on its surface. That is, upon adsorption, we observed the loss of the protein’s interior hydrogen bonds and the native contacts, which were related with unwinding events in the protein’s helices. The protein/C3N nanosheet interacting process was dominated by vdW contributions to the energy and the stepwise changes observed in the values of this energy term suggested a gradual unfolding pattern of HP35 during the absorption event. Furthermore, we also found that the interaction energy showed a linear correlation with the native Q ratio of HP35, suggesting that the degree of HP35 unfolding was linearly time-dependent to the interaction energy. Our findings shed light on the underlying molecular mechanism of the potential consequences of C3N-based nanostructures to proteins, which might delineate the future applications of these nanomaterials in biomedicine.

P1199THE IMPORTANCE OF DETERMINING ALPHA-2-MACROGLOBULIN CONCENTRATITON IN PATIENTS ON PERITONEAL DIALYSIS

Background and Aims To determine the concentration of Alpha-2-macroglobulin (α2M) and its activity in patients treated with peritoneal dialysis (PD) and healthy subjects (H). Method a cross-sectional study included 50 PD and 30 H, in which the concentration of active α2M molecules was determined. The measurement is based on covalent binding of α2M and trypsin and zymography with gelatin incorporated into the electrophoretic gel. Results The intensity of proteolytic bands seen with native α2M was analyzed in relation to the concentration of α2M in patients with PD with less and / or more than three peritonitis. Linear dependence was only visible in the case of the upper range, which originates from the intact α2M-trypsin complex. The highest coefficient of variation is calculated for the smallest α2M concentration (4.2%). The signal intensities originating from α2M (H) were higher compared to PD, with the PD group being lower in patients with higher peritonitis, and the difference was more pronounced as concentration increased, resulting in a lower slope of dependence. The proposed method can reliably measure the amount of α2M originating in a group H, while the quantification of α2M in PD groups that may have structurally altered α2M can be seen more for condition estimation than for accurate measurements. The zymographic test in this experiment allows us to investigate the relationship of the concentration, structure and function of α2M in different pathological conditions, as well as in the presence of modifying agents that can accompany them. Conclusion A simple method for the determination of α2M concentration and function by zymography has been developed, which can be used to examine residual α2M activity after partial denaturation or structural modification due to disease.

Study of gentamicin deposition in cellulose with albumin

Methods of binding antibacterial drugs to the surface of cellulose without the use of oxidizing agents to prevent the occurrence of wound infections have been studied. The immobilization of gentamicin in the complex of partially denatured albumin in the composition with bacterial cellulose has been analyzed. The study was carried out on samples of cellulose synthesized by Gluconacetobacter hansenii. Albumin served as a binding agent, which was used to impregnate cellulose samples, which were then denatured. Using PCR amplifi cation CFX (BioRad), the optimal denaturation temperature was selected. The effectiveness of the immobilization of albumin in the thickness of the cellulose was assessed by staining it with the luminescent dye SYPRO® Ruby Protein Gel Stain, followed by transilluminator detection. Bacterial cellulose impregnated with undenatured albumin was used as a control. Albumin immobilization in bacterial cellulose was observed at temperatures of 65– 95 °C. The antibacterial activity of the complex “cellulose + albumin + gentamicin” was evaluated using a test strain of bacteria Staphylococcus aureus ATCC 25923. The growth inhibition of the test strain of bacteria was observed in all tests with bacterial cellulose in combination with partially denatured albumin and gentamicin. In control samples, in which gentamicin was not immobilized as part of partially denatured albumin, growth inhibition zones of Staphylococcus aureus ATCC 25923 were not noted. It was concluded that by partial denaturation of albumin it is possible to delay antibacterial drugs in the thickness of bacterial cellulose for their further release. A new version of the material suitable for the production of implants and bandages based on bacterial cellulose gel with antibacterial properties is proposed. Dressings based on a composite of bacterial cellulose, albumin and gentamicin are most relevant for the treatment of burns. The presence of gentamicin in their composition is also relevant for the prevention of bacterial infections.

KARAKTERISTIK FISIKOKIMIA GELATIN KULIT IKAN AYAM-AYAM ( Abaliste stellaris ) DENGAN PRA-PERLAKUAN KONSENTRASI ASAM SITRAT

Gelatin is a fibrous protein obtained by partial denaturation of collagen. Traditionally, gelatin derived from mammals’ skins and bones, such as cow and pork. However, both mammals’ gelatin could risk for bovine spongiform encephalopathy (BSE) and foot mouth disease (FMD), besides, gelatin extracted from pork is prohibited in Islam rules. Therefore, fish processing waste is potential as a source of gelatin in terms of starry triggerfish (Abalistes stellaris) skin gelatin. The aims of this study is to characterize physichochemical of gelatin extracted from skin of starry triggerfish. The methods used in this research, experiment with completely randomized design (CDR) by soaking different concentrations of citric acid (0.2; 0.4; and 0.6 M). The results showed that the concentrations of citric acid had significantly different (P<0.05) on the yield, viscosity, gel strength, and fat content of starry triggerfish skin gelatin. On the other hand, the pH, melting point, galling point, protein, moisture, and ash value of starry triggerfish skin gelatin did not perform significantly different (P>0.05). The most properties of starry triggerfish skin gelatin meet the commercial gelatin, and it is able to as a potential alternative of halal gelatin.

Reduction of Deoxynivalenol in Wheat with Superheated Steam and Its Effects on Wheat Quality

Deoxynivalenol (DON) is the most commonly found mycotoxin in scabbed wheat. In order to reduce the DON concentration in scabbed wheat with superheated steam (SS) and explore the feasibility to use the processed wheat as crisp biscuit materials, wheat kernels were treated with SS to study the effects of SS processing on DON concentration and the quality of wheat. Furthermore, the wheat treated with SS were used to make crisp biscuits and the texture qualities of biscuits were measured. The results showed that DON in wheat kernels could be reduced by SS effectively. Besides, the reduction rate raised significantly with the increase of steam temperature and processing time and it was also affected significantly by steam velocity. The reduction rate in wheat kernels and wheat flour could reach 77.4% and 60.5% respectively. In addition, SS processing might lead to partial denaturation of protein and partial gelatinization of starch, thus affecting the rheological properties of dough and pasting properties of wheat flour. Furthermore, the qualities of crisp biscuits were improved at certain conditions of SS processing.

Development of Reduced-Fat, Reduced-Sodium Semi-Hard Sheep Milk Cheese

This paper examines the effects of the incorporation of denatured whey proteins along with salting in NaCl/KCl brine on the characteristics and ripening of sheep milk reduced-fat (RF), semi-hard cheese. Incorporation of denatured whey proteins was carried out by: i. adding commercial microparticulated whey protein (MWP) in reduced-fat cheese milk (RFM), or ii. by ‘in situ’ heat-induced partial denaturation of whey proteins of reduced-fat cheese milk (RFD). The implemented cheesemaking conditions included curd washing, moderate clotting, scalding temperatures, and ripening of cheeses packed in plastic bags under vacuum at 10 °C. Full-fat cheeses (FF) were manufactured in parallel. Physicochemical composition, textural profile, and proteolysis were assessed throughout 60 days of ripening. The mean moisture, fat on dry matter (FDM), moisture on non-fat substances (MNFS), protein on dry matter (PDM), salt, and salt-in-moisture (S/M) content of the RF cheeses were 47.4%, 32.8%, 57.3%, 54.3%, 1.63%, and 3.36%, respectively; pH ≈ 5.0, aw ≈ 0.977, Ca ≈ 1000 mg/100 g cheese. The MNFS of FF and RF cheeses were similar. Proteolysis indices were not affected by any of the treatments, and they were similar to the FF counterparts. The applied cheesemaking technology was adequate for the production of semi-hard reduced-fat and reduced-sodium cheeses. Ripening under packaging hindered moisture loss without impairing the evolution of proteolysis and textural parameters. The same holds true for salting in NaCl/KCl brine. The high pasteurization of cheese milk was more effective for the increase of moisture and MNFS than the addition of MWP, without exhibiting any adverse effects.

Basic Kirkwood – Buff Theory of Solution Structure and Appropriate Application of Wyman Linkage Equation to Biochemical Phenomena

Background: Researchers who have shown interest in the consequence of introducing dry biomolecules or a solution of it into cosolvents generally known as osmolyte, have applied many models for the elucidation of the scientific basis of the results obtained. The Kirkwood and Buff theory (KBT) or its reverse form has been the basis for the interpretation of the effect of the osmolyte. There seems to be no generally acceptable definition of terms in the basic KBT mathematical formalism. There is also error in stated equations describing solution structure and misapplication of Wyman linkage relation. Therefore, the objectives of this research are 1) to show how the equation of preferential interaction parameter is derived based on KBT, 2) to show the appropriate way in which Wyman linkage relation can be applied, 3) to apply biochemical approach (using generated data) to the equation of preferential interaction parameter (preferential interaction parameter is symbolised as ) for its calculation and calculation of parameters linked to KBT derived equations. Methods: The research is mainly theoretical and partly experimental. The experiment entails Bernfeld method of enzyme assay for the generation of data. Results and Discussion: The change in solvation preference upon the ethanol partial denaturation of the enzyme and the corresponding change in preferential interaction parameter were respectively positive and negative in sign. Unexpectedly ethanol was preferentially excluded from the enzyme. Conclusion: The equations of preferential interaction parameters were derived. The appropriate way is either by calculation or measurement of preferential interaction parameter. Therefore, or for the change, cannot be a constant (or slope) and an instrumentation–based measurable parameter at the same time. Based on Wyman linkage relation, purely biochemical thermodynamic parameter is linked to preferential interaction parameters which are therefore, thermodynamic parameters.

Quantitation of the active alpha-2-macroglobulin by trypsin protease zymography

A simple and reliable method for the determination of the concentration and function of alpha-2-macroglobulin (?2M) by zymography was developed. The method is based on the covalent binding of ?2M and trypsin followed by non-reducing PAGE and zymography with gelatine incorporated in the electrophoretic gel. The results showed that ?2M binds trypsin in a concentration-dependent manner exhibiting a linear relation. The sensitivity of the method is 125 nM and the intra-assay coefficient of variation 4.2 %. Freezing of ?2M induces its partial denaturation, which could be seen as the reduction in the amount of functional molecule and its reactivity with trypsin. The reported method enables measurement of ?2M taking into consideration both its quantity and function, stressing the importance of the determination of the amount of physiologically active molecules and not just their presence in the sample. The method was further confirmed using ?2M from patients with end-stage renal disease who are known to be under increased oxidative stress and inflammation, which are expected to modify the structure of proteins.