RESUMEN
Peptide gemini-surfactant (PG-surfactant), a kind of lipopeptide, is composed of a short linker peptide (X) between two alkyl-chain-modified Cys residues and peripheral peptides at the N-terminal (Y) and the C-terminal (Z) sides, respectively, of the alkylated Cys residues. In this study, we developed and examined a series of PG-surfactants containing two C12 saturated alkanes and oligo-Lys, arranged at the X-, Y-, or Z-positions. To arrange oligo-Lys at the Y- or Z-positions, a repeat sequence of -Asp-Lys-Asp-Lys- was used at the X-position. All of the PG-surfactants exhibited high antimicrobial activity against both Gram-positive and -negative bacteria. In addition to high antimicrobial activity, a low hemolysis activity is prerequisite for efficient intravenous administration. Among the synthesized PG-surfactants, those having -(Lys)3- at the Y- or Z-positions, i.e. K3-DKDKC12 and DKDKC12K3, showed reasonably low hemolytic activities. This combination of high antimicrobial activity along with low hemolytic activity is an essential and unique property and has not been previously reported for the synthesized lipopeptides. Further, using scanning electron microscopy (SEM) and N-phenyl-1-naphthylamine (NPN) uptake assay we showed that the antimicrobial activity of these PG-surfactants may be attributed to membrane disruptive mechanisms. Although the PG-surfactants with low hemolytic activity could interact and localize onto red blood cell surfaces and cause slight expansion of cell morphologies, no subsequent penetration occurred. In summary, we describe here the successful development of PG-surfactants having high antibacterial and low hemolytic activity, thus providing a significant molecular platform to develop novel antimicrobial agents.
Asunto(s)
Antiinfecciosos/síntesis química , Antiinfecciosos/farmacología , Lisina/química , Oligopéptidos/química , Tensoactivos/química , Administración Intravenosa , Animales , Antiinfecciosos/administración & dosificación , Antiinfecciosos/química , Cationes , Eritrocitos/efectos de los fármacos , Bacterias Gramnegativas , Bacterias Grampositivas/efectos de los fármacos , Hemólisis/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica de Rastreo , ConejosRESUMEN
The development of additional extraction surfactants for membrane proteins is necessary for membrane protein research, since optimal combinations for the successful extraction of target membrane proteins from biological membranes that minimize protein denaturation are hard to predict. In particular, those that have a unique basal molecular framework are quite attractive and highly desired in this research field. In this study, we successfully constructed a new extraction surfactant for membrane proteins, NPDGC12KK, from the peptide-gemini-surfactant (PG-surfactant) molecular framework. The PG-surfactant is a U-shaped lipopeptide scaffold, consisting of a short linker peptide (-X-) between two long alkyl-chain-modified Cys residues and a peripheral peptide (Y-) at the N-terminal side of long alkyl-chain-modified Cys residues. Using photosystem I (PSI) and photosystem II (PSII) derived from Thermosynecoccus vulcanus as representative membrane proteins, we evaluated whether NPDGC12KK could solubilize membrane proteins while maintaining structure and functions. Neither the membrane integral domain nor the cytoplasmic domain of PSI and PSII suffered any damage upon the use of NPDGC12KK based on detailed photophysical measurements. Using thylakoid membranes of T. vulcanus as a representative biological membrane sample, we performed experiments to extract membrane proteins, such as PSI and PSII. Based on the extraction efficiency and maintenance of protein supramolecular structure established using clear native-PAGE analyses, we proved that NPDGC12KK functions as a novel class of peptide-containing extraction surfactants for membrane proteins.
Asunto(s)
Proteínas de la Membrana/química , Proteínas de la Membrana/aislamiento & purificación , Tensoactivos/química , Fraccionamiento Químico/métodos , Cisteína/química , Lipopéptidos/química , Micelas , Péptidos/química , Complejo de Proteína del Fotosistema I/química , Complejo de Proteína del Fotosistema II/química , Ingeniería de Proteínas/métodos , Espectrometría de Fluorescencia , Synechocystis/química , Tilacoides/químicaRESUMEN
Protein-encapsulated fibermats are an attractive platform for protein-based bioactive materials. However, the choice of methods is still limited and not applicable to a wide range of proteins. In this study, we studied new polymeric materials for constructing protein-encapsulated fibermats, in which protein molecules are encapsulated within the nanofibers of fibermats without causing deleterious changes to protein structure or function. We constructed a protein-encapsulated fibermat using the poly(γ-glutamate) (PGA)/(3-glycidyloxypropyl)-trimethoxysilane (GPTMS) hybrid as a precursor for electrospinning. Because the PGA/GPTMS hybrid is water-soluble, protein molecules can be added to the precursor in an aqueous solution, significantly enhancing protein stability. Polycondensation during electrospinning (in-flight polycondensation) makes the obtained fibermats water-insoluble, which stabilizes the fibermat structure such that it is resistant to degradation in aqueous buffer. The molecular structure of the PGA/GPTMS hybrid gives rise to unique molecular permeability, which alters the selectivity and specificity of biochemical reactions involving the encapsulated enzymes; lower molecular-weight (MW) substrates can permeate the nanofibers, promoting enzyme activity, but higher MW substrates such as inhibitor peptides cannot permeate the nanofibers, suppressing enzyme activity. We present an effective method of encapsulating bioactive molecules while maintaining their structure and function, increasing the versatility of electrospun fibermats for constructing various bioactive materials.
Asunto(s)
Ácido Poliglutámico/análogos & derivados , Proteínas/química , Dióxido de Silicio/química , Nanofibras/química , Ácido Poliglutámico/química , Silanos/químicaRESUMEN
For the first time, we have successfully fabricated microfiltration (MF) hollow fiber membranes by the thermally induced phase separation (TIPS) and non-solvent induced phase separation (NIPS) methods using cellulose acetate benzoate (CBzOH), which is a cellulose derivative with considerable chemical resistance. To obtain an appropriate CBzOH TIPS membrane, a comprehensive solvent screening was performed to choose the appropriate solvent to obtain a membrane with a porous structure. In parallel, the CBzOH membrane was prepared by the NIPS method to compare and evaluate the effect of membrane structure using the same polymer material. Prepared CBzOH membrane by TIPS method showed high porosity, pore size around 100 nm or larger and high pure water permeability (PWP) with slightly low rection performance compared to that by NIPS. On the contrary, CBzOH membranes prepared with the NIPS method showed three times lower PWP with higher rejection. The chemical resistance of the prepared CBzOH membranes was compared with that of cellulose triacetate (CTA) hollow fiber membrane, which is a typical cellulose derivative as a control membrane, using a 2000 ppm sodium hypochlorite (NaClO) solution. CBzOH membranes prepared with TIPS and NIPS methods showed considerable resistance against the NaClO solution regardless of the membrane structure, porosity and pore size. On the other hand, when the CTA membrane, as the control membrane, was subjected to the NaClO solution, membrane mechanical strength sharply decreased over the exposure time to NaClO. It is interesting that although the CBzOH TIPS membrane showed three times higher pure water permeability than other membranes with slightly lower rejection and considerably higher NaClO resistance, the mechanical strength of this membrane is more than two times higher than other membranes. While CBzOH samples showed no change in chemical structure and contact angle, CTA showed considerable change in chemical structure and a sharp decrease in contact angle after treatment with NaClO. Thus, CBzOH TIPS hollow fiber membrane is noticeably interesting considering membrane performance in terms of filtration performance, mechanical strength and chemical resistance on the cost of slightly losing rejection performance.