Reductive Alkylation of Candida Rugosa Lipase: Structural Approaches

The properties of alkylated lipase are successfully explored through experimental and molecular modelling methods. Alkylation was done using aldehydes with different degree of modification to represent different levels of hydrophobicity which is important for enzymes to work in nonaqueous environment. Far ultraviolet circular dichroism (CD) spectroscopy of the lipase in aqueous solvent shows that increasing the degree of modification from 49% to 86% resulted in loss in secondary structure which is attributed to the enzyme unfolding. The secondary structure elements of the CD spectra of native and modified lipase were analysed using the CDPro software and the K2D program. Both methods yield the same results in that the ratio of α-helical structure is lost. This result explains why alkylated lipases have much lower activity in an aqueous environment. Molecular modelling simulations were performed to study the structural and dynamical changes of the lipase upon different levels of modification. Simulations were run for 1 ns (300 K) with five different initial velocities to obtain better conformational sampling. Two solvent systems were used: TIP3P water model and carbon tetrachloride (CCl4) solvent model in periodic boundary condition (PBC). Generally, lipases simulated in water are less deviated in term of root mean square deviations (rmsd) compared to lipases simulated in CCl4. Lid movements are essential for lipase function, both in water and waterlipid environments. Analyses of lid dynamics were done using timecorrelation function and second-order Legendre polynomial function. Lipase in water and CCl4 shows different properties of dynamics. Without alkylation, the time correlation function of lipase in water shows one slow exponential decay with a correlation time of τ = 92.8 ps. In contrast, for simulations in CCl4 the lid has a more complex dynamics. Exponential fit of open CRL in CCl4 results in two different τ values: a fast motion τ1 = 5.6 ps and a slow motion τ2 = 163.8 ps.Upon alkylation, different levels of modification show different properties of lid motions. In CCl4, lid region is highly stabilised upon 95% alkylation with slow motion mode of τ1 = 4.1 ps and τ2 = 577.8 ps. Slow motion effect of lid region is also observed at 63% with τ1 = 2.9 ps and τ2 = 209.2 ps and 43% modification with τ1 = 3.4 ps and τ2 = 117.9 ps. In water, 43% and 95% modification show similar motion with unmodified lipase, with one slow exponential decay of τ = 142.8 ps and 133.6 ps, respectively. However, 63% modification shows more complex dynamics with different τ values which mimics the dynamics properties in CCl4. A novel lid-locking mechanism which stabilises the opening form of lid region has been observed during simulations of unmodified CRL in CCl4, i.e. a salt bridge between Lys85 and Asp284. This salt bridge is highly stabilised on unmodified lipase with a distance of 3.3 Å compared with lipase simulated in water with a distance of 15.25 Å. Alkylation at 43% causes the salt bridge to be deformed in CCl4 with a distance of 6.03 Å; however, 63% modification stabilises the salt bridge with a distance of 3.88 and 95% modification shows the most stabilising effect with a distance of 3.19 Å

LAPORAN INDIVIDU PRAKTEK PENGALAMAN LAPANGAN (PPL) SMP NEGERI 1 WONOSARI

Program PPL yang diselenggarakan oleh Universitas Negeri Yogyakarta merupakan program praktek pengalaman lapangan yang telah dirancang sebagai bagian dari implementasi, pengabdian, tanggung jawab serta loyalitas perguruan tinggi. Dengan PPL ini praktikan memperoleh kesempatan menghadapi kondisi riil dalam proses belajar mengajar. Selain itu program ini sangat berguna untuk penguasaan kompetensi keilmuan dan ketrampilan bidang studi, ketrampilan pengembangan profesi, dan kompetensi dalam pembentukan kepribadian sebagai pendidikan yang profesional. Kegiatan PPL ini dilaksanakan bersama dengan KKN (Kuliah Kerja Nyata) selama dua bulan di laksanakan pada hari senin sampai jumat dan jumat langsung di lanjutkan dengan terjun lokasi KKN, yang dilaksanakan mulai tanggal 15 Juli 2016 sampai dengan 15 September 2016 di SMP Negeri 1 Wonosari dan KKN di bertempat di Dukuh Ringinsari dan Madusari Wonosari, Wonosari Gunung kidul Yogyakarta . Kegiatan yang dilakukan dalam rangka PPL di SMP Negeri 1 Wonosari antar lain: observasi lingkungan pembelajaran dan lingkungan fisik sekolah, persiapan mengejar, pembutan rencana pembelajaran, kegiatan praktek mengajar di kelas, pembuatan media pembelajaran, evaluasi pembelajaran, analisis hasil evaluasi, dan pembuatan laporan sebagai kegiatan akhir dalam rangkaian Praktek Pengalaman Lapangan (PPL) di SMP Negeri 1 Wonosari. Hasil Pratek Pengalaman Lapangan (PPL) mengungkapkan bahwa praktekan dapat menerapkan ilmu pengetahuan dan praktek keguruan dengan teori kependidikan secara terpadu, yang digunakan sebagai bekal membentuk calon pendidikn profesional. Namun dalam pelaksanaannya masih terdapat hambatan PPL. Hambatan itu diantarnya rasa canggung pada saat pertama kali mengajar, kurang percaya diri dan kurang menguasai kelas kadang peserta didik kurang memperhatikan. Hambatan itu dapat teratasi seiringnya berjalanya waktu. Dengan semua program kegiatan yang dilaksanakan oleh praktikan tersebut, praktikan berharap dapat menjadikan kegiatan tersebut sebagai wadah untuk memaksimalkan semua potensi yang praktikan miliki. Selain itu juga membantu upanya pemerintah dalam meningkatkan kualitas SDM melaui pembangunan pendidikan

KETERAMPILAN TEKNIK DASAR BOLA VOLI MELALUI RUBRIK PENILAIAN DI SMP N 2 WERU SUKOHARJO

Penelitian ini bertujuan untuk mengetahui keterampilan teknik dasar dalam permainan bola voli di SMP N 2 Weru, Kabupaten Sukoharjo. Jenis penelitian ini adalah penelitian deskriptif. Metode yang digunakan adalah survei. Populasi dalam penelitian ini adalah siswa SMP Negeri 2 Weru, Sukoharjo yang berjumlah 35 siswa. Instrumen yang digunakan adalah lembar observasi/rubrik penilaian yang terdiri atas teknik servis atas, passing atas, passing bawah, dan smash. Teknik analisis data menggunakan analisis deskriptif kuantitatif yang disajikan dalam bentuk persentase. Hasil penelitian menunjukkan bahwa kemampuan teknik dasar bolavoli siswa SMP Negeri 2 Weru, Sukoharjo berada pada kategori “sangat kurang” sebesar 0% (0 siswa), “kurang” sebesar 11,43% (4 siswa), “sedang” sebesar 51,43% (18 siswa), “baik” sebesar 37,14% (13 siswa), dan “sangat baik” sebesar 0% (0 siswa). Berdasarkan nilai rata-rata, yaitu 43,49, kemampuan teknik dasar bolavoli siswa SMP Negeri 2 Weru, Sukoharjo dalam kategori “sedang”, layak sebagai instrumen penilain untuk teknik dasar bola voli

Structure and dynamics of Candida rugosa lipase: the role of organic solvent

The effect of organic solvent on the structure and dynamics of proteins was investigated by multiple molecular dynamics simulations (1 ns each) of Candida rugosa lipase in water and in carbon tetrachloride. The choice of solvent had only a minor structural effect. For both solvents the open and the closed conformation of the lipase were near to their experimental X-ray structures (Cα rms deviation 1-1.3 Å). However, the solvents had a highly specific effect on the flexibility of solvent-exposed side chains: polar side chains were more flexible in water, but less flexible in organic solvent. In contrast, hydrophobic residues were more flexible in organic solvent, but less flexible in water. As a major effect solvent changed the dynamics of the lid, a mobile element involved in activation of the lipase, which fluctuated as a rigid body about its average position. While in water the deviations were about 1.6 Å, organic solvent reduced flexibility to 0.9 Å. This increase rigidity was caused by two salt bridges (Lys85-Asp284, Lys75-Asp79) and a stable hydrogen bond (Lys75-Asn 292) in organic solvent. Thus, organic solvents stabilize the lid but render the side chains in the hydrophobic substrate-binding site more mobile. © Springer-Verlag 2004

Solution structure of a novel T-cell adhesion inhibitor derived from the fragment of ICAM-1 receptor: cyclo(1,8)-Cys-Pro-Arg-Gly-Gly-Ser-Val-Cys

This study is aimed at elucidating the structure of a novel T-cell adhesion inhibitor, cyclo(1,8)-CPRGGSVC using one- and two-dimensional 1H NMR and molecular dynamics (MD) simulation. The peptide is derived from the sequence of its parent peptide cIBR (cyclo(1,12)-PenPRGGSVLVTGC), which is a fragment of intercellular adhesion molecule-1 (ICAM-1). Our previous results show that the cyclo(1,8)-CPRGGSVC peptide binds to the LFA-1 I-domain and inhibits heterotypic T-cell adhesion, presumably by blocking the LFA-1/ICAM-1 interactions. The structure of the peptide was determined using NMR and MD simulation in aqueous solution. Our results indicate that the peptide adopts type-I β-turn conformation at the Pro2-Arg3-Gly4-Gly5 (PRGG) sequence. The β-turn structure at the PRGG motif is well conserved in cIBR peptide and ICAM-1 receptor, which suggests the importance of the PRGG motif for the biological activity of cyclo(1,8)-CPRGGSVC peptide. Meanwhile, the Gly5-Ser6-Val7-Cys8-Cys1 (GSVCC) sequence forms a “turn-like” random coil structure that does not belong to any structured motif. Therefore, cyclo(1,8)-CPRGGSVC peptide has only one structured region at the PRGG sequence, which may play an important role in the binding of the peptide to the LFA-1 I-domain. The conserved β-turn conformation of the PRGG motif in ICAM-1, cIBR, and cyclo(1,8)-CPRGGSVC peptides can potentially be used to design peptidomimetics

Structure-based and ligand-based virtual screening of novel methyltransferase inhibitors of the dengue virus.

The dengue virus is the most significant arthropod-borne human pathogen, and an increasing number of cases have been reported over the last few decades. Currently neither vaccines nor drugs against the dengue virus are available. NS5 methyltransferase (MTase), which is located on the surface of the dengue virus and assists in viral attachment to the host cell, is a promising antiviral target. In order to search for novel inhibitors of NS5 MTase, we performed a computer-aided virtual screening of more than 5 million commercially available chemical compounds using two approaches: i) structure-based screening using the crystal structure of NS5 MTase and ii) ligand-based screening using active ligands of NS5 MTase. Structure-based screening was performed using the LIDAEUS (LIgand Discovery At Edinburgh UniverSity) program. The ligand-based screening was carried out using the EDULISS (EDinburgh University LIgand Selection System) program. The selection of potential inhibitors of dengue NS5 MTase was based on two criteria: the compounds must bind to NS5 MTase with a higher affinity than that of active NS5 MTase ligands, such as ribavirin triphosphate (RTP) and S-adenosyl-L-homocysteine (SAH); and the compounds must interact with residues that are catalytically important for the function of NS5 MTase. We found several compounds that bind strongly to the RNA cap site and the S-adenosyl-L-methionine (SAM) binding site of NS5 MTase with better binding affinities than that of RTP and SAH. We analyzed the mode of binding for each compound to its binding site, and our results suggest that all compounds bind to their respective binding sites by interacting with, and thus blocking, residues that are vital for maintaining the catalytic activity of NS5 MTase. We discovered several potential compounds that are active against dengue virus NS5 MTase through virtual screening using structure-based and ligand-based methods. These compounds were predicted to bind into the SAM binding site and the RNA cap site with higher affinities than SAH and RTP. These compounds are commercially available and can be purchased for further biological activity tests

Effect of modification of the physicochemical properties of ICAM-1-derived peptides on internalization and intracellular distribution in the human leukemic cell line HL-60

The objective of this work is to test the hypothesis that increasing the hydrophilicity of DOX−peptide conjugates may modify their entry mechanisms into HL-60 cells from passive diffusion to receptor-mediated uptake. To test this hypothesis, the entry mechanisms and the intracellular disposition of DOX−cIBR7, DOX−PEGcIBR7, FITC−cIBR, and FITC−cIBR7 were evaluated in HL-60 cells. To increase the hydrophilicity of the peptide, the cIBR peptide (cyclo(1,12)PenPRGGSVLVTGC) was modified to cIBR7 peptide (cyclo(1,8)CPRGGSVC) by removing the hydrophobic residues at the C-terminus. DOX−cIBR7 conjugate, which has higher hydrophilicity than DOX−cIBR, was synthesized. Second, a hydrophilic linker (11-amino-3,6,9-trioxaundecanate linker) was incorporated between DOX and cIBR7 to generate DOX−PEGcIBR7 with higher hydrophilicity than DOX−cIBR7. As controls, FITC−cIBR and FITC−cIBR7 were used to check for any endocytic uptake process of the peptide. As previously found with DOX−cIBR, DOX−cIBR7, and DOX−PEGcIBR7, conjugates enter the cells via passive diffusion and not via the energy-dependent endocytic process. This result suggests that an increase in hydrophilicity does not influence the entry mechanism of the DOX−peptide conjugates. In contrast to the DOX−cIBR7 conjugate, the FITC−cIBR7 conjugate showed energy-dependent cellular entry into the cells and followed an endocytic pathway similar to that for dextran. Finally, the entry of DOX−cIBR7 and DOX−PEGcIBR into the cell cytosol was shown to be due to the properties of DOX and not to those of the peptide

CPB1 of Aedes aegypti Interacts with DENV2 E Protein and Regulates Intracellular Viral Accumulation and Release from Midgut Cells

Aedes aegypti is a principal vector responsible for the transmission of dengue viruses (DENV). To date, vector control remains the key option for dengue disease management. To develop new vector control strategies, a more comprehensive understanding of the biological interactions between DENV and Ae. aegypti is required. In this study, a cDNA library derived from the midgut of female adult Ae. aegypti was used in yeast two-hybrid (Y2H) screenings against DENV2 envelope (E) protein. Among the many interacting proteins identified, carboxypeptidase B1 (CPB1) was selected, and its biological interaction with E protein in Ae. aegypti primary midgut cells was further validated. Our double immunofluorescent assay showed that CPB1-E interaction occurred in the endoplasmic reticulum (ER) of the Ae. aegypti primary midgut cells. Overexpression of CPB1 in mosquito cells resulted in intracellular DENV2 genomic RNA or virus particle accumulation, with a lower amount of virus release. Therefore, we postulated that in Ae. aegypti midgut cells, CPB1 binds to the E protein deposited on the ER intraluminal membranes and inhibits DENV2 RNA encapsulation, thus inhibiting budding from the ER, and may interfere with immature virus transportation to the trans-Golgi network

Discovery of Mycobacterium tuberculosis CYP121 new inhibitor via structure-based drug repurposing

Tuberculosis (TB) remains a serious threat to human health with the advent of multi-drug resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB). The urge to find novel drugs to deal with the appearance of drug-resistant TB and its variants is highly needed. This study aims to find new CYP121 inhibitors by screening 8,773 compounds from the drug repositioning database RepoDB. The selection of CYP121 potential inhibitors was based on two criteria: the new inhibitor should bind to CYP121 with higher affinity than its original ligand and interact with catalytically important residues for the function of CYP121. The ligands were docked onto CYP121 using AutoDock Vina, and the molecular dynamics simulation of the selected ligand was conducted using YASARA Structure. We found that antrafenine, an anti-inflammatory and analgesic agent with high CYP inhibitory promiscuity, was bound to CYP121 with a binding affinity of -12.6 kcal/mol and interacted with important residues at the CYP121 binding site. Molecular dynamics analysis of CYP121 bound to the original ligand and antrafenine showed that both ligands affected the dynamics of residues located distantly from the active site. Antrafenine caused more structural changes to CYP121 than the original ligand, as indicated by a significantly higher number of affected residues and rigid body movements caused by the binding of antrafenine to CYP121

Enantioselectivity investigation of short polar peptides with different positions in the Michael reaction

This work reports the effectiveness of short polar peptides as asymmetric catalysts in Michael reactions to attain good yields of enantio- and diastereoselective isomers. In a comparison, glutamic acid and histidine produced greater ee and yields when they were applied as the second amino acid in short trimeric peptides. These short polar peptides were found to be efficient organocatalysts for the asymmetric Michael addition reaction in water