BEGIN:VCALENDAR VERSION:2.0 PRODID:-//CERN//INDICO//EN BEGIN:VEVENT SUMMARY:Regulation of actin-binding proteins mediated by lipid-protein int eractions DTSTART;VALUE=DATE-TIME:20260326T130500Z DTEND;VALUE=DATE-TIME:20260326T132500Z DTSTAMP;VALUE=DATE-TIME:20260425T214620Z UID:indico-contribution-804-10345@events.saip.org.za DESCRIPTION:Speakers: Yosuke Senju ()\nThe actin cytoskeleton drives membr ane deformation during many cellular processes\, such as migration\, morph ogenesis\, and endocytosis. Phosphatidylinositol 4\,5-bisphosphate [PI(4\, 5)P2]\, one of the phosphoinositides\, regulates the activities of many ac tin-binding proteins (ABPs)\, including profilin\, cofilin\, Dia2\, N-WASP \, ezrin\, and moesin\; however\, the underlying molecular mechanisms rema in elusive. Here\, we applied a combination of biophysical assays and atom istic molecular dynamics simulations to uncover the molecular principles u nderlying ABP interactions with phosphoinositide-containing membranes. Our results reveal that these proteins show significant differences in membra ne interaction dynamics and in the ranges of phosphoinositide densities th ey can sense. Profilin and cofilin show transient\, low-affinity interacti ons with membranes\, whereas F-actin assembly factors Dia2 and N-WASP stay on membranes longer to perform their functions. Ezrin and moesin\, which link the actin cytoskeleton to the plasma membrane\, bind to membranes wit h high affinity and slow dissociation kinetics\, regulating PI(4\,5)P2 lat eral diffusion. Unlike profilin\, cofilin\, Dia2\, and N-WASP\, they do no t require a high ‘stimulus-responsive’ phosphoinositide density for me mbrane binding. Together\, these findings demonstrate that the membrane-in teraction mechanisms of ABPs have evolved to precisely fulfill their speci fic cellular functions in cytoskeletal dynamics.\n\nhttps://events.saip.or g.za/event/272/contributions/10345/ LOCATION: URL:https://events.saip.org.za/event/272/contributions/10345/ END:VEVENT BEGIN:VEVENT SUMMARY:Linker switch mutations between canonical Plasmodium falciparum Hs p70-1 and PfHsp70-z reveal the role of this motif in regulating the functi onal specialisation of the two chaperones DTSTART;VALUE=DATE-TIME:20260326T104000Z DTEND;VALUE=DATE-TIME:20260326T110000Z DTSTAMP;VALUE=DATE-TIME:20260425T214620Z UID:indico-contribution-804-10336@events.saip.org.za DESCRIPTION:Speakers: Addmore Shonhai (University of Venda)\nOf the five m alaria-causing species\, Plasmodium falciparum accounts for most malaria-r elated deaths. Central to the survival and infectivity of the parasite is rapid replication coupled to upregulated protein production. Thus\, the de velopment of this malaria parasite is supported by the role of several hea t shock proteins (Hsps)\, which facilitate protein folding. P. falciparum Hsp70-1 (PfHsp70-1) and PfHsp70-z are essential molecular chaperones (mole cules that assist proteins to fold correctly) that are cytosol-localized. PfHsp70-z belongs to the Hsp110 cluster of Hsp70-like proteins. Whereas P fHsp70-1 serves as a refolding chaperone\, PfHsp70-z is restricted to prev enting aggregation of proteins in the cell. The structural features underp inning the functional specialization of these chaperones remain elusive. P fHsp70-z possesses a unique linker segment. In the current study\, we expl ored the role of the linker in regulating the functional specialization of the two P. falciparum Hsp70s. Using recombinant forms of PfHsp70-1\, PfHs p70-z\, and E. coli Hsp70 (DnaK) as well as their linker switch mutant for ms\, we explored the effects of the linker mutations using circular dichro ism\, intrinsic and extrinsic fluorescence coupled to biochemical and in c ellulo analyses. Our findings demonstrate that the linker of PfHsp70-z mod ulates global conformation of the chaperone\, regulating several functions such as client protein binding\, chaperone\, and ATPase activities. In ad dition\, as opposed to the flexible linker of PfHsp70-1\, the PfHsp70-z li nker is rigid\, conferring notable conformational stability to this chaper one\, making it an effective holdase chaperone. Our findings highlight the role of the linker in regulating the functional specificity of Hsp70. We discuss the implications of our findings to the development of the malaria parasite at the blood stages of the parasite.\n\nhttps://events.saip.org. za/event/272/contributions/10336/ LOCATION: URL:https://events.saip.org.za/event/272/contributions/10336/ END:VEVENT BEGIN:VEVENT SUMMARY:Production of Leishmania spp. ∆24-sterol methyltransferases for the development of antiparasitic therapies DTSTART;VALUE=DATE-TIME:20260326T124500Z DTEND;VALUE=DATE-TIME:20260326T130500Z DTSTAMP;VALUE=DATE-TIME:20260425T214620Z UID:indico-contribution-804-10325@events.saip.org.za DESCRIPTION:Speakers: Bernadette Belter ()\nParasitic diseases\, such as l eishmaniasis\, pose a growing global health threat\, particularly in regio ns with high HIV/AIDS prevalence [1]. The limited efficacy and growing res istance to current treatments necessitate the discovery of novel therapeut ic targets. ∆24-sterolmethyltransferase (SMT) is an attractive target as an essential enzyme in the ergosterol biosynthetic pathway of protozoa an d has no mammalian homologue [2]. SMT catalyses the transfer of a methyl g roup from S-adenosyl-methionine to the C24 position of zymosterol or lanos terol [3]. Structural characterisation of SMTs via X-ray crystallography i s crucial for structure-based drug discovery approaches. This study aims t o produce and structurally characterise SMTs from *Leishmania* *donovani* and *Leishmania major* to identify lead compounds for antiparasitic therap ies.\nThe *ERG6* genes\, encoding SMT\, from *L. donovani* and *L. major* were expressed in *E. coli*\, but resulted in insoluble protein. We then f used ERG6 to genes encoding mCherry or SUMO and successfully expressed the fusions as soluble proteins. The SMTs were cleaved from the fusion partne rs and purified using column chromatography methods. Furthermore\, the p urified SMTs have been shown to methylate lanosterol using GC-MS. Biotrans formation optimisation is underway to improve conversion yields.\nCrystall isation trials using the vapour-diffusion sitting-drop method are currentl y ongoing. Future work includes collecting X-ray diffraction data of the S MT crystals to solve their three-dimensional structures. The structural in formation\, as well as a robust crystal system\, is a prerequisite for X-r ay crystallographic fragment screening\, which will be used to map SMT bin ding pockets for inhibitor design. \nReferences\n[1] Dangarembizi\, R.\, W asserman\, S.\, Hoving\, J. C.\, et al. Parasite Immunology\, (2023) e1295 3. \n[2] Sakyi\, P. O.\, Kwofie\, S. K.\, Tuekpe\, J. K.\, et al. Pharmace uticals\, (2023) 16(3)\, 330.\n[3] Nes\, W.D.\, Chaudhuri\, M.\, Leaver\, D.J. Biomolecules\, (2024) 14:249.\n\nhttps://events.saip.org.za/event/272 /contributions/10325/ LOCATION: URL:https://events.saip.org.za/event/272/contributions/10325/ END:VEVENT BEGIN:VEVENT SUMMARY:Objective clustering algorithm applied to single-molecule FCP data DTSTART;VALUE=DATE-TIME:20260326T102000Z DTEND;VALUE=DATE-TIME:20260326T104000Z DTSTAMP;VALUE=DATE-TIME:20260425T214620Z UID:indico-contribution-804-10323@events.saip.org.za DESCRIPTION:Speakers: Michael Lovemore (Univeristy of the Witwatersrand)\n Bulk spectroscopic measurements of photosynthetic light-harvesting complex es report ensemble-averaged properties that often obscure the heterogeneit y and dynamic behaviour present at the level of individual complexes. Sing le-molecule fluorescence spectroscopy provides access to this hidden compl exity through measurements of fluorescence intensity and lifetime\; howeve r\, interpreting raw intensity–lifetime distributions can be challenging because broad\, overlapping populations frequently appear visually as onl y one or two states. Fucoxanthin chlorophyll protein (FCP) is the major li ght-harvesting complex of diatoms and contains Lhcx subunits that are impl icated in photoprotection under high-light conditions. We investigated the fluorescence dynamics of FCP complexes under different environmental cond itions using single-molecule intensity and lifetime measurements\, perform ing a comparative\, pH-dependent study of two FCP types to examine how Lhc x modulates the photoprotective behaviour. To objectively extract the unde rlying emissive states\, Gaussian mixture model (GMM) clustering was appli ed to the intensity–lifetime distributions. The optimal number of cluste rs was objectively determined using information criteria (AIC\, BIC\, ICL) and cluster-quality metrics to ensure statistical robustness. This approa ch revealed multiple emissive states beyond the simple two-state (quenched /unquenched) interpretation suggested by visual inspection\, enabling dire ct comparison across datasets and highlighting how environmental condition s and Lhcx content influence the accessibility and stability of the variou s photophysical states within FCP.\n\nhttps://events.saip.org.za/event/272 /contributions/10323/ LOCATION: URL:https://events.saip.org.za/event/272/contributions/10323/ END:VEVENT BEGIN:VEVENT SUMMARY:Multi-timescale fluorescence correlation spectroscopy of the main plant light-harvesting complex during aggregation stages DTSTART;VALUE=DATE-TIME:20260326T100000Z DTEND;VALUE=DATE-TIME:20260326T102000Z DTSTAMP;VALUE=DATE-TIME:20260425T214620Z UID:indico-contribution-804-10322@events.saip.org.za DESCRIPTION:Speakers: Francois Conradie ()\nLight-Harvesting Complex II (L HCII) is the most abundant photosynthetic membrane pigment-protein complex in higher plants\, enabling extremely efficient solar energy harvesting. Their light-harvesting function is finely controlled by processes that swi tch the complexes into a photoprotective state. LHCII aggregates\, that fe ature strong quenching of excitation light\, are often believed to be enha nce a plant’s photoprotective capability. Finding new ways to study thes e aggregates can lead to important insights into the fundamental mechanism s by which plants protect themselves against high sunlight intensities\, w hich could\, for example\, inform efforts to improve crop yields in a chan ging climate. In our project\, we have developed techniques that combine f luorescence correlation spectroscopy (FCS) and time-correlated single-phot on counting (TCSPC). The aggregation of LHCII was investigated at increasi ng levels by step-wise removal of detergent from low-concentration purifie d samples. Applying pulse-interleaved excitation (PIE) enabled advanced FC S to accurately measure translational and estimated rotational diffusion c oefficients\, yielding the hydrodynamic radii of LHCII during aggregation. Furthermore\, extending measurement times and using both pulsed and conti nuous excitation unveiled photophysics from microseconds down to picosecon d timescales. The results show a rich combination of dimensional informati on and excitation dynamics in these photosynthetic aggregates\, highlighti ng the importance of singlet-triplet annihilation when studying quenching in LHCII.\n\nhttps://events.saip.org.za/event/272/contributions/10322/ LOCATION: URL:https://events.saip.org.za/event/272/contributions/10322/ END:VEVENT END:VCALENDAR