Biology subjects

Lima, L. M. T.

Publications and source records attributed to Lima, L. M. T..

7 recordsLinked to original sources

bioRxiv2025

Pinpointing protein structures over broad temperature range using hydrophobic protection

Protein interactions are dynamic processes influenced by chemical and physical variables. While variable temperatures play an important role in understanding biological processes, their use in protein crystallography has been limited due to the loss of diffraction power. Here, we report the selection of a cryo-protectant that enables X-ray diffraction data collection across a continuum of temperatures, from cryogenic to room temperature. Although several hydrophobic materials effectively preserved samples at 100 K, only a few compounds allowed data collection at 300 K. We identified a hydrophobic grease suitable for both home-source and synchrotron applications, supporting ultrafast and slow diffraction, with exposure times as long as 60 seconds per image at atomic resolution. Data collected between 100 K and 300 K showed no significant effect of exposure time (ranging from 70 msec to 1 second) but revealed an exponential temperature dependence on the overall B-factor. These findings highlight the importance of hydrophobic grease in protecting against environmental variables, enabling optimized data collection across an ultra-wide temperature range. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/662548v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@102a42eorg.highwire.dtl.DTLVardef@68facaorg.highwire.dtl.DTLVardef@2905d5org.highwire.dtl.DTLVardef@13e219b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Amyloid Folding: an Origami-Based Approach

Amyloid is an ordered folding pattern that involves a cross-beta arrangement, with hydrogen-bonding between adjacent chains in the fiber elongation axis, and side-chain interactions perpendicular to it, forming steric and/or polar zippers intra- and/or inter-chains. The arrangement of the polypeptide backbone and the side-chain interactions can be arranged with different morphologies that are repeated along the fiber growth axis as stacking units. A simple representation of amyloid folding of proteins using paper origami is reported here, which can be used to visualize the {beta}-sheet along fiber axis and the perpendicular variability in topological arrangement. The model uses regular office paper, in the form of multiple linearized polypeptide chains, arranged in parallel or anti-parallel forms, connected by hydrogen bonds. Alternating mountain/valley creases along the growth axis of the fibrils and, over the front/back side-chains, results in a pleated sheet that can be used to study the topological arrangement of amyloid fibrils and the polar/steric zippers. The present models can be used as teaching tools for understanding the structural and molecular basis of amyloid folding, chain growth, homo- and cross-seeding, and other features of amyloid function in health, disease and biotechnology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/659339v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@888d0eorg.highwire.dtl.DTLVardef@e4b027org.highwire.dtl.DTLVardef@1e8ff0forg.highwire.dtl.DTLVardef@1c160ed_HPS_FORMAT_FIGEXP M_FIG C_FIG Key featuresO_LIIntroduces a physical origami model of amyloid {beta}-sheet structure using paper folding. C_LIO_LIDemonstrates how hydrogen bonding and side-chain interactions can be conceptualized in 3D. C_LIO_LIModels morphological polymorphism and parallel/antiparallel alignment. C_LIO_LIUseful for teaching complex biophysical concepts like cross-seeding, fibril growth, and molecular stability in amyloids. C_LIO_LIHighlights the educational value for biochemistry, structural biology, biomedical and material sciences. C_LI

biophysics↗

bioRxiv2025

Dietary zinc restriction induces irreversible allodynia in adult mice

IntroductionNeuropathic pain is a debilitating condition highly prevalent worldwide. One of the main factors related to its development is poor nutrition and micronutrient deficiency in the diet. Essential metals, especially divalent ions as zinc, play crucial roles in the neurobiology of the nervous system, including pain signaling and transmission mechanisms, and metabolism control. The present study aimed to understand the impact of zinc in neuropathic pain, evaluating whether restriction and direct repletion of a dietary zinc could change nociceptive behavior and metabolic parameters in adult mice. MethodsAdult male Swiss mice received a zinc-restricted diet for eight weeks. The repletion group received the restricted diet for four weeks followed by normal zinc diet for another four weeks. Mechanical and heat thermal pain sensitivity were assessed using the von Frey filaments and Hargreaves tests, respectively. ResultsZinc restriction resulted in decreased body weight gain and led to an increased mechanical and thermal sensitivity to heat. Dietary zinc repletion reversed the thermal allodynia and increased weight gain, abdominal adipose tissue, and liver weight compared to a normal zinc diet. No changes were observed concerning food and water intake, glycemic profile, pancreatic morphology and plasma amylin, ConclusionsThe reduction in the bioavailability of dietary zinc promotes metabolic and nociceptive changes in adult mice, inducing allodynia characteristic of neuropathic pain.

biochemistry↗

bioRxiv2025

Temperature-Resolved Crystallography Reveals Rigid-Body Dominance Over Local Flexibility in B-Factors

The crystallographic B-factor (Bf), also known as the Debye-Waller factor (DWF) or temperature factor, relates to the mean square displacement of atoms (X2). The X2 may be composed of individual contributions from lattice disorder (LT), static conformational heterogeneity (H) throughout the lattice, rigid body vibration (RB), local conformational vibration (V), and zero-point atomic fluctuation (A). The Bf has been widely employed as a surrogate measure of local protein flexibility, although such relation has not been confirmed. In addition, reproducibility of the absolute B-factor is difficult to achieve, hampering the understanding of their individual contribution. Here we report the crystallographic investigation of the enzyme-ligand complex of trypsin with benzamidine from cryo to room temperature, through a 200 K range (9-point triplicate design), by crystal stabilization with hydrophobic grease. The extent of temperature-induced conformational changes showed no connection with their respective B-factors. The B-factor variation due to temperature was constant for all atoms of the system, of about 0.005 K-1. The results caution against interpreting absolute, normalized or zero-point B-factors as direct proxies for protein dynamics, which is further supported by structural analysis of data from independent groups with trypsin-benzamidine complexes obtained under dissimilar experimental conditions. The similar thermal dependence of B-factor for all atoms of the system suggests a major contribution of this physical variable over uniform rigid body vibration.

biophysics↗

bioRxiv2023

Oral delivery of the amylin receptor agonist pramlintide

AimThe aims of this study were to design and characterize polymeric microparticles for oral delivery of pramlintide, a triple proline human amylin analogue clinically proved efficient in diabetes therapy. MethodsThe microparticles were prepared with gastric-resistant polymer Eudragit S100 by double-emulsion and solvent evaporation technique. The study responses were repeatability, encapsulation efficiency, yield, morphology, particle size, response to acidic and alkaline milieu and pharmacokinetics. ResultsWe obtained spherical microcapsules, with particle size of 66 m {+/-} 11, with 83.2 % {+/-} 2.7 efficiency for pramlintide entrapment and 67.6 % {+/-} 2.1 yield. Intra-venous pramlintide free in solution showed a plasmatic half-life of 6.8 min in mice. In contrast, oral delivery of acid-resistant pramlintide-loaded microparticles in mice showed a protracted release for 120 min compared to 30 min obtained for pramlintide in solution. ConclusionsOral route is an alternative for therapeutic development of pramlintide formulations. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/566408v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@d75002org.highwire.dtl.DTLVardef@1c5e914org.highwire.dtl.DTLVardef@492823org.highwire.dtl.DTLVardef@ea171b_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2023

Spatially resolved distribution of pancreatic hormones proteoforms by MALDI-imaging mass spectrometry

Zinc plays crucial role in the immune system and endocrine processes. Dietary zinc restriction leads to the degeneration of the endocrine pancreas resulting in hormonal imbalance in {beta}-cell. Proteostasis may vary depending on the stage of a pathophysiological process, motivating the development of tools aimed at the direct analysis of biological status. Among proteomics methods, MALDI-ToF-MS can serve as a rapid peptidomics tool from analysis of extracts or by histological imaging. Here we report the optimization of MALDI imaging mass spectrometry analysis of thin histological sections from mouse pancreas, allowing the identification of major islet peptide hormones and major accumulated precursors and/or proteolytic products of peptide hormones. Cross-validation of the identified peptide hormones was performed by LC-ESI-MS from pancreatic islet extracts. Mice fed a zinc-restricted diet had a relatively lower amount of peptide intermediates in comparison with the control group. These data provide evidence for complex modulation of proteostasis by an imbalance of micronutrients, directly accessed by MALDI-MSI.

biochemistry↗

bioRxiv2019

Zinc restriction promotes β-cell hyper-hormonemia and endocrine pancreas degeneration in mice

Zinc is a key component of proteins, including interaction with varying pancreatic hormones, including insulin and amylin. Zinc is key in insulin crystallinity in ZnT8 knock-out mice models, although the dietary role of zinc restriction over both energetic metabolism and {beta}-pancreatic hormonemia and morphology remained unexplored. We aimed to test whether dietary zinc restriction on swiss male mice would impact over endocrine pancreas and metabolic phenotype. We evaluated the role of dietary zinc restriction on {beta}-pancreatic hormonemia on non-transgenic Swiss male mice weaned onto a control or low-zinc diet for 4 weeks. Growth, glycemia, insulinemia, amylinemia and pancreatic islet were smaller in intervention group despite insulin crystallinity in secretory granules. We have found overlabelling for insulin, amylin and toxic oligomers in apoptotic pancreatic islet. High production of {beta}-pancreatic hormones in zinc-restricted animals counteract the decreasing islet size due to their apoptotic cells. We conclude that zinc deficiency is sufficient to promote islet {beta}-cell hormonal disruption and degeneration.

biochemistry↗