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

Zinc plays a crucial role both in the immune system and endocrine processes. Zinc restriction in the diet has been shown to lead to degeneration of the endocrine pancreas, resulting in hormonal imbalance within the ß-cells. Proteostasismay vary depending on the stage of a pathophysiological process, which underscores the need for tools aimed at directly analyzing biological status. Among proteomics methods, MALDI-ToF-MS can serve as a rapid peptidomics tool for analyzing extracts or by histological imaging. Here we report the optimization of MALDI imaging mass spectrometry analysis of histological thin sections from mouse pancreas. This optimization enables the identification of the major islet peptide hormones as well as the 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 subjected to a zinc-restricted diet exhibited a relatively lower amount of peptide intermediates compared to the control group. These findings provide evidence for a complex modulation of proteostasis by micronutrients imbalance, a phenomenon directly accessed by MALDI-MSI.

Applications of Mass Spectrometry in the Characterization, Screening, Diagnosis, and Prognosis of COVID-19.

Mass spectrometry (MS) is a powerful analytical technique that plays a central role in modern protein analysis and the study of proteostasis. In the field of advanced molecular technologies, MS-based proteomics has become a cornerstone that is making a significant impact in the post-genomic era and as precision medicine moves from the research laboratory to clinical practice. The global dissemination of COVID-19 has spurred collective efforts to develop effective diagnostics, vaccines, and therapeutic interventions. This chapter highlights how MS seamlessly integrates with established methods such as RT-PCR and ELISA to improve viral identification and disease progression assessment. In particular, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) takes the center stage, unraveling intricate details of SARS-CoV-2 proteins, revealing modifications such as glycosylation, and providing insights critical to formulating therapies and assessing prognosis. However, high-throughput analysis of MALDI data presents challenges in manual interpretation, which has driven the development of programmatic pipelines and specialized packages such as MALDIquant. As we move forward, it becomes clear that integrating proteomic data with various omic findings is an effective strategy to gain a comprehensive understanding of the intricate biology of COVID-19 and ultimately develop targeted therapeutic paradigms.

Mutations in asparaginase II from E. coli and implications for inactivation and PEGylation.

All clinically-used asparaginases convert L-asparagine (L-Asn) to l-aspartate (L-Asp) and l-glutamine (L-Gln) to L-glutamate (L-Glu), which has been useful in reducing bioavailable asparagine and glutamine in patients under treatment for acute lymphoblastic leukemia. The E. coli type 2 L-asparaginase (EcA2) can present different sequences among varying bacterial strains, which we hypothesized that might affect their biological function, stability and interchangeability. Here we report the analysis of two EcA2 provided by the public health system of a middle-income country. These enzymes were reported to have similar specific activity in vitro, whereas they differ in vivo. Protein sequencing by LC-MS-MS and peptide mapping by MALDI-ToF-MS of their tryptic digests revealed that Aginasa™ share similar sequence to EcA2 from E. coli strain BL21(DE3), while Leuginase™ has sequence equivalent to EcA2 from E. coli strain AS1.357. The two amino acid differences between Aginasa™ (64D and 252 T) and Leuginase™ (64 N and 252S) resulted in structural divergences in solution as accessed by small-angle X-ray scattering and molecular dynamics simulation trajectories. The conformational variability further results in dissimilar surface accessibility with major consequences for PEGylation, as well as different susceptibility to degradation by limited proteolysis. The present results reveal that the sequence variations between these two EcA2 variants results in conformational changes associated with differential conformational plasticity, potentially affecting physico-chemical and biological properties, including proteolytic and immunogenic silent inactivation.

Recombinant Bacillus Calmette-Guérin Expressing SARS-CoV-2 Chimeric Protein Protects K18-hACE2 Mice against Viral Challenge.

COVID-19 has accounted for more than 6 million deaths worldwide. Bacillus Calmette-Guérin (BCG), the existing tuberculosis vaccine, is known to induce heterologous effects over other infections due to trained immunity and has been proposed to be a potential strategy against SARS-CoV-2 infection. In this report, we constructed a recombinant BCG (rBCG) expressing domains of the SARS-CoV-2 nucleocapsid and spike proteins (termed rBCG-ChD6), recognized as major candidates for vaccine development. We investigated whether rBCG-ChD6 immunization followed by a boost with the recombinant nucleocapsid and spike chimera (rChimera), together with alum, provided protection against SARS-CoV-2 infection in K18-hACE2 mice. A single dose of rBCG-ChD6 boosted with rChimera associated with alum elicited the highest anti-Chimera total IgG and IgG2c Ab titers with neutralizing activity against SARS-CoV-2 Wuhan strain when compared with control groups. Importantly, following SARS-CoV-2 challenge, this vaccination regimen induced IFN-γ and IL-6 production in spleen cells and reduced viral load in the lungs. In addition, no viable virus was detected in mice immunized with rBCG-ChD6 boosted with rChimera, which was associated with decreased lung pathology when compared with BCG WT-rChimera/alum or rChimera/alum control groups. Overall, our study demonstrates the potential of a prime-boost immunization system based on an rBCG expressing a chimeric protein derived from SARS-CoV-2 to protect mice against viral challenge.

Oral formulation of Wnt inhibitor complex reduces inflammation and fibrosis in intraperitoneal implants in vivo.

The use of implantable biomaterials to replace physiological and anatomical functions has been widely investigated in the clinic. However, the selection of biomaterials is crucial for long-term function, and the implantation of certain biomaterials can cause inflammatory and fibrotic processes, triggering a foreign body reaction that leads to loss of function and consequent need for removal. Specifically, the Wnt signaling pathway controls the healing process of the human body, and its dysregulation can result in inflammation and fibrosis, such as in peritoneal fibrosis. Here, we assessed the effects of daily oral administration of a Wnt pathway inhibitor complex (CD:LGK974) to reduce the inflammatory, fibrotic, and angiogenic processes caused by intraperitoneal implants. CD:LGK974 significantly reduced the infiltration of immune cells and release of inflammatory cytokines in the implant region compared to the control groups. Furthermore, CD:LGK974 inhibited collagen deposition and reduced the expression of pro-fibrotic α-SMA and TGF-ß1, confirming fibrosis reduction. Finally, the CD:LGK974 complex decreased VEGF levels and both the number and area of blood vessels formed, suggesting decreased angiogenesis. This work introduces a potential new application of the Wnt inhibitor complex to reduce peritoneal fibrosis and the rejection of implants at the intraperitoneal site, possibly allowing for longer-term functionality of existing clinical biomaterials.

Rad5 HIRAN domain: Structural insights into its interaction with ssDNA through molecular modeling approaches.

The Rad5 protein is an SWI/SNF family ubiquitin ligase that contains an N-terminal HIRAN domain and a RING C3HC4 motif. The HIRAN domain is critical for recognition of the stalled replication fork during the replication process and acts as a sensor to initiate the damaged DNA checkpoint. It is a conserved domain widely distributed in eukaryotic organisms and is present in several DNA-binding proteins from all kingdoms. Here we showed that distant species have important differences in key residues that affect affinity for ssDNA. Based on these findings, we hypothesized that different HIRAN domains might affect fork reversal and translesion synthesis through different metabolic processes. To address this question, we predicted the tertiary structure of both yeast and human HIRAN domains using molecular modeling. Structural dynamics experiments showed that the yeast HIRAN domain exhibited higher structural denaturation than its human homolog, although both domains became stable in the presence of ssDNA. Analysis of atomic contacts revealed that a greater number of interactions between the ssDNA nucleotides and the Rad5 domain are electrostatic. Taken together, these results provide new insights into the molecular mechanism of the HIRAN domain of Rad5 and may guide us to further elucidate differences in the ancient eukaryotes HIRAN sequences and their DNA affinity.Communicated by Ramaswamy H. Sarma.

Toxicity of spike fragments SARS-CoV-2 S protein for zebrafish: A tool to study its hazardous for human health?

Despite the significant increase in the generation of SARS-CoV-2 contaminated domestic and hospital wastewater, little is known about the ecotoxicological effects of the virus or its structural components in freshwater vertebrates. In this context, this study evaluated the deleterious effects caused by SARS-CoV-2 Spike protein on the health of Danio rerio, zebrafish. We demonstrated, for the first time, that zebrafish injected with fragment 16 to 165 (rSpike), which corresponds to the N-terminal portion of the protein, presented mortalities and adverse effects on liver, kidney, ovary and brain tissues. The conserved genetic homology between zebrafish and humans might be one of the reasons for the intense toxic effects followed inflammatory reaction from the immune system of zebrafish to rSpike which provoked damage to organs in a similar pattern as happen in severe cases of COVID-19 in humans, and, resulted in 78,6% of survival rate in female adults during the first seven days. The application of spike protein in zebrafish was highly toxic that is suitable for future studies to gather valuable information about ecotoxicological impacts, as well as vaccine responses and therapeutic approaches in human medicine. Therefore, besides representing an important tool to assess the harmful effects of SARS-CoV-2 in the aquatic environment, we present the zebrafish as an animal model for translational COVID-19 research.

Epimers l- and d-Phenylseptin: How the relative stereochemistry affects the peptide-membrane interactions.

In recent decades, several epimers of peptides containing d-amino acids have been identified in antimicrobial sequences, a feature which has been associated with post-translational modification. Generally, d-isomers present similar or inferior antimicrobial activity, only surpassing their epimers in resistance to peptidases. The naturally occurring l-Phenylseptin (l-Phes) and d-Phenylseptin (d-Phes) peptides (FFFDTLKNLAGKVIGALT-nh2) were reported with d-epimer showing higher activity against Staphylococcus aureus and Xanthomonas axonopodis in comparison with the l-epimer. In this study, we combine structural (CD, solution NMR), orientational (solid-state NMR) and biophysical (ITC, DSC and DLS) studies to understand the role of the d-phenylalanine in the increase of the antimicrobial activity. Although both peptides are structurally similar in the helical region ranging from D4 to the C-terminus, significant structural differences were observed near the peptides' N-termini (which encompasses the FFF motif). Specific aromatic interactions involving the phenylalanine side chains of d-Phes is responsible to maintaining the F1-F3 residues on the hydrophobic face of the peptide, increasing its amphipathicity when compared to the l-epimer. The higher capability of d-Phes to exert an efficient anchoring in the hydrophobic core of the phospholipid bilayer indicates a pivotal role of the N-terminus in enhancing the interaction between the d-peptide and the membrane interface in relation to its epimer.

High-resolution structural profile of hylaseptin-4: Aggregation, membrane topology and pH dependence of overall membrane binding process.

Hylaseptin-4 (HSP-4, GIGDILKNLAKAAGKAALHAVGESL-NH2) is an antimicrobial peptide originally isolated from Hypsiboas punctatus tree frog. The peptide has been chemically synthetized for structural investigations by CD and NMR spectroscopies. CD experiments reveal the high helical content of HSP-4 in biomimetic media. Interestingly, the aggregation process seems to occur at high peptide concentrations either in aqueous solution or in presence of biomimetic membranes, indicating an increase in the propensity of the peptide for adopting a helical conformation. High-resolution NMR structures determined in presence of DPC-d38 micelles show a highly ordered α-helix from amino acid residues I2 to S24 and a smooth bend near G14. A large separation between hydrophobic and hydrophilic residues occurs up to the A16 residue, from which a shift in the amphipathicity is noticed. Oriented solid-state NMR spectroscopy show a roughly parallel orientation of the helical structure along the POPC lipid bilayer surface, with an insertion of the hydrophobic N-terminus into the bilayer core. Moreover, a noticeable pH dependence of the aggregation process in both aqueous and in biomimetic membrane environments is attributed to a single histidine residue (H19). The protonation degree of the imidazole side-chain might help in modulating the peptide-peptide or peptide-lipid interactions. Finally, molecular dynamics simulations confirm the orientation and preferential helical conformation and in addition, show that HSP-4 tends to self-aggregate in order to stabilize its active conformation in aqueous or phospholipid bilayer environments.

Biophysical characterization of two commercially available preparations of the drug containing Escherichia coli L-Asparaginase 2.

The hydrolysis of asparagine and glutamine by L-asparaginase has been used to treat acute lymphoblastic leukemia for over four decades. Each L-asparaginase monomer has a long loop that closes over the active site upon substrate binding, acting as a lid. Here we present a comparative study of two commercially available preparations of the drug containing Escherichia coli L-Asparaginase 2 (EcA2), performed by a comprehensive array of biophysical and biochemical approaches. We report the oligomeric landscape and conformational and dynamic plasticity of E. coli type 2 L-asparaginase present in two different formulations, and its relationship with L-aspartic acid, which is present in Aginasa, but not in Leuginase. The L-Asp present in Aginasa formulation was found to provide to EcA2 a resistance to in vitro proteolysis. EcA2 shows a composition of monomers and oligomers up to tetramers, which is mostly not altered in the presence of L-Asp. Ion-mobility spectrometry-mass spectrometry reveals two conformers for the monomeric EcA2, and that monomeric species has sufficient capacity for selective binding to L-Asp and L-Glu. The N-terminal loop of the EcA2 present in Leuginase, which is part of the active site is disordered, but it gets ordered in the presence of L-Asp, while L-Glu only does so to a limited extent. These data provide new insights on the mechanistic of ligand recognition by EcA2, and the impact of formulation in its conformational diversity landscape.

Propedia: a database for protein-peptide identification based on a hybrid clustering algorithm.

BACKGROUND: Protein-peptide interactions play a fundamental role in a wide variety of biological processes, such as cell signaling, regulatory networks, immune responses, and enzyme inhibition. Peptides are characterized by low toxicity and small interface areas; therefore, they are good targets for therapeutic strategies, rational drug planning and protein inhibition. Approximately 10% of the ethical pharmaceutical market is protein/peptide-based. Furthermore, it is estimated that 40% of protein interactions are mediated by peptides. Despite the fast increase in the volume of biological data, particularly on sequences and structures, there remains a lack of broad and comprehensive protein-peptide databases and tools that allow the retrieval, characterization and understanding of protein-peptide recognition and consequently support peptide design. RESULTS: We introduce Propedia, a comprehensive and up-to-date database with a web interface that permits clustering, searching and visualizing of protein-peptide complexes according to varied criteria. Propedia comprises over 19,000 high-resolution structures from the Protein Data Bank including structural and sequence information from protein-peptide complexes. The main advantage of Propedia over other peptide databases is that it allows a more comprehensive analysis of similarity and redundancy. It was constructed based on a hybrid clustering algorithm that compares and groups peptides by sequences, interface structures and binding sites. Propedia is available through a graphical, user-friendly and functional interface where users can retrieve, and analyze complexes and download each search data set. We performed case studies and verified that the utility of Propedia scores to rank promissing interacting peptides. In a study involving predicting peptides to inhibit SARS-CoV-2 main protease, we showed that Propedia scores related to similarity between different peptide complexes with SARS-CoV-2 main protease are in agreement with molecular dynamics free energy calculation. CONCLUSIONS: Propedia is a database and tool to support structure-based rational design of peptides for special purposes. Protein-peptide interactions can be useful to predict, classifying and scoring complexes or for designing new molecules as well. Propedia is up-to-date as a ready-to-use webserver with a friendly and resourceful interface and is available at: https://bioinfo.dcc.ufmg.br/propedia.

S. mansoni SmKI-1 Kunitz-domain: Leucine point mutation at P1 site generates enhanced neutrophil elastase inhibitory activity.

The Schistosoma mansoni SmKI-1 protein is composed of two domains: a Kunitz-type serine protease inhibitor motif (KD) and a C-terminus domain with no similarity outside the genera. Our previous work has demonstrated that KD plays an essential role in neutrophil elastase (NE) binding blockage, in neutrophil influx and as a potential anti-inflammatory molecule. In order to enhance NE blocking capacity, we analyzed the KD sequence from a structure-function point of view and designed specific point mutations in order to enhance NE affinity. We substituted the P1 site residue at the reactive site for a leucine (termed RL-KD), given its central role for KD's inhibition to NE. We have also substituted a glutamic acid that strongly interacts with the P1 residue for an alanine, to help KD to be buried on NE S1 site (termed EA-KD). KD and the mutant proteins were evaluated in silico by molecular docking to human NE, expressed in Escherichia coli and tested towards its NE inhibitory activity. Both mutated proteins presented enhanced NE inhibitory activity in vitro and RL-KD presented the best performance. We further tested RL-KD in vivo in an experimental model of monosodium urate (MSU)-induced acute arthritis. RL-KD showed reduced numbers of total cells and neutrophils in the mouse knee cavity when compared to KD. Nevertheless, both RL-KD and KD reduced mice hypernociception in a similar fashion. In summary, our results demonstrated that both mutated proteins showed enhanced NE inhibitory activity in vitro. However, RL-KD had a prominent effect in diminishing inflammatory parameters in vivo.

Immunoinformatic Analysis of SARS-CoV-2 Nucleocapsid Protein and Identification of COVID-19 Vaccine Targets.

COVID-19 is a worldwide emergency; therefore, there is a critical need for foundational knowledge about B and T cell responses to SARS-CoV-2 essential for vaccine development. However, little information is available defining which determinants of SARS-CoV-2 other than the spike glycoprotein are recognized by the host immune system. In this study, we focus on the SARS-CoV-2 nucleocapsid protein as a suitable candidate target for vaccine formulations. Major B and T cell epitopes of the SARS-CoV-2 N protein are predicted and resulting sequences compared with the homolog immunological domains of other coronaviruses that infect human beings. The most dominant of B cell epitope is located between 176-206 amino acids in the SRGGSQASSRSSSRSRNSSRNSTPGSSRGTS sequence. Further, we identify sequences which are predicted to bind multiple common MHC I and MHC II alleles. Most notably there is a region of potential T cell cross-reactivity within the SARS-CoV-2 N protein position 102-110 amino acids that traverses multiple human alpha and betacoronaviruses. Vaccination strategies designed to target these conserved epitope regions could generate immune responses that are cross-reactive across human coronaviruses, with potential to protect or modulate disease. Finally, these predictions can facilitate effective vaccine design against this high priority virus.

Vaccines for COVID-19: perspectives from nucleic acid vaccines to BCG as delivery vector system.

This article discusses standard and new disruptive strategies in the race to develop an anti-COVID-19 vaccine. We also included new bioinformatic data from our group mapping immunodominant epitopes and structural analysis of the spike protein. Another innovative approach reviewed here is the use of BCG vaccine as priming strategy and/or delivery system expressing SARS-CoV-2 antigens.

Structural and immunological characterization of a new nucleotidyltransferase-like antigen from Paracoccidioides brasiliensis.

Pb27 antigen is an interesting alternative to immunological diagnosis of Paracoccidioidomycosis (PCM) and has demonstrated to be protective in experimental PCM. Its tertiary structure and possible function remained unknown till now. To study Pb27 at the atomic level, the recombinant protein was expressed in Escherichia coli BL21(DE3), purified, and its three-dimensional structure was solved by X-ray crystallography. Based on this structure, we performed a residue correlation analysis and in silico ligand search assays to address a possible biological function to Pb27. We identified Pb27 as a member of the extensive nucleotidyltransferase superfamily. The protein has an αßαßαß topology with two domains (N- and C-terminal domains) and adopts a monomeric form as its biological unit in solution. Structural comparisons with similar members of the superfamily clearly indicate Pb27 C-terminal domain is singular and may play an important role in its biological function. Bioinformatics analysis suggested that Pb27 might bind to ATP and CTP. This suggestion is corroborated by the fact that a magnesium cation is coordinated by two aspartic acid residues present at the active site (between N- and C-terminal domains), as evidenced by X-ray diffraction data. Besides, NMR assays (1H-15N HSQC spectra) confirmed the binding of CTP to Pb27, demonstrating for the first time an interaction between a nucleotide and this protein. Moreover, we evaluated the reactivity of sera from patients with Paracoccidioides brasiliensis infection against the recombinant form of Pb27 and showed that it was recognized by sera from infected and treated patients. Predicted B and T cell epitopes were synthesized and further evaluated against sera of PCM patients, providing information of the most reactive peptides in Pb27 primary structure which interact with specific Pb27 antibodies.

Oligomeric transition and dynamics of RNA binding by the HuR RRM1 domain in solution.

Human antigen R (HuR) functions as a major post-transcriptional regulator of gene expression through its RNA-binding activity. HuR is composed by three RNA recognition motifs, namely RRM1, RRM2, and RRM3. The two N-terminal RRM domains are disposed in tandem and contribute mostly to HuR interaction with adenine and uracil-rich elements (ARE) in mRNA. Here, we used a combination of NMR and electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) to characterize the structure, dynamics, RNA recognition, and dimerization of HuR RRM1. Our solution structure reveals a canonical RRM fold containing a 19-residue, intrinsically disordered N-terminal extension, which is not involved in RNA binding. NMR titration results confirm the primary RNA-binding site to the two central ß-strands, ß1 and ß3, for a cyclooxygenase 2 (Cox2) ARE I-derived, 7-nucleotide RNA ligand. We show by 15N relaxation that, in addition to the N- and C-termini, the ß2-ß3 loop undergoes fast backbone dynamics (ps-ns) both in the free and RNA-bound state, indicating that no structural ordering happens upon RNA interaction. ESI-IMS-MS reveals that HuR RRM1 dimerizes, however dimer population represents a minority. Dimerization occurs via the α-helical surface, which is oppositely orientated to the RNA-binding ß-sheet. By using a DNA analog of the Cox2 ARE I, we show that DNA binding stabilizes HuR RRM1 monomer and shifts the monomer-dimer equilibrium toward the monomeric species. Altogether, our results deepen the current understanding of the mechanism of RNA recognition employed by HuR.

Schistosoma mansoni SmKI-1 serine protease inhibitor binds to elastase and impairs neutrophil function and inflammation.

Protease inhibitors have important function during homeostasis, inflammation and tissue injury. In this study, we described the role of Schistosoma mansoni SmKI-1 serine protease inhibitor in parasite development and as a molecule capable of regulating different models of inflammatory diseases. First, we determine that recombinant (r) SmKI-1 and its Kunitz domain but not the C-terminal region possess inhibitory activity against trypsin and neutrophil elastase (NE). To better understand the molecular basis of NE inhibition by SmKI-1, molecular docking studies were also conducted. Docking results suggest a complete blockage of NE active site by SmKI-1 Kunitz domain. Additionally, rSmKI-1 markedly inhibited the capacity of NE to kill schistosomes. In order to further investigate the role of SmKI-1 in the parasite, we designed specific siRNA to knockdown SmKI-1 in S. mansoni. SmKI-1 gene suppression in larval stage of S. mansoni robustly impact in parasite development in vitro and in vivo. To determine the ability of SmKI-1 to interfere with neutrophil migration and function, we tested SmKI-1 anti-inflammatory potential in different murine models of inflammatory diseases. Treatment with SmKI-1 rescued acetaminophen (APAP)-mediated liver damage, with a significant reduction in both neutrophil recruitment and elastase activity. In the model of gout arthritis, this protein reduced neutrophil accumulation, IL-1ß secretion, hypernociception, and overall pathological score. Finally, we demonstrated the ability of SmKI-1 to inhibit early events that trigger neutrophil recruitment in pleural cavities of mice in response to carrageenan. In conclusion, SmKI-1 is a key protein in S. mansoni survival and it has the ability to inhibit neutrophil function as a promising therapeutic molecule against inflammatory diseases.

Serine protease inhibitors containing a Kunitz domain: their role in modulation of host inflammatory responses and parasite survival.

Proteins containing a Kunitz domain have the typical serine protease inhibition function ranging from sea anemone to man. Protease inhibitors play major roles in infection, inflammation disorders and cancer. This review discusses the role of serine proteases containing a Kunitz domain in immunomodulation induced by helminth parasites. Helminth parasites are associated with protection from inflammatory conditions. Therefore, interest has raised whether worm parasites or their products hold potential as drugs for treatment of immunological disorders. Finally, we also propose the use of recombinant SmKI-1 from Schistosoma mansoni as a potential therapeutic molecule to treat inflammatory diseases.

Structure and membrane interactions of the homodimeric antibiotic peptide homotarsinin.

Antimicrobial peptides (AMPs) from amphibian skin are valuable template structures to find new treatments against bacterial infections. This work describes for the first time the structure and membrane interactions of a homodimeric AMP. Homotarsinin, which was found in Phyllomedusa tarsius anurans, consists of two identical cystine-linked polypeptide chains each of 24 amino acid residues. The high-resolution structures of the monomeric and dimeric peptides were determined in aqueous buffers. The dimer exhibits a tightly packed coiled coil three-dimensional structure, keeping the hydrophobic residues screened from the aqueous environment. An overall cationic surface of the dimer assures enhanced interactions with negatively charged membranes. An extensive set of biophysical data allowed us to establish structure-function correlations with antimicrobial assays against Gram-positive and Gram-negative bacteria. Although both peptides present considerable antimicrobial activity, the dimer is significantly more effective in both antibacterial and membrane biophysical assays.

Allosteric Transmission along a Loosely Structured Backbone Allows a Cardiac Troponin C Mutant to Function with Only One Ca2+ Ion.

Hypertrophic cardiomyopathy (HCM) is one of the most common cardiomyopathies and a major cause of sudden death in young athletes. The Ca2+ sensor of the sarcomere, cardiac troponin C (cTnC), plays an important role in regulating muscle contraction. Although several cardiomyopathy-causing mutations have been identified in cTnC, the limited information about their structural defects has been mapped to the HCM phenotype. Here, we used high-resolution electron-spray ionization mass spectrometry (ESI-MS), Carr-Purcell-Meiboom-Gill relaxation dispersion (CPMG-RD), and affinity measurements of cTnC for the thin filament in reconstituted papillary muscles to provide evidence of an allosteric mechanism in mutant cTnC that may play a role to the HCM phenotype. We showed that the D145E mutation leads to altered dynamics on a µs-ms time scale and deactivates both of the divalent cation-binding sites of the cTnC C-domain. CPMG-RD captured a low populated protein-folding conformation triggered by the Glu-145 replacement of Asp. Paradoxically, although D145E C-domain was unable to bind Ca2+, these changes along its backbone allowed it to attach more firmly to thin filaments than the wild-type isoform, providing evidence for an allosteric response of the Ca2+-binding site II in the N-domain. Our findings explain how the effects of an HCM mutation in the C-domain reflect up into the N-domain to cause an increase of Ca2+ affinity in site II, thus opening up new insights into the HCM phenotype.