Structure-Based Rational Design of Small α-Helical Peptides with Broad-Spectrum Activity against Multidrug-Resistant Pathogens

A series of small (7–12 mer) amphipathic cationic peptides were designed and synthesized to create short helical peptides with broad-range bactericidal activity and selectivity toward the bacterial cells. The analysis identified a lead 12-mer peptide 8b with broad-spectrum activity against Gram-positive (MIC = 3.1–6.2 μg/mL) and Gram-negative (MIC = 6.2–12.5 μg/mL) bacteria and selectivity toward prokaryotic versus eukaryotic cells (HC50 = 280 μg/mL, \u3e75% cell viability at 150 μg/mL). The rapid membranolytic action of 8b was demonstrated by a calcein dye leakage assay and confirmed using scanning electron microscopy. According to circular dichroism and NMR spectroscopy, the peptides have an irregular spatial structure in water. A lipid bilayer induced an amphipathic helix only in 12-mer peptides, including 8b. Molecular dynamics simulations provided detailed information about the interaction of 8b and its closest analogues with bacterial and mammalian membranes and revealed the roles of particular amino acids in the activity and selectivity of peptides

Nuclear Magnetic Resonance Solution Structure and Functional Behavior of the Human Proton Channel

The human voltage-gated proton channel [Hv1(1) or VSDO(2)] plays an important role in the human innate immune system. Its structure differs considerably from those of other cation channels. It is built solely of a voltage-sensing domain and thus lacks the central pore domain, which is essential for other cation channels. Here, we determined the solution structure of an N- and C-terminally truncated human Hv1 (Δ-Hv1) in the resting state by nuclear magnetic resonance (NMR) spectroscopy. Δ-Hv1 comprises the typical voltage-sensing antiparallel four-helix bundle (S1–S4) preceded by an amphipathic helix (S0). The solution structure corresponds to an intermediate state between resting and activated forms of voltage-sensing domains. Furthermore, Zn2+-induced closing of proton channel Δ-Hv1 was studied with two-dimensional NMR spectroscopy, which showed that characteristic large scale dynamics of open Δ-Hv1 are absent in the closed state of the channel. Additionally, pH titration studies demonstrated that a higher H+ concentration is required for the protonation of side chains in the Zn2+-induced closed state than in the open state. These observations demonstrate both structural and dynamical changes involved in the process of voltage gating of the Hv1 channel and, in the future, may help to explain the unique properties of unidirectional conductance and the exceptional ion selectivity of the channel

Preparation and Characterization of Stable α-Synuclein Lipoprotein Particles

Multiple neurodegenerative diseases are caused by the aggregation of the human α-Synuclein (α-Syn(6)) protein. α-Syn possesses high structural plasticity and the capability of interacting with membranes. Both features are not only essential for its physiological function but also play a role in the aggregation process. Recently it has been proposed that α-Syn is able to form lipid-protein particles reminiscent of high-density lipoproteins. Here, we present a method to obtain a stable and homogeneous population of nanometer-sized particles composed of α-Syn and anionic phospholipids. These particles are called α-Syn lipoprotein (nano)particles to indicate their relationship to high-density lipoproteins formed by human apolipoproteins in vivo and of in vitro self-assembling phospholipid bilayer nanodiscs. Structural investigations of the α-Syn lipoprotein particles by circular dichroism (CD) and magic angle solid-state nuclear magnetic resonance (MAS SS-NMR) spectroscopy establish that α-Syn adopts a helical secondary structure within these particles. Based on cryo-electron microscopy (cryo-EM) and dynamic light scattering (DLS) α-Syn lipoprotein particles have a defined size with a diameter of ~23 nm. Chemical cross-linking in combination with solution-state NMR and multiangle static light scattering (MALS) of α-Syn particles reveal a high-order protein-lipid entity composed of approximately 8-10 α-Syn molecules. The close resemblance in size between cross-linked in vitro-derived α-Syn lipoprotein particles and a cross-linked species of endogenous α-Syn from SH-SY5Y human neuroblastoma cells indicates a potential functional relevance of α-Syn lipoprotein nanoparticles

A Novel Micropeptide, \u3cem\u3eSlitharin\u3c/em\u3e, Exerts Cardioprotective Effects in Myocardial Infarction

Purpose: Micropeptides are an emerging class of proteins that play critical roles in cell signaling. Here, we describe the discovery of a novel micropeptide, dubbed slitharin (Slt), in conditioned media from Cardiosphere-derived cells (CDCs), a therapeutic cardiac stromal cell type. Experimental design: We performed mass spectrometry of peptide-enriched fractions from the conditioned media of CDCs and a therapeutically inert cell type (human dermal fibrobasts). We then evaluated the therapeutic capacity of the candidate peptide using an in vitro model of cardiomyocyte injury and a rat model of myocardial infarction. Results: We identified a novel 24-amino acid micropeptide (dubbed Slitharin [Slt]) with a non-canonical leucine start codon, arising from long intergenic non-coding (LINC) RNA 2099. Neonatal rat ventricular myocytes (NRVMs) exposed to Slt were protected from hypoxic injury in vitro compared to a vehicle or scrambled control. Transcriptomic analysis of cardiomyocytes exposed to Slt reveals cytoprotective capacity, putatively through regulation of stress-induced MAPK-ERK. Slt also exerted cardioprotective effects in rats with myocardial infarction as shown by reduced infarct size 48 h post-injury. Conclusions and clinical relavance: Thus, Slt is a non-coding RNA-derived micropeptide, identified in the extracellular space, with a potential cardioprotective function

The Desensitization Gating of the MthK K+ Channel Is Governed by Its Cytoplasmic Amino Terminus

The RCK-containing MthK channel undergoes two inactivation processes: activation-coupled desensitization and acid-induced inactivation. The acid inactivation is mediated by the C-terminal RCK domain assembly. Here, we report that the desensitization gating is governed by a desensitization domain (DD) of the cytoplasmic N-terminal 17 residues. Deletion of DD completely removes the desensitization, and the process can be fully restored by a synthetic DD peptide added in trans. Mutagenesis analyses reveal a sequence-specific determinant for desensitization within the initial hydrophobic segment of DD. Proton nuclear magnetic resonance (1H NMR) spectroscopy analyses with synthetic peptides and isolated RCK show interactions between the two terminal domains. Additionally, we show that deletion of DD does not affect the acid-induced inactivation, indicating that the two inactivation processes are mutually independent. Our results demonstrate that the short N-terminal DD of MthK functions as a complete moveable module responsible for the desensitization. Its interaction with the C-terminal RCK domain may play a role in the gating process

In Situ Formation of an Azo Bridge on Proteins Controllable by Visible Light

Optical modulation of proteins provides superior spatiotemporal resolution for understanding biological processes, and photoswitches built on light-sensitive proteins have been significantly advancing neuronal and cellular studies. Small molecule photoswitches could complement protein-based switches by mitigating potential interference and affording high specificity for modulation sites. However, genetic encodability and responsiveness to nonultraviolet light, two desired properties possessed by protein photoswitches, are challenging to be engineered into small molecule photoswitches. Here we developed a small molecule photoswitch that can be genetically installed onto proteins in situ and controlled by visible light. A pentafluoro azobenzene-based photoswitchable click amino acid (F-PSCaa) was designed to isomerize in response to visible light. After genetic incorporation into proteins via the expansion of the genetic code, F-PSCaa reacts with a nearby cysteine within the protein generating an azo bridge in situ. The resultant bridge is switchable by visible light and allows conformation and binding of CaM to be regulated by such light. This photoswitch should prove valuable in optobiology for its minimal interference, site flexibility, genetic encodability, and response to the more biocompatible visible light

NMR spectroscopic and analytical ultracentrifuge analysis of membrane protein detergent complexes

Background Structural studies of integral membrane proteins (IMPs) are hampered by inherent difficulties in their heterologous expression and in the purification of solubilized protein-detergent complexes (PDCs). The choice and concentrations of detergents used in an IMP preparation play a critical role in protein homogeneity and are thus important for successful crystallization. Results Seeking an effective and standardized means applicable to genomic approaches for the characterization of PDCs, we chose 1D-NMR spectroscopic analysis to monitor the detergent content throughout their purification: protein extraction, detergent exchange, and sample concentration. We demonstrate that a single NMR measurement combined with a SDS-PAGE of a detergent extracted sample provides a useful gauge of the detergent's extraction potential for a given protein. Furthermore, careful monitoring of the detergent content during the process of IMP production allows for a high level of reproducibility. We also show that in many cases a simple sedimentation velocity measurement provides sufficient data to estimate both the oligomeric state and the detergent-to-protein ratio in PDCs, as well as to evaluate the homogeneity of the samples prior to crystallization screening. Conclusion The techniques presented here facilitate the screening and selection of the extraction detergent, as well as help to maintain reproducibility in the detergent exchange and PDC concentration procedures. Such reproducibility is particularly important for the optimization of initial crystallization conditions, for which multiple purifications are routinely required.ISSN:1472-680

In Situ Formation of an Azo Bridge on Proteins Controllable by Visible Light

Optical modulation of proteins provides superior spatiotemporal resolution for understanding biological processes, and photoswitches built on light-sensitive proteins have been significantly advancing neuronal and cellular studies. Small molecule photoswitches could complement protein-based switches by mitigating potential interference and affording high specificity for modulation sites. However, genetic encodability and responsiveness to nonultraviolet light, two desired properties possessed by protein photoswitches, are challenging to be engineered into small molecule photoswitches. Here we developed a small molecule photoswitch that can be genetically installed onto proteins in situ and controlled by visible light. A pentafluoro azobenzene-based photoswitchable click amino acid (F-PSCaa) was designed to isomerize in response to visible light. After genetic incorporation into proteins via the expansion of the genetic code, F-PSCaa reacts with a nearby cysteine within the protein generating an azo bridge in situ. The resultant bridge is switchable by visible light and allows conformation and binding of CaM to be regulated by such light. This photoswitch should prove valuable in optobiology for its minimal interference, site flexibility, genetic encodability, and response to the more biocompatible visible light