Semisynthesis of functional transmembrane proteins in GUVs

Cellular transmembrane (TM) proteins are essential sentries of the cell facilitating cell-cell communication, internal signaling, and solute transport. Reconstituting functional TM proteins into model membranes remains a challenge due to the difficulty of expressing hydrophobic TM domains and the required use of detergents. Herein, we use a intein-mediated ligation strategy to semisynthesize bitopic TM proteins in synthetic membranes. We have adapted the trans splicing capabilities of split inteins for a native peptide ligation between a synthetic TM peptide embedded in the membrane of giant unilamellar vesicles (GUVs) and an expressed soluble protein. We demonstrate that the extracellular domain of programmed cell death protein 1 (PD-1), a mammalian transmembrane immune checkpoint receptor, retains its function for binding its ligand PD-L1 at a reconstituted membrane interface after ligation to a synthetic TM peptide in GUV membranes. We envision that the construction of full-length TM proteins using orthogonal split intein-mediated semisynthetic protein ligations will expand applications of membrane protein reconstitution in pharmacology, biochemistry, biophysics, and artificial cell development.

Mitochondrial energetics with transmembrane electrostatically localized protons: do we have a thermotrophic feature?

Transmembrane electrostatically localized protons (TELP) theory has been recently recognized as an important addition over the classic Mitchell’s chemiosmosis; thus, the proton motive force (pmf) is largely contributed from TELP near the membrane. As an extension to this theory, a novel phenomenon of mitochondrial thermotrophic function is now characterized by biophysical analyses of pmf in relation to the TELP concentrations at the liquid-membrane interface. This leads to the conclusion that the oxidative phosphorylation also utilizes environmental heat energy associated with the thermal kinetic energy (kBT) of TELP in mitochondria. The local pmf is now calculated to be in a range from 300 to 340 mV while the classic pmf (which underestimates the total pmf) is in a range from 60 to 210 mV in relation to a range of membrane potentials from 50 to 200 mV. Depending on TELP concentrations in mitochondria, this thermotrophic function raises pmf significantly by a factor of 2.6 to sixfold over the classic pmf. Therefore, mitochondria are capable of effectively utilizing the environmental heat energy with TELP for the synthesis of ATP, i.e., it can lock heat energy into the chemical form of energy for cellular functions.

Structural basis of the membrane intramolecular transacylase reaction responsible for lyso-form lipoprotein synthesis

Lipoproteins serve diverse functions in the bacterial cell and some are essential for survival. Some lipoproteins are adjuvants eliciting responses from the innate immune system of the host. The growing list of membrane enzymes responsible for lipoprotein synthesis includes the recently discovered lipoprotein intramolecular transacylase, Lit. Lit creates a lipoprotein that is less immunogenic, possibly enabling the bacteria to gain a foothold in the host by stealth. Here, we report the crystal structure of the Lit enzyme from Bacillus cereus and describe its mechanism of action. Lit consists of four transmembrane helices with an extracellular cap. Conserved residues map to the cap-membrane interface. They include two catalytic histidines that function to effect unimolecular transacylation. The reaction involves acyl transfer from the sn-2 position of the glyceryl moiety to the amino group on the N-terminal cysteine of the substrate via an 8-membered ring intermediate. Transacylation takes place in a confined aromatic residue-rich environment that likely evolved to bring distant moieties on the substrate into proximity and proper orientation for catalysis.

The surprising structural and mechanistic dichotomy of membrane-associated phosphoglycosyl transferases

Phosphoglycosyl transferases (PGTs) play a pivotal role at the inception of complex glycoconjugate biosynthesis pathways across all domains of life. PGTs promote the first membrane-committed step in the en bloc biosynthetic strategy by catalyzing the transfer of a phospho-sugar from a nucleoside diphospho-sugar to a membrane-resident polyprenol phosphate. Studies on the PGTs have been hampered because they are integral membrane proteins, and often prove to be recalcitrant to expression, purification and analysis. However, in recent years exciting new information has been derived on the structures and the mechanisms of PGTs, revealing the existence of two unique superfamilies of PGT enzymes that enact catalysis at the membrane interface. Genome neighborhood analysis shows that these superfamilies, the polytopic PGT (polyPGT) and monotopic PGT (monoPGT), may initiate different pathways within the same organism. Moreover, the same fundamental two-substrate reaction is enacted through two different chemical mechanisms with distinct modes of catalysis. This review highlights the structural and mechanistic divergence between the PGT enzyme superfamilies and how this is reflected in differences in regulation in their varied glycoconjugate biosynthesis pathways.

Estudo de tecnologias de investigação ambiental em alta resolução para refinamento do modelo conceitual — estudo de caso: Duque de Caxias (RJ), Brasil

RESUMO Este trabalho apresenta os resultados da utilização de duas técnicas de investigação geoambiental em alta resolução: investigação passiva de vapores do solo e investigação com o uso de Membrane Interface Probe (MIP), em uma área contaminada por hidrocarbonetos de petróleo, localizada no município de Duque de Caxias, Rio de Janeiro. O processo de gerenciamento ambiental da área iniciou-se em 2012 e contemplou as etapas preconizadas nas legislações vigentes. Os resultados obtidos a partir das investigações tradicionais foram insuficientes para a compreensão das características físicas e geoquímicas necessária para o sucesso das fases de diagnóstico e, consequentemente, da remediação. Portanto, duas investigações em alta resolução foram conduzidas com o objetivo de refinar o modelo conceitual de forma a atender adequadamente à Resolução CONAMA nº 420/09 e à Resolução CONEMA nº 44/12, permitindo ações futuras mais eficientes. A investigação passiva de vapores do solo utilizou amostradores compostos por materiais adsorventes granulares, encapsulados em uma membrana microporosa hidrofóbica e quimicamente inerte que permite a difusão dos vapores presentes no meio. Os resultados representam qualitativamente a presença de contaminação no subsolo. O MIP é uma ferramenta de direct push com medição em tempo real, que detecta a presença da contaminação tanto em meios insaturados quanto saturados inconsolidados. A partir dos resultados integrados, foi possível constatar que a distribuição de compostos orgânicos voláteis (volatile organic compounds (VOCs)) em água subterrânea ocorre de forma descontínua ao longo da área, sendo possível identificar cinco hotspots distintos e suas diferentes áreas fonte, incluindo uma região com presença de fase livre.

Membrane insertion of Cavin1 facilitates caveolae assembly

Caveolae are small cell surface invaginations, important for control of membrane tension, signaling cascades and lipid sorting. Their formation is coupled to the lipid-dependent oligomerization of the proteins Caveolin1 and Cavin1, which are essential for membrane curvature generation. Yet, the mechanistic understanding of how Cavin1 assembles at the membrane interface is lacking. Here, we used model membranes combined with biophysical techniques to show that Cavin1 inserts into membranes. We found that the helical region 1 (HR1) initiated membrane binding via electrostatic interactions, which is further enforced by partial helical insertion in a PI(4,5)P2-dependent process mediated by the disordered region 1 (DR1). In agreement with this, the DR1 was found important for the co-assembly of Cavin1 with Caveolin1 in living cells. We propose that DR1 and HR1 of Cavin1 constitute a novel membrane interacting unit facilitating dynamic rounds of assembly and disassembly of Cavin1 at the membrane.