Reconstitution of the Melibiose Permease of serovar Typhimurium (MelB) into Lipid Nanodiscs.

Membrane proteins play critical roles in cell physiology and pathology. The conventional way to study membrane proteins at protein levels is to use optimal detergents to extract proteins from membranes. Identification of the optimal detergent is tedious , and in some cases, the protein functions are compromised.

Distinct roles of the major binding residues in the cation-binding pocket of the melibiose transporter MelB.

Salmonella enterica serovar Typhimurium melibiose permease (MelB) is a prototype of the major facilitator superfamily (MFS) transporters, which play important roles in human health and diseases. MelB catalyzed the symport of galactosides with Na, Li, or H but prefers the coupling with Na. Previously, we determined the structures of the inward- and outward-facing conformation of MelB and the molecular recognition for galactoside and Na.

Distinct roles of the major binding residues in the cation-binding pocket of MelB.

serovar Typhimurium melibiose permease (MelB) is a prototype of the major facilitator superfamily (MFS) transporters, which play important roles in human health and diseases. MelB catalyzed the symport of galactosides with either H, Li, or Na, but prefers the coupling with Na. Previously, we determined the structures of the inward- and outward-facing conformation of MelB, as well as the molecular recognition for galactoside and Na.

Mobile barrier mechanisms for Na-coupled symport in an MFS sugar transporter.

While many 3D structures of cation-coupled transporters have been determined, the mechanistic details governing the obligatory coupling and functional regulations still remain elusive. The bacterial melibiose transporter (MelB) is a prototype of major facilitator superfamily transporters. With a conformation-selective nanobody, we determined a low-sugar affinity inward-facing Na-bound cryoEM structure.

Rational Approach to Improve Detergent Efficacy for Membrane Protein Stabilization.

Membrane protein structures are essential for the molecular understanding of diverse cellular processes and drug discovery. Detergents are not only widely used to extract membrane proteins from membranes but also utilized to preserve native protein structures in aqueous solution. However, micelles formed by conventional detergents are suboptimal for membrane protein stabilization, necessitating the development of novel amphiphilic molecules with enhanced protein stabilization efficacy.

Mobile barrier mechanisms for Na-coupled symport in an MFS sugar transporter.

While many 3D structures of cation-coupled transporters have been determined, the mechanistic details governing the obligatory coupling and functional regulations still remain elusive. The bacterial melibiose transporter (MelB) is a prototype of the Na-coupled major facilitator superfamily transporters. With a conformational nanobody (Nb), we determined a low-sugar affinity inward-facing Na-bound cryoEM structure.

The insertase YidC chaperones the polytopic membrane protein MelB inserting and folding simultaneously from both termini.

The insertion and folding of proteins into membranes is crucial for cell viability. Yet, the detailed contributions of insertases remain elusive. Here, we monitor how the insertase YidC guides the folding of the polytopic melibiose permease MelB into membranes.

In vivo and in vitro characterizations of melibiose permease (MelB) conformation-dependent nanobodies reveal sugar-binding mechanisms.

Salmonella enterica serovar Typhimurium melibiose permease (MelB) is a prototype of the Na-coupled major facilitator superfamily transporters, which are important for the cellular uptake of molecules including sugars and small drugs. Although the symport mechanisms have been well-studied, mechanisms of substrate binding and translocation remain enigmatic. We have previously determined the sugar-binding site of outward-facing MelB by crystallography.

Direct determination of oligomeric organization of integral membrane proteins and lipids from intact customizable bilayer.

Hierarchical organization of integral membrane proteins (IMP) and lipids at the membrane is essential for regulating myriad downstream signaling. A quantitative understanding of these processes requires both detections of oligomeric organization of IMPs and lipids directly from intact membranes and determination of key membrane components and properties that regulate them. Addressing this, we have developed a platform that enables native mass spectrometry (nMS) analysis of IMP-lipid complexes directly from intact and customizable lipid membranes.

Tris(hydroxymethyl)aminomethane Linker-Bearing Triazine-Based Triglucosides for Solubilization and Stabilization of Membrane Proteins.

High-resolution membrane protein structures are essential for a fundamental understanding of the molecular basis of diverse cellular processes and for drug discovery. Detergents are widely used to extract membrane-spanning proteins from membranes and maintain them in a functional state for downstream characterization. Due to limited long-term stability of membrane proteins encapsulated in conventional detergents, development of novel agents is required to facilitate membrane protein structural study.

Substrate-binding guides individual melibiose permeases MelB to structurally soften and to destabilize cytoplasmic middle-loop C3.

The melibiose permease MelB is a well-studied Na-coupled transporter of the major facilitator superfamily. However, the symport mechanism of galactosides and cations is still not fully understood, especially at structural levels. Here, we use single-molecule force spectroscopy to investigate substrate-induced structural changes of MelB from Salmonella typhimurium.

3,4-Bis(hydroxymethyl)hexane-1,6-diol-based Maltosides (HDMs) for Membrane-Protein Study: Importance of Detergent Rigidity-Flexibility Balance in Protein Stability.

Detergents have been major contributors to membrane-protein structural study for decades. However, membrane proteins solubilized in conventional detergents tend to aggregate or denature over time. Stability of large eukaryotic membrane proteins with complex structures tends to be particularly poor, necessitating development of novel detergents with improved properties.

Molecular Basis for the Cation Selectivity of Salmonella typhimurium Melibiose Permease.

Cation selectivity and coupling are important attributes of cation-coupled symporters. Salmonella typhimurium melibiose permease (MelB) catalyzes the co-transport of galactosides with cations (H, Li, or Na). 3-D crystal structures of MelB have revealed the molecular recognition for sugar substrates, but the cation binding and coupling mechanisms have not been defined to atomic levels.

Maltose-bis(hydroxymethyl)phenol (MBPs) and Maltose-tris(hydroxymethyl)phenol (MTPs) Amphiphiles for Membrane Protein Stability.

Membrane protein structures provide a fundamental understanding of their molecular actions and are of importance for drug development. Detergents are widely used to solubilize, stabilize, and crystallize membrane proteins, but membrane proteins solubilized in conventional detergents are prone to denaturation and aggregation. Thus, developing novel detergents with enhanced efficacy for protein stabilization remains important.

Complete cysteine-scanning mutagenesis of the Salmonella typhimurium melibiose permease.

The melibiose permease of Salmonella typhimurium (MelB) catalyzes the stoichiometric symport of galactopyranoside with a cation (H, Li, or Na) and is a prototype for Na-coupled major facilitator superfamily (MFS) transporters presenting from bacteria to mammals. X-ray crystal structures of MelB have revealed the molecular recognition mechanism for sugar binding; however, understanding of the cation site and symport mechanism is still vague. To further investigate the transport mechanism and conformational dynamics of MelB, we generated a complete single-Cys library containing 476 unique mutants by placing a Cys at each position on a functional Cys-less background.

X-ray crystallography reveals molecular recognition mechanism for sugar binding in a melibiose transporter MelB.

Major facilitator superfamily_2 transporters are widely found from bacteria to mammals. The melibiose transporter MelB, which catalyzes melibiose symport with either Na, Li, or H, is a prototype of the Na-coupled MFS transporters, but its sugar recognition mechanism has been a long-unsolved puzzle. Two high-resolution X-ray crystal structures of a Salmonella typhimurium MelB mutant with a bound ligand, either nitrophenyl-α-D-galactoside or dodecyl-β-D-melibioside, were refined to a resolution of 3.

Cooperative binding ensures the obligatory melibiose/Na+ cotransport in MelB.

MelB catalyzes the obligatory cotransport of melibiose with Na+, Li+, or H+. Crystal structure determination of the Salmonella typhimurium MelB (MelBSt) has revealed a typical major facilitator superfamily (MFS) fold at a periplasmic open conformation. Cooperative binding of Na+ and melibiose has been previously established.

Diastereomeric Cyclopentane-Based Maltosides (CPMs) as Tools for Membrane Protein Study.

Amphiphilic agents, called detergents, are invaluable tools for studying membrane proteins. However, membrane proteins encapsulated by conventional head-to-tail detergents tend to denature or aggregate, necessitating the development of structurally distinct molecules with improved efficacy. Here, a novel class of diastereomeric detergents with a cyclopentane core unit, designated cyclopentane-based maltosides (CPMs), were prepared and evaluated for their ability to solubilize and stabilize several model membrane proteins.

Structure, function, and ion-binding properties of a K channel stabilized in the 2,4-ion-bound configuration.

Here, we present the atomic resolution crystallographic structure, the function, and the ion-binding properties of the KcsA mutants, G77A and G77C, that stabilize the 2,4-ion-bound configuration (i.e., water, K, water, K-ion-bound configuration) of the K channel's selectivity filter.

Conformationally Restricted Monosaccharide-Cored Glycoside Amphiphiles: The Effect of Detergent Headgroup Variation on Membrane Protein Stability.

Detergents are widely used to isolate membrane proteins from lipid bilayers, but many proteins solubilized in conventional detergents are structurally unstable. Thus, there is major interest in the development of novel amphiphiles to facilitate membrane protein research. In this study, we have designed and synthesized novel amphiphiles with a rigid scyllo-inositol core, designated scyllo-inositol glycosides (SIGs).

Self-Assembly Behavior and Application of Terphenyl-Cored Trimaltosides for Membrane-Protein Studies: Impact of Detergent Hydrophobic Group Geometry on Protein Stability.

Amphipathic agents are widely used in various fields including biomedical sciences. Micelle-forming detergents are particularly useful for in vitro membrane-protein characterization. As many conventional detergents are limited in their ability to stabilize membrane proteins, it is necessary to develop novel detergents to facilitate membrane-protein research.

Self-Assembly Behaviors of a Penta-Phenylene Maltoside and Its Application for Membrane Protein Study.

We prepared an amphiphile with a penta-phenylene lipophilic group and a branched trimaltoside head group. This new agent, designated penta-phenylene maltoside (PPM), showed a marked tendency to self-assembly into micelles via strong aromatic-aromatic interactions in aqueous media, as evidenced by H NMR spectroscopy and fluorescence studies. When utilized for membrane protein studies, this new agent was superior to DDM, a gold standard conventional detergent, in stabilizing multiple proteins long term.

Asymmetric maltose neopentyl glycol amphiphiles for a membrane protein study: effect of detergent asymmetricity on protein stability.

Maintaining protein stability in an aqueous solution is a prerequisite for protein structural and functional studies, but conventional detergents have increasingly showed limited ability to maintain protein integrity. A representative novel agent, maltose neopentyl glycol-3 (MNG-3), has recently substantially contributed to membrane protein structural studies. Motivated by the popular use of this novel agent, we prepared asymmetric versions of MNG-3 and evaluated these agents with several membrane proteins including two G protein-coupled receptors in this study.

A comparative study of branched and linear mannitol-based amphiphiles on membrane protein stability.

The study of membrane proteins is extremely challenging, mainly because of the incompatibility of the hydrophobic surfaces of membrane proteins with an aqueous medium. Detergents are essential agents used to maintain membrane protein stability in non-native environments. However, conventional detergents fail to stabilize the native structures of many membrane proteins.

Structural and functional characterization of protein-lipid interactions of the Salmonella typhimurium melibiose transporter MelB.

Background: Membrane lipids play critical roles in the structure and function of membrane-embedded transporters. Salmonella typhimurium MelB (MelB) is a symporter coupling melibiose translocation with a cation (Na, Li, or H). We present an extensive study on the effects of specific phospholipids on the structure of MelB and the melibiose transport catalyzed by this protein.