Dendronic trimaltoside amphiphiles (DTMs) for membrane protein study

Sadaf, Aiman,Du, Yang,Santillan, Claudia,Mortensen, Jonas S.,Molist, Iago,Seven, Alpay B.,Hariharan, Parameswaran,Skiniotis, Georgios,Loland, Claus J.,Kobilka, Brian K.,Guan, Lan,Byrne, Bernadette,Cha Royal Society of Chemistry 2017 Chemical Science Vol.8 No.12

<▼1><P>A novel amphiphile with a dendronic hydrophobic group (DTM-A6) was markedly effective at stabilizing and visualizing a GPCR-G<SUB>s</SUB> complex.</P></▼1><▼2><P>The critical contribution of membrane proteins in normal cellular function makes their detailed structure and functional analysis essential. Detergents, amphipathic agents with the ability to maintain membrane proteins in a soluble state in aqueous solution, have key roles in membrane protein manipulation. Structural and functional stability is a prerequisite for biophysical characterization. However, many conventional detergents are limited in their ability to stabilize membrane proteins, making development of novel detergents for membrane protein manipulation an important research area. The architecture of a detergent hydrophobic group, that directly interacts with the hydrophobic segment of membrane proteins, is a key factor in dictating their efficacy for both membrane protein solubilization and stabilization. In the current study, we developed two sets of maltoside-based detergents with four alkyl chains by introducing dendronic hydrophobic groups connected to a trimaltoside head group, designated dendronic trimaltosides (DTMs). Representative DTMs conferred enhanced stabilization to multiple membrane proteins compared to the benchmark conventional detergent, DDM. One DTM (<I>i.e.</I>, DTM-A6) clearly outperformed DDM in stabilizing human β<SUB>2</SUB> adrenergic receptor (β<SUB>2</SUB>AR) and its complex with G<SUB>s</SUB> protein. A further evaluation of this DTM led to a clear visualization of β<SUB>2</SUB>AR-G<SUB>s</SUB> complex <I>via</I> electron microscopic analysis. Thus, the current study not only provides novel detergent tools useful for membrane protein study, but also suggests that the dendronic architecture has a role in governing detergent efficacy for membrane protein stabilization.</P></▼2>

Conformationally Preorganized Diastereomeric Norbornane-Based Maltosides for Membrane Protein Study: Implications of Detergent Kink for Micellar Properties

Das, Manabendra,Du, Yang,Ribeiro, Orquidea,Hariharan, Parameswaran,Mortensen, Jonas S.,Patra, Dhabaleswar,Skiniotis, Georgios,Loland, Claus J.,Guan, Lan,Kobilka, Brian K.,Byrne, Bernadette,Chae, Pil S American Chemical Society 2017 JOURNAL OF THE AMERICAN CHEMICAL SOCIETY - Vol.139 No.8

<P>Detergents are essential tools for functional and structural studies of membrane proteins. However, conventional detergents are limited in their scope and utility, particularly for eukaryotic membrane proteins. Thus, there are major efforts to develop new amphipathic agents with enhanced properties. Here, a novel class of diastereomeric agents with a preorganized conformation, designated norbornane-based maltosides (NBMs), were prepared and evaluated for their ability to solubilize and stabilize membrane proteins. Representative NBMs displayed enhanced behaviors compared to n-dodecyl-beta-D-maltoside (DDM) for all membrane proteins tested. Efficacy of the individual NBMs varied depending on the overall detergent shape and alkyl chain length. Specifically, NBMs with no kink in the lipophilic region conferred greater stability to the proteins than NBMs with a kink. In addition, long alkyl chain NBMs were generally better at stabilizing membrane proteins than short alkyl chain agents. Furthermore, use of one well-behaving NBM enabled us to attain a marked stabilization and clear visualization of a challenging membrane protein complex using electron microscopy. Thus, this study not only describes novel maltoside detergents with enhanced protein stabilizing properties but also suggests that overall detergent geometry has an important role in determining membrane protein stability. Notably, this is the first systematic study on the effect of detergent kinking on micellar properties and associated membrane protein stability.</P>

New penta-saccharide-bearing tripod amphiphiles for membrane protein structure studies

Ehsan, Muhammad,Ghani, Lubna,Du, Yang,Hariharan, Parameswaran,Mortensen, Jonas S.,Ribeiro, Orquidea,Hu, Hongli,Skiniotis, Georgios,Loland, Claus J.,Guan, Lan,Kobilka, Brian K.,Byrne, Bernadette,Chae, Royal Society of Chemistry 2017 The Analyst Vol.142 No.20

<P>Integral membrane proteins either alone or as complexes carry out a range of key cellular functions. Detergents are indispensable tools in the isolation of membrane proteins from biological membranes for downstream studies. Although a large number of techniques and tools, including a wide variety of detergents, are available, purification and structural characterization of many membrane proteins remain challenging. In the current study, a new class of tripod amphiphiles bearing two different penta-saccharide head groups, designated TPSs, were developed and evaluated for their ability to extract and stabilize a range of diverse membrane proteins. Variations in the structures of the detergent head and tail groups allowed us to prepare three sets of the novel agents with distinctive structures. Some TPSs (TPS-A8 and TPS-E7) were efficient at extracting two proteins in a functional state while others (TPS-E8 and TPS-E10L) conferred marked stability to all membrane proteins (and membrane protein complexes) tested here compared to a conventional detergent. Use of TPS-E10L led to clear visualization of a receptor-Gs complex using electron microscopy, indicating profound potential in membrane protein research.</P>

Vitamin E-based glycoside amphiphiles for membrane protein structural studies

Ehsan, Muhammad,Du, Yang,Molist, Iago,Seven, Alpay B.,Hariharan, Parameswaran,Mortensen, Jonas S.,Ghani, Lubna,Loland, Claus J.,Skiniotis, Georgios,Guan, Lan,Byrne, Bernadette,Kobilka, Brian K.,Chae, The Royal Society of Chemistry 2018 Organic & Biomolecular Chemistry Vol.16 No.14

<P>Membrane proteins play critical roles in a variety of cellular processes. For a detailed molecular level understanding of their biological functions and roles in disease, it is necessary to extract them from the native membranes. While the amphipathic nature of these bio-macromolecules presents technical challenges, amphiphilic assistants such as detergents serve as useful tools for membrane protein structural and functional studies. Conventional detergents are limited in their ability to maintain the structural integrity of membrane proteins and thus it is essential to develop novel agents with enhanced properties. Here, we designed and characterized a novel class of amphiphiles with vitamin E (<I>i.e.</I>, α-tocopherol) as the hydrophobic tail group and saccharide units as the hydrophilic head group. Designated vitamin E-based glycosides (VEGs), these agents were evaluated for their ability to solubilize and stabilize a set of membrane proteins. VEG representatives not only conferred markedly enhanced stability to a diverse range of membrane proteins compared to conventional detergents, but VEG-3 also showed notable efficacy toward stabilization and visualization of a membrane protein complex. In addition to hydrophile-lipophile balance (HLB) of detergent molecules, the chain length and molecular geometry of the detergent hydrophobic group seem key factors in determining detergent efficacy for membrane protein (complex) stability.</P>

Structural and Functional Analysis of a β₂-Adrenergic Receptor Complex with GRK5

Komolov, Konstantin E.,Du, Yang,Duc, Nguyen Minh,Betz, Robin M.,Rodrigues, Joã,o P.G.L.M.,Leib, Ryan D.,Patra, Dhabaleswar,Skiniotis, Georgios,Adams, Christopher M.,Dror, Ron O.,Chung, Ka Young Cell Press 2017 Cell Vol. No.

<P><B>Summary</B></P> <P>The phosphorylation of agonist-occupied G-protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) functions to turn off G-protein signaling and turn on arrestin-mediated signaling. While a structural understanding of GPCR/G-protein and GPCR/arrestin complexes has emerged in recent years, the molecular architecture of a GPCR/GRK complex remains poorly defined. We used a comprehensive integrated approach of cross-linking, hydrogen-deuterium exchange mass spectrometry (MS), electron microscopy, mutagenesis, molecular dynamics simulations, and computational docking to analyze GRK5 interaction with the β<SUB>2</SUB>-adrenergic receptor (β<SUB>2</SUB>AR). These studies revealed a dynamic mechanism of complex formation that involves large conformational changes in the GRK5 RH/catalytic domain interface upon receptor binding. These changes facilitate contacts between intracellular loops 2 and 3 and the C terminus of the β<SUB>2</SUB>AR with the GRK5 RH bundle subdomain, membrane-binding surface, and kinase catalytic cleft, respectively. These studies significantly contribute to our understanding of the mechanism by which GRKs regulate the function of activated GPCRs.</P> <P><B>PaperClip</B></P> <P>Display Omitted</P> <P><B>Highlights</B></P> <P> <UL> <LI> GRK5-β<SUB>2</SUB>AR binding is enhanced by receptor and kinase ligands and acidic lipids </LI> <LI> GRK5 binding to the β<SUB>2</SUB>AR involves a multi-site interaction </LI> <LI> Receptor binding triggers substantial conformational changes in GRK5 </LI> <LI> RH/catalytic domain separation in GRK5 is essential for receptor phosphorylation </LI> </UL> </P> <P><B>Graphical Abstract</B></P> <P>[DISPLAY OMISSION]</P>

Context dependency of Set1/COMPASS-mediated histone H3 Lys4 trimethylation

Thornton, Janet L.,Westfield, Gerwin H.,Takahashi, Yoh-hei,Cook, Malcolm,Gao, Xin,Woodfin, Ashley R.,Lee, Jung-Shin,Morgan, Marc A.,Jackson, Jessica,Smith, Edwin R.,Couture, Jean-Francois,Skiniotis, G Cold Spring Harbor Laboratory Press 2014 Genes & development Vol.28 No.2

<P>The stimulation of H3K4 trimethylation (H3K4me3) by H2B monoubiquitination (H2Bub) has been widely studied, and multiple mechanisms have been proposed for this form of histone cross-talk. Thornton et al. combine biochemical, structural, and in vivo approaches to provide a novel mechanism for the role of H2B ubiquitination machinery in the regulation of histone H3K4 methylation by COMPASS. This study demonstrates that the H2Bub machinery and Cps35/Swd2 function to focus the H3K4me3 activity of COMPASS at promoter-proximal regions in a context-dependent manner.</P>