scholarly journals Identification of recombinant Fabs for structural and functional characterization of HIV-host factor complexes

PLoS ONE ◽  
2021 ◽  
Vol 16 (5) ◽  
pp. e0250318
Author(s):  
Natalia Sevillano ◽  
Evan M. Green ◽  
Jörg Votteler ◽  
Dong Young Kim ◽  
Xuefeng Ren ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Functional Characterization ◽  
Phage Display Library ◽  
Host Factor ◽  
Host Protein ◽  
Rapid Screening ◽  
Functional Studies ◽  
Negative Stain ◽  
Immunization Strategies

Viral infection and pathogenesis is mediated by host protein—viral protein complexes that are important targets for therapeutic intervention as they are potentially less prone to development of drug resistance. We have identified human, recombinant antibodies (Fabs) from a phage display library that bind to three HIV-host complexes. We used these Fabs to 1) stabilize the complexes for structural studies; and 2) facilitate characterization of the function of these complexes. Specifically, we generated recombinant Fabs to Vif-CBF-β-ELOB-ELOC (VCBC); ESCRT-I complex and AP2-complex. For each complex we measured binding affinities with KD values of Fabs ranging from 12–419 nM and performed negative stain electron microscopy (nsEM) to obtain low-resolution structures of the HIV-Fab complexes. Select Fabs were converted to scFvs to allow them to fold intracellularly and perturb HIV-host protein complex assembly without affecting other pathways. To identify these recombinant Fabs, we developed a rapid screening pipeline that uses quantitative ELISAs and nsEM to establish whether the Fabs have overlapping or independent epitopes. This pipeline approach is generally applicable to other particularly challenging antigens that are refractory to immunization strategies for antibody generation including multi-protein complexes providing specific, reproducible, and renewable antibody reagents for research and clinical applications. The curated antibodies described here are available to the scientific community for further structural and functional studies on these critical HIV host-factor proteins.

scholarly journals Deletion of small ankyrin 1 (sAnk1) isoforms results in structural and functional alterations in aging skeletal muscle fibers

2015 ◽  
Vol 308 (2) ◽  
pp. C123-C138 ◽  
Author(s):  
E. Giacomello ◽  
M. Quarta ◽  
C. Paolini ◽  
R. Squecco ◽  
P. Fusco ◽  
...  
Keyword(s):  
Structural Damage ◽  
Skeletal Muscles ◽  
Functional Characterization ◽  
Sr Proteins ◽  
Endurance Test ◽  
Tubular Aggregates ◽  
Functional Studies ◽  
Old Mice ◽  
Contractile Performance

Muscle-specific ankyrins 1 (sAnk1) are a group of small ankyrin 1 isoforms, of which sAnk1.5 is the most abundant. sAnk1 are localized in the sarcoplasmic reticulum (SR) membrane from where they interact with obscurin, a myofibrillar protein. This interaction appears to contribute to stabilize the SR close to the myofibrils. Here we report the structural and functional characterization of skeletal muscles from sAnk1 knockout mice (KO). Deletion of sAnk1 did not change the expression and localization of SR proteins in 4- to 6-mo-old sAnk1 KO mice. Structurally, the main modification observed in skeletal muscles of adult sAnk1 KO mice (4–6 mo of age) was the reduction of SR volume at the sarcomere A band level. With increasing age (at 12–15 mo of age) extensor digitorum longus (EDL) skeletal muscles of sAnk1 KO mice develop prematurely large tubular aggregates, whereas diaphragm undergoes significant structural damage. Parallel functional studies revealed specific changes in the contractile performance of muscles from sAnk1 KO mice and a reduced exercise tolerance in an endurance test on treadmill compared with control mice. Moreover, reduced Qγcharge and L-type Ca2+current, which are indexes of affected excitation-contraction coupling, were observed in diaphragm fibers from 12- to 15-mo-old mice, but not in other skeletal muscles from sAnk1 KO mice. Altogether, these findings show that the ablation of sAnk1, by altering the organization of the SR, renders skeletal muscles susceptible to undergo structural and functional alterations more evident with age, and point to an important contribution of sAnk1 to the maintenance of the longitudinal SR architecture.

Functional characterization of an abnormal factor XII molecule (F XII Bern)

Blood ◽  
1991 ◽  
Vol 78 (4) ◽  
pp. 997-1004
Author(s):  
WA Wuillemin ◽  
I Huber ◽  
M Furlan ◽  
B Lammle
Keyword(s):  
Dextran Sulfate ◽  
Functional Characterization ◽  
Healthy Woman ◽  
Chain Molecule ◽  
Molecular Defect ◽  
Factor Xii ◽  
Single Chain ◽  
Functional Studies ◽  
Functional Defect

An 18-year-old healthy woman was found to have cross-reacting material (CRM)-positive factor XII (F XII) deficiency, F XII clotting activity was less than 0.01 U/mL, whereas F XII antigen was 0.11 U/mL. An F XII inhibitor was excluded. To partially characterize the molecular defect of the abnormal F XII, immunologic and functional studies were performed on the proposita's plasma. The abnormal F XII was a single chain molecule with the same molecular weight (80 Kd) and the same isoelectric points (pl, 5.9 to 6.8) as normal F XII. Dextran sulfate activation of the proposita's plasma showed no proteolytic cleavage of F XII even after 120 minutes, whereas F XII in pooled normal plasma, diluted 1:10 with CRM-negative F XII-deficient plasma, was completely cleaved after 40 minutes. Adsorption to kaolin was identical for both abnormal and normal F XII. In the presence of dextran sulfate and exogenous plasma kallikrein, the abnormal F XII was cleaved with the same rate as normal F XII. However, kallikrein-cleaved abnormal F XII was not able to cleave factor XI and plasma prekallikrein, in contrast to activated normal F XII. Thus, these studies show that the functional defect of this abnormal F XII, denoted as F XII Bern, is due to the lack of protease activity of the kallikrein-cleaved molecule. Therefore, the structural defect is likely to be located in the light chain region of F XII, containing the enzymatic active site.

scholarly journals Rapid transfer of overexpressed integral membrane protein from the host membrane into soluble lipid nanodiscs without previous purification

2015 ◽  
Vol 396 (8) ◽  
pp. 903-915 ◽  
Author(s):  
Nazhat Shirzad-Wasei ◽  
Jenny van Oostrum ◽  
Petra H.M. Bovee-Geurts ◽  
Lisanne J.A. Kusters ◽  
Giel J.C.G.M. Bosman ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Functional Characterization ◽  
Integral Membrane Protein ◽  
Partial Purification ◽  
Functional Studies ◽  
Host Membrane ◽  
Lipid Nanodiscs ◽  
Membrane Scaffold ◽  
Rapid Transfer

Abstract Structural and functional characterization of integral membrane proteins in a bilayer environment is strongly hampered by the requirement of detergents for solubilization and subsequent purification, as detergents commonly affect their structure and/or activity. Here, we describe a rapid procedure with minimal exposure to detergent to directly assemble an overexpressed integral membrane protein into soluble lipid nanodiscs prior to purification. This is exemplified with recombinant his-tagged rhodopsin, which is rapidly extracted from its host membrane and directly assembled into membrane scaffold protein (MSP) nanodiscs. We further demonstrate that, even when the MSP was his-tagged as well, partial purification of the rhodopsin-nanodiscs could be achieved exploiting immobilized-metal chromatography. Recoveries of rhodopsin up to 80% were achieved in the purified nanodisc fraction. Over 95% of contaminating membrane protein and his-tagged MSP could be removed from the rhodopsin-nanodiscs using a single Ni2+-affinity chromatography step. This level of purification is amply sufficient for functional studies. We provide evidence that the obtained rhodopsin-nanodisc preparations are fully functional both photochemically and in their ability to bind the cognate G-protein.

scholarly journals Structural and functional characterization of the mitochondrial complex IV assembly factor Coa6

2019 ◽  
Vol 2 (5) ◽  
pp. e201900458 ◽  
Author(s):  
Shadi Maghool ◽  
N Dinesha G Cooray ◽  
David A Stroud ◽  
David Aragão ◽  
Michael T Ryan ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Protein Complexes ◽  
Functional Characterization ◽  
Copper Binding ◽  
Loss Of Function ◽  
Structural Explanation ◽  
Complex Iv ◽  
Assembly Factor ◽  
Bundle Structure

Assembly factors play key roles in the biogenesis of many multi-subunit protein complexes regulating their stability, activity, and the incorporation of essential cofactors. The human assembly factor Coa6 participates in the biogenesis of the CuA site in complex IV (cytochrome c oxidase, COX). Patients with mutations in Coa6 suffer from mitochondrial disease due to complex IV deficiency. Here, we present the crystal structures of human Coa6 and the pathogenic W59CCoa6-mutant protein. These structures show that Coa6 has a 3-helical bundle structure, with the first 2 helices tethered by disulfide bonds, one of which likely provides the copper-binding site. Disulfide-mediated oligomerization of the W59CCoa6 protein provides a structural explanation for the loss-of-function mutation.

The study of vacuolar-type ATPases by single particle electron microscopy

2014 ◽  
Vol 92 (6) ◽  
pp. 460-466 ◽  
Author(s):  
Jianhua Zhao ◽  
John L. Rubinstein
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Catalytic Mechanism ◽  
Protein Complexes ◽  
Protein Assemblies ◽  
Functional Studies ◽  
Large Structures ◽  
Negative Stain ◽  
And Function ◽  
Complex Activities

Nature’s molecular machines often work through the concerted action of many different protein subunits, which can give rise to large structures with complex activities. Vacuolar-type ATPases (V-ATPases) are membrane-embedded protein assemblies with a unique rotary catalytic mechanism. The dynamic nature and instability of V-ATPases make structural and functional studies of these enzymes challenging. Electron microscopy (EM) techniques, especially single particle electron cryomicroscopy (cryo-EM) and negative-stain EM, have provided extensive insight into the structure and function of these protein complexes. This minireview outlines what has been learned about V-ATPases using electron microscopy, highlights current challenges for their structural study, and discusses what cryo-EM will allow us to learn about these fascinating enzymes in the future.

scholarly journals FAMOSS, a conserved 41-aa peptide involved in plant tip growth regulation

2021 ◽  
Author(s):  
Anna Mamaeva ◽  
Andrey Kniazev ◽  
Ilia Sedlov ◽  
Nina Golub ◽  
Daria Kharlampieva ◽  
...  
Keyword(s):  
Growth Regulation ◽  
Opposite Effect ◽  
Transmembrane Domain ◽  
Functional Characterization ◽  
Small Gtpase ◽  
Reading Frame ◽  
Functional Studies ◽  
Non Coding Rnas ◽  
Small Open Reading Frame

Recent evidence shows that small open reading frame (smORF; <100 codons)-encoded peptides (SEPs) containing transmembrane domains are preadapted to be progenitors of novel functional genes. A dozen of such SEPs translated from long non-coding RNAs (lncRNAs) are already functionally characterised in animals. However, functional plant lncRNA-smORF-coded peptides are not yet described. Here, we report detailed functional characterization of a 41-aa peptide encoded by lncRNA-smORFs in the moss Physcomitrium patens, which was named "FAst-growing MOSS" (FAMOSS). We found that the FAMOSS interacts with the Rab-type small GTPase proteins and its overexpression leads to faster moss growth rate and more intensive vesicular transport in apical cells, while its knockout results in the opposite effect. The FAMOSS contains a predicted transmembrane domain and possible orthologs from streptophyta algae to flowering plants have a very conserved structure. Thus, the FAMOSS peptide is a previously unknown conserved player of Rab-mediated processes in plants. Our findings are in line with functional studies of transmembrane SEPs in animals and prove the principles of SEPs evolution. This study provides new insights into functions of plant lncRNA-smORFs.

scholarly journals Short Communication: Identification of Mildew Locus O (MLO) genes in Durio zibethinus genome corresponding with the Powdery Mildew disease

2018 ◽  
Vol 19 (6) ◽  
pp. 2204-2212
Author(s):  
RAHMAT AZHARI KEMAL ◽  
ERIC BERNARDUS L. SANDJAJA ◽  
AUDI PUTRA SANTOSA ◽  
JEREMIAS IVAN
Keyword(s):  
Powdery Mildew ◽  
Short Communication ◽  
Functional Characterization ◽  
Functional Studies ◽  
Durio Zibethinus ◽  
Common Motif ◽  
Powdery Mildew Disease ◽  
Intracellular Domains

Kemal RA, Sandjaja EBL, Santosa AP, Ivan J. 2018. Short Communication: Identification of Mildew Locus O (MLO) genes in Durio zibethinus genome corresponding with the Powdery Mildew disease. Biodiversitas 19: 2204-2212. Mildew Locus O (MLO) is a protein consisting of seven transmembrane domains and appears in the various type of plants. MLO proteins are classified into seven clades. It is known that specific clades have different roles in a plant. MLOs from Clades IV and V have been linked to plant's susceptibility to Powdery Mildew (PM) disease. This study aimed to provide an overview of MLO genes present in durian (Durio zibethinus) genome. Bioinformatic analyses were conducted to analyze the phylogeny and structure of MLO genes and proteins in durian. The result showed that there were 20 putative DzMLO genes in durian, encoding 39 putative DzMLO proteins. Durian MLOs belong to Clade I-VI with one protein belongs to Clade IV and five proteins belong to Clade V. Those six MLO proteins shared a common motif in C-terminal and second intracellular domains. Putative alternative splicing and differential expressions were observed among Clade V DzMLO genes. These findings will facilitate the functional characterization of MLO genes and proteins in durian. Functional studies, especially on C-terminal and second intracellular domains, need to be conducted to elucidate the role of MLO in PM susceptibility in durian.

scholarly journals Beyond classic editing: innovative CRISPR approaches for functional studies of long non-coding RNA

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Dahlia A Awwad
Keyword(s):  
Genetic Manipulation ◽  
Functional Characterization ◽  
The Novel ◽  
Functional Studies ◽  
Non Coding Rna ◽  
Rna Targeting ◽  
Potential Applications ◽  
And Function ◽  
Long Non Coding Rna

Abstract Long non-coding RNAs (lncRNAs) makeup a considerable part of the non-coding human genome and had been well-established as crucial players in an array of biological processes. In spite of their abundance and versatile roles, their functional characteristics remain largely undiscovered mainly due to the lack of suitable genetic manipulation tools. The emerging CRISPR/Cas9 technology has been widely adapted in several studies that aim to screen and identify novel lncRNAs as well as interrogate the functional properties of specific lncRNAs. However, the complexity of lncRNAs genes and the regulatory mechanisms that govern their transcription, as well as their unique functionality pose several limitations the utilization of classic CRISPR methods in lncRNAs functional studies. Here, we overview the unique characteristics of lncRNAs transcription and function and the suitability of the CRISPR toolbox for applications in functional characterization of lncRNAs. We discuss some of the novel variations to the classic CRISPR/Cas9 system that have been tailored and applied previously to study several aspects of lncRNAs functionality. Finally, we share perspectives on the potential applications of various CRISPR systems, including RNA-targeting, in the direct editing and manipulation of lncRNAs.

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