Transient local secondary structure in the intrinsically disordered C-term of the Albino3 insertase.

Albino3 (Alb3) is an integral membrane protein fundamental to the targeting and insertion of light-harvesting complex (LHC) proteins into the thylakoid membrane. Alb3 contains a stroma-exposed C-terminus (Alb3-Cterm) that is responsible for binding the LHC-loaded transit complex before LHC membrane insertion. Alb3-Cterm has been reported to be intrinsically disordered, but precise mechanistic details underlying how it recognizes and binds to the transit complex are lacking, and the functional roles of its four different motifs have been debated.

An Effective Electric Dipole Model for Voltage-induced Gating Mechanism of Lysenin.

Lysenin is a pore-forming toxin, which self-inserts open channels into sphingomyelin containing membranes and is known to be voltage regulated. The mechanistic details of its voltage gating mechanism, however, remains elusive despite much recent efforts. Here, we have employed a novel combination of experimental and computational techniques to examine a model for voltage gating, that is based on the existence of an "effective electric dipole" inspired by recent reported structures of lysenin.

Adhesive force between graphene nanoscale flakes and living biological cells.

We report on a measurement technique that quantifies the adhesive force between multi-layers of graphene flakes and the cell wall of live Escherichia coli cells using atomic force microscopy (AFM) in-fluid Peak Force- Quantitative Nanomechanical Mapping mode. To measure the adhesive force, we made use of the negative charge of E. coli cells to allow them to stick to positively charged surfaces, such as glass or silicon, that were covered by poly-L-Lysine.

Domain Organization in the 54-kDa Subunit of the Chloroplast Signal Recognition Particle.

Chloroplast signal recognition particle (cpSRP) is a heterodimer composed of an evolutionarily conserved 54-kDa GTPase (cpSRP54) and a unique 43-kDa subunit (cpSRP43) responsible for delivering light-harvesting chlorophyll binding protein to the thylakoid membrane. While a nearly complete three-dimensional structure of cpSRP43 has been determined, no high-resolution structure is yet available for cpSRP54. In this study, we developed and examined an in silico three-dimensional model of the structure of cpSRP54 by homology modeling using cytosolic homologs.

Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms.

Regulation of Structural Dynamics within a Signal Recognition Particle Promotes Binding of Protein Targeting Substrates.

Protein targeting is critical in all living organisms and involves a signal recognition particle (SRP), an SRP receptor, and a translocase. In co-translational targeting, interactions among these proteins are mediated by the ribosome. In chloroplasts, the light-harvesting chlorophyll-binding protein (LHCP) in the thylakoid membrane is targeted post-translationally without a ribosome.

First human study of a chimeric anti-methamphetamine monoclonal antibody in healthy volunteers.

This first-in-human study examined the safety and pharmacokinetics of ch-mAb7F9, an anti-methamphetamine monoclonal antibody, in healthy volunteers. Single, escalating doses of ch-mAb7F9 over the range of 0.2 to 20 mg/kg were administered to 42 subjects who were followed for 147 d.

Preclinical characterization of an anti-methamphetamine monoclonal antibody for human use.

Ch-mAb7F9, a human-mouse chimeric monoclonal antibody (mAb) designed to bind (+)-methamphetamine (METH) with high affinity and specificity, was produced as a treatment medication for METH abuse. In these studies, we present the preclinical characterization that provided predictive evidence that ch-mAb7F9 may be safe and effective in humans. In vitro ligand binding studies showed that ch-mAb7F9 is specific for and only binds its target ligands (METH, (+)-amphetamine, and 3,4-methylenedioxy-N-methylamphetamine) with high affinity.

A model for the hysteresis observed in gating of lysenin channels.

The pore-forming toxin lysenin self-inserts to form conductance channels in natural and artificial lipid membranes containing sphingomyelin. The inserted channels exhibit voltage regulation and hysteresis of the macroscopic current during the application of positive periodic voltage stimuli. We explored the bi-stable behavior of lysenin channels and present a theoretical approach for the mechanism of the hysteresis to explain its static and dynamic components.

Radiation-induced galectin-1 by endothelial cells: a promising molecular target for preferential drug delivery to the tumor vasculature.

The present study reports on a new strategy for selective, radiation therapy-amplified drug delivery using an antiangiogenic 33-a.a., tumor vasculature-targeting ligand, anginex, to improve the therapeutic ratio for strategies developed against solid tumors.

Bi-stability, hysteresis, and memory of voltage-gated lysenin channels.

Lysenin, a 297 amino acid pore-forming protein extracted from the coelomic fluid of the earthworm E. foetida, inserts constitutively open large conductance channels in natural and artificial lipid membranes containing sphingomyelin. The inserted channels show voltage regulation and slowly close at positive applied voltages.

Potential analytical applications of lysenin channels for detection of multivalent ions.

Transmembrane protein transporters possessing binding sites for ions, toxins, pharmaceutical drugs, and other molecules constitute excellent candidates for developing sensitive and selective biosensing devices. Their attractiveness for analytical purposes is enhanced by the intrinsic amplification capabilities shown when the binding event leads to major changes in the transportation of ions or molecules other than the analyte itself. The large-scale implementation of such transmembrane proteins in biosensing devices is limited by the difficulties encountered in inserting functional transporters into artificial bilayer lipid membranes and by the limitations in understanding and exploiting the changes induced by the interaction with the analyte for sensing purposes.

A dynamic cpSRP43-Albino3 interaction mediates translocase regulation of chloroplast signal recognition particle (cpSRP)-targeting components.

The chloroplast signal recognition particle (cpSRP) and its receptor, chloroplast FtsY (cpFtsY), form an essential complex with the translocase Albino3 (Alb3) during post-translational targeting of light-harvesting chlorophyll-binding proteins (LHCPs). Here, we describe a combination of studies that explore the binding interface and functional role of a previously identified cpSRP43-Alb3 interaction. Using recombinant proteins corresponding to the C terminus of Alb3 (Alb3-Cterm) and various domains of cpSRP43, we identify the ankyrin repeat region of cpSRP43 as the domain primarily responsible for the interaction with Alb3-Cterm.

Multivalent ions control the transport through lysenin channels.

We report the effect of different ions on the conducting properties of lysenin channels inserted into planar lipid bilayer membranes. Our observations indicated that multivalent ions inhibited the lysenin channels conductance in a concentration dependent manner. The analysis performed on single channels revealed that multivalent ions induced reversible sub-conducting or closed states depending on the ionic charge and size.

Identification and characterization of native proteins of Escherichia coli BL-21 that display affinity towards Immobilized Metal Affinity Chromatography and Hydrophobic Interaction Chromatography Matrices.

The purpose of this study was to identify and characterize Escherichia coli proteins which display affinity towards both Immobilized Metal Affinity Chromatography (IMAC) and Hydrophobic Interaction Chromatography (HIC). Co(II) IMAC was chosen as the primary capture step, followed by HIC employing different concentrations of salt to promote adsorption. Results provided insight on this rather small pool of E.

Controlled gating of lysenin pores.

Lysenin forms unitary large conductance pores in artificial bilayer membranes containing sphingomyelin. A population of lysenin pores inserted into such a bilayer membrane exhibited a dynamic negative conductance region, as predicted by a simple two-state model for voltage-gated channels. The recorded I-V curves demonstrated that lysenin pores inserted into the bilayer are uniformly oriented.

1H, 13C and 15N resonance assignments of the C-terminal domain of the 43 kDa subunit of the chloroplast signal recognition particle.

We report the assignment of a 109 amino acid C-terminal chromo domain of the chloroplast signal recognition particle cpSRP43 subunit. cpSRP43 plays a crucial role in the targeting of light harvesting chlorophyll proteins to the thylakoids.

The membrane-binding motif of the chloroplast signal recognition particle receptor (cpFtsY) regulates GTPase activity.

The chloroplast signal recognition particle (cpSRP) and its receptor (cpFtsY) function in thylakoid biogenesis to target integral membrane proteins to thylakoids. Unlike cytosolic SRP receptors in eukaryotes, cpFtsY partitions between thylakoid membranes and the soluble stroma. Based on sequence alignments, a membrane-binding motif identified in Escherichia coli FtsY appears to be conserved in cpFtsY, yet whether the proposed motif is responsible for the membrane-binding function of cpFtsY has yet to be shown experimentally.

H, C and N resonance assignments of the C-terminal domain of the 43 kDa subunit of the chloroplast signal recognition particle.

We report the assignment of a 109 amino acid C-terminal chromo domain of the chloroplast signal recognition particle cpSRP43 subunit. cpSRP43 plays a crucial role in the targeting of light harvesting chlorophyll proteins to the thylakoids.

Assembly of chloroplast signal recognition particle involves structural rearrangement in cpSRP43.

Signal recognition particle in chloroplasts (cpSRP) exhibits the unusual ability to bind and target full-length proteins to the thylakoid membrane. Unlike cytosolic SRPs in prokaryotes and eukaryotes, cpSRP lacks an RNA moiety and functions as a heterodimer composed of a conserved 54-kDa guanosine triphosphatase (cpSRP54) and a unique 43-kDa subunit (cpSRP43). Assembly of the cpSRP heterodimer is a prerequisite for post-translational targeting activities and takes place through interactions between chromatin modifier domain 2 (CD2) of cpSRP43 and a unique 10-amino-acid region in cpSRP54 (cpSRP54(pep)).

Dynamics of saxitoxin binding to saxiphilin c-lobe reveals conformational change.

Thermodynamic parameters (DeltaG, DeltaH, DeltaS, DeltaC(p)) have been determined to evaluate the dynamics of binding of saxitoxin to the c-lobe of saxiphilin. We have developed an improved method to rapidly express and purify recombinant saxiphilin c-lobe, and fully characterized it by mass spectrometry for the first time. Surface plasmon resonance (SPR) was used to characterize the interaction between saxitoxin and immobilized c-lobe.

Canonical signal recognition particle components can be bypassed for posttranslational protein targeting in chloroplasts.

The chloroplast signal recognition particle (cpSRP) and its receptor (cpFtsY) target proteins both cotranslationally and posttranslationally to the thylakoids. This dual function enables cpSRP to utilize its posttranslational activities for targeting a family of nucleus-encoded light-harvesting chlorophyll binding proteins (LHCPs), the most abundant membrane proteins in plants. Previous in vitro experiments indicated an absolute requirement for all cpSRP pathway soluble components.

Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse.

When generating monoclonal antibodies (mAb) against small molecules, the chemical composition and molecular orientation of the drug-like hapten on the antigen is a crucial determinant. This is especially important when attempting to discover therapeutic mAb against the drugs of abuse (+)-methamphetamine [(+)-METH], (+)-amphetamine [(+)-AMP], and the related compound (+)-3,4-methylenedioxymethamphetamine [(+)-MDMA, the plus isomer in the racemic mixture known as MDMA or ecstasy]. The goal of these studies was to design and synthesize (+)-METH-like haptens with structural attributes that could make them effective for generating monoclonal antibodies for treating medical problems associated with these stimulant drugs of abuse.

Monoclonal antibody form and function: manufacturing the right antibodies for treating drug abuse.

Drug abuse continues to be a major national and worldwide problem, and effective treatment strategies are badly needed. Antibodies are promising therapies for the treatment of medical problems caused by drug abuse, with several candidates in preclinical and early clinical trials. Monoclonal antibodies can be designed that have customized affinity and specificity against drugs of abuse, and because antibodies can be designed in various forms, in vivo pharmacokinetic characteristics can be tailored to suit specific clinical applications (eg, long-acting for relapse prevention, or short-acting for overdose).