Refining the Quality of Monoclonal Antibodies: Grafting Unique Peptide-Binding Site in the Fab Framework.

Monoclonal antibodies (mAbs) are a major therapeutic modality. Grafting the meditope binding site onto mAbs, also known as meditope-enabling, can extend the usefulness of mAbs by providing an additional protein-protein interaction surface without altering the stability or antigen binding. We have previously used this site for attaching dyes, cytotoxic drugs, and entire proteins.

Template-Catalyzed, Disulfide Conjugation of Monoclonal Antibodies Using a Natural Amino Acid Tag.

The high specificity and favorable pharmacological properties of monoclonal antibodies (mAbs) have prompted significant interest in re-engineering this class of molecules to add novel functionalities for enhanced therapeutic and diagnostic potential. Here, we used the high affinity, meditope-Fab interaction to template and drive the rapid, efficient, and stable site-specific formation of a disulfide bond. We demonstrate that this template-catalyzed strategy provides a consistent and reproducible means to conjugate fluorescent dyes, cytotoxins, or "click" chemistry handles to meditope-enabled mAbs (memAbs) and memFabs.

Photoactivation and relaxation studies on the cyanobacterial orange carotenoid protein in the presence of copper ion.

Photosynthesis starts with absorption of light energy by light-harvesting antenna complexes with subsequent production of energy-rich organic compounds. However, all photosynthetic organisms face the challenge of excess photochemical conversion capacity. In cyanobacteria, non-photochemical quenching (NPQ) performed by the orange carotenoid protein (OCP) is one of the most important mechanisms to regulate the light energy captured by light-harvesting antennas.

Engineered holocytochrome synthases that biosynthesize new cytochromes .

Cytochrome (cyt ), required for electron transport in mitochondria, possesses a covalently attached heme cofactor. Attachment is catalyzed by holocytochrome synthase (HCCS), leading to two thioether bonds between heme and a conserved CXXCH motif of cyt In cyt , histidine (His19) of CXXCH acts as an axial ligand to heme iron and upon release of holocytochrome from HCCS, folding leads to formation of a second axial interaction with methionine (Met81). We previously discovered mutations in human HCCS that facilitate increased biosynthesis of cyt in recombinant Focusing on HCCS E159A, novel cyt variants in quantities that are sufficient for biophysical analysis are biosynthesized.

Native Mass Spectrometry Analysis of Oligomerization States of Fluorescence Recovery Protein and Orange Carotenoid Protein: Two Proteins Involved in the Cyanobacterial Photoprotection Cycle.

The orange carotenoid protein (OCP) and fluorescence recovery protein (FRP) are present in many cyanobacteria and regulate an essential photoprotection cycle in an antagonistic manner as a function of light intensity. We characterized the oligomerization states of OCP and FRP by using native mass spectrometry, a technique that has the capability of studying native proteins under a wide range of protein concentrations and molecular masses. We found that dimeric FRP is the predominant state at protein concentrations ranging from 3 to 180 μM and that higher-order oligomers gradually form at protein concentrations above this range.

Transcriptomic analysis illuminates genes involved in chlorophyll synthesis after nitrogen starvation in Acaryochloris sp. CCMEE 5410.

Acaryochloris species are a genus of cyanobacteria that utilize chlorophyll (chl) d as their primary chlorophyll molecule during oxygenic photosynthesis. Chl d allows Acaryochloris to harvest red-shifted light, which gives them the ability to live in filtered light environments that are depleted in visible light. Although genomes of multiple Acaryochloris species have been sequenced, their analysis has not revealed how chl d is synthesized.

Dramatic Domain Rearrangements of the Cyanobacterial Orange Carotenoid Protein upon Photoactivation.

Photosynthetic cyanobacteria make important contributions to global carbon and nitrogen budgets. A protein known as the orange carotenoid protein (OCP) protects the photosynthetic apparatus from damage by dissipating excess energy absorbed by the phycobilisome, the major light-harvesting complex in many cyanobacteria. OCP binds one carotenoid pigment, but the color of this pigment depends on conditions.

Chemical activation of the cyanobacterial orange carotenoid protein.

The effects of the Hofmeister series of ions on the activation of the orange carotenoid protein (OCP) from the inactive orange form to the active red form were tested. Kosmotropes led to lower OCP activation, whereas chaotropes led to greater OCP activation. Concentrations of thiocyanate exceeding 1.

Site-directed mutagenesis of the highly perturbed copper site of auracyanin D.

Type-1 copper proteins participate in redox reactions and biological catalysis. Significant variation exists within the electronic structure of type-1 copper sites, producing both blue and green proteins. Classical, "blue" sites have been extensively studied, but "green" sites have been poorly characterized.

Mass spectrometry footprinting reveals the structural rearrangements of cyanobacterial orange carotenoid protein upon light activation.

The orange carotenoid protein (OCP), a member of the family of blue light photoactive proteins, is required for efficient photoprotection in many cyanobacteria. Photoexcitation of the carotenoid in the OCP results in structural changes within the chromophore and the protein to give an active red form of OCP that is required for phycobilisome binding and consequent fluorescence quenching. We characterized the light-dependent structural changes by mass spectrometry-based carboxyl footprinting and found that an α helix in the N-terminal extension of OCP plays a key role in this photoactivation process.

Structural analysis of the homodimeric reaction center complex from the photosynthetic green sulfur bacterium Chlorobaculum tepidum.

The reaction center (RC) complex of the green sulfur bacterium Chlorobaculum tepidum is composed of the Fenna-Matthews-Olson antenna protein (FMO) and the reaction center core (RCC) complex. The RCC complex has four subunits: PscA, PscB, PscC, and PscD. We studied the FMO/RCC complex by chemically cross-linking the purified sample followed by biochemical and spectroscopic analysis.

Metalloproteins diversified: the auracyanins are a family of cupredoxins that stretch the spectral and redox limits of blue copper proteins.

The metal sites of electron transfer proteins are tuned for function. The type 1 copper site is one of the most utilized metal sites in electron transfer reactions. This site can be tuned by the protein environment from +80 mV to +680 mV in typical type 1 sites.

Alternative Complex III from phototrophic bacteria and its electron acceptor auracyanin.

Alternative Complex III (ACIII) is a multisubunit integral membrane protein electron transfer complex that is proposed to be an energy-conserving functional replacement for the bacterial cytochrome bc1 or b6f complexes. Clues to the structure and function of this novel complex come from its relation to other bacterial enzyme families. The ACIII complex has menaquinone: electron acceptor oxidoreductase activity and contains protein subunits with multiple Fe-S centers and c-type hemes.

Redirecting gaze to improve the cosmetic appearance of strabismus.

Purpose: We studied a behavioral technique in which strabismics can reduce the detectability of their eye turn by appropriately redirecting their gaze. We tested the efficacy of this technique for various amounts of strabismus and different positions of gaze.

Methods: We took digital images of a model gazing in different lateral directions and digitally edited them to simulate fixation with one eye on different lateral locations whereas the other eye was misaligned from the first by +/-0, 6, 12, 18, 24, and 30 Delta.