Impact of stirring material on formation of submicron and subvisible aggregates in mAbs by quantitative laser diffraction, dynamic light scattering and background membrane imaging.

Aggregation of monoclonal antibodies (mAbs) is the driving force for their undesirable immunogenic effects. There are multiple factors responsible for aggregation in therapeutic proteins. One significant cause is the process-related shear and interfacial stress generated due to impellers and stirrers. This investigation focuses on understanding the possible aggregation arising upon stirring mAb formulations using stirrers made of different materials. We used quantitative laser diffraction (qLD) to monitor and quantify the stirring induced formation of submicron and subvisible aggregates in the size range from 100 nm to 10 µm. We analysed various aspects of aggregate generation, such as onset of aggregation, particle size distribution, and concentration of aggregates generated using stirrers of different materials. We observed that mixing with stainless steel stirrers resulted in a quicker onset of aggregation and led to significantly higher concentrations of aggregates. Analysis of the stirred samples using dynamic light scattering (DLS) and background imaging technique (BMI) were conducted to complement the qLD analysis. All the three techniques resulted in a similar trend, showing presence of larger and higher quantities of aggregates in steel stirred samples, as compared to those stirred using PEEK and glass. Additionally, we performed SEC-HPLC to quantify the soluble fraction of monomer and recorded that the least amount was present in the steel stirred samples. This work highlights the need for optimizing the materials used for fabricating the stirrers/impellers.

Global protein dynamics as communication sensors in peptide synthetase domains.

Biological activity is governed by the timely redistribution of molecular interactions, and static structural snapshots often appear insufficient to provide the molecular determinants that choreograph communication. This conundrum applies to multidomain enzymatic systems called nonribosomal peptide synthetases (NRPSs), which assemble simple substrates into complex metabolites, where a dynamic domain organization challenges rational design to produce new pharmaceuticals. Using a nuclear magnetic resonance (NMR) atomic-level readout of biochemical transformations, we demonstrate that global structural fluctuations help promote substrate-dependent communication and allosteric responses, and impeding these global dynamics by a point-site mutation hampers allostery and molecular recognition. Our results establish global structural dynamics as sensors of molecular events that can remodel domain interactions, and they provide new perspectives on mechanisms of allostery, protein communication, and NRPS synthesis.

Minimizing Pervasive Artifacts in 4D Covariance Maps for Protein Side Chain NMR Assignments.

Nuclear magnetic resonance (NMR) is a mainstay of biophysical studies that provides atomic level readouts to formulate molecular mechanisms. Side chains are particularly important to derive mechanisms involving proteins as they carry functional groups, but NMR studies of side chains are often limited by challenges in assigning their signals. Here, we designed a novel computational method that combines spectral derivatives and matrix square-rooting to produce reliable 4D covariance maps from routinely acquired 3D spectra and facilitates side chain resonance assignments. Thus, we generate two 4D maps from 3D-HcccoNH and 3D-HCcH-TOCSY spectra that each help overcome signal overlap or sensitivity losses. These 4D maps feature HC-HSQCs of individual side chains that can be paired to assigned backbone amide resonances of individual aliphatic signals, and both are obtained from a single modified covariance calculation. Further, we present 4D maps produced using conventional triple resonance experiments to easily assign asparagine side chain amide resonances. The 4D covariance maps encapsulate the lengthy manual pattern recognition used in traditional assignment methods and distill the information as correlations that can be easily visualized. We showcase the utility of the 4D covariance maps with a 10 kDa peptidyl carrier protein and a 52 kDa cyclization domain from a nonribosomal peptide synthetase.

Covariance nuclear magnetic resonance methods for obtaining protein assignments and novel correlations.

Protein NMR resonance assignment can be a tedious and error prone process, and it is often a limiting factor in biomolecular NMR studies. Challenges are exacerbated in larger proteins, disordered proteins, and often alpha-helical proteins, owing to an increase in spectral complexity and frequency degeneracies. Here, several multi-dimensional spectra must be inspected and compared in an iterative manner before resonances can be assigned with confidence. Over the last two decades, covariance NMR has evolved to become applicable to protein multi-dimensional spectra. The method, previously used to generate new correlations from spectra of small organic molecules, can now be used to recast assignment procedures as mathematical operations on NMR spectra. These operations result in multidimensional correlation maps combining all information from input spectra and providing direct correlations between moieties that would otherwise be compared indirectly through reporter nuclei. Thus, resonances of sequential residues can be identified and side-chain signals can be assigned by visual inspection of 4D arrays. This review highlights advances in covariance NMR that permitted to generate reliable 4D arrays and describes how these arrays can be obtained from conventional NMR spectra.

A Disulfide Stabilized ß-Sandwich Defines the Structure of a New Cysteine Framework M-Superfamily Conotoxin.

The structure of a new cysteine framework (-C-CC-C-C-C-) "M"-superfamily conotoxin, Mo3964, shows it to have a ß-sandwich structure that is stabilized by inter-sheet cross disulfide bonds. Mo3964 decreases outward K(+) currents in rat dorsal root ganglion neurons and increases the reversal potential of the NaV1.2 channels. The structure of Mo3964 (PDB ID: 2MW7 ) is constructed from the disulfide connectivity pattern, i.e., 1-3, 2-5, and 4-6, that is hitherto undescribed for the "M"-superfamily conotoxins. The tertiary structural fold has not been described for any of the known conus peptides. NOE (549), dihedral angle (84), and hydrogen bond (28) restraints, obtained by measurement of (h3)JNC' scalar couplings, were used as input for structure calculation. The ensemble of structures showed a backbone root mean square deviation of 0.68 ± 0.18 Å, with 87% and 13% of the backbone dihedral (ϕ, ψ) angles lying in the most favored and additional allowed regions of the Ramachandran map. The conotoxin Mo3964 represents a new bioactive peptide fold that is stabilized by disulfide bonds and adds to the existing repertoire of scaffolds that can be used to design stable bioactive peptide molecules.

Mutations that alter the regulation of the chb operon of Escherichia coli allow utilization of cellobiose.

Wild-type strains of Escherichia coli are normally unable to metabolize cellobiose. However, cellobiose-positive (Cel(+)) mutants arise upon prolonged incubation on media containing cellobiose as the sole carbon source. We show that the Cel(+) derivatives carry two classes of mutations that act concertedly to alter the regulation of the chb operon involved in the utilization of N,N'-diacetylchitobiose. These consist of mutations that abrogate negative regulation by the repressor NagC as well as single base-pair changes in the transcriptional regulator chbR that translate into single-amino-acid substitutions. Introduction of chbR from two Cel(+) mutants resulted in activation of transcription from the chb promoter at a higher level in the presence of cellobiose, in reporter strains carrying disruptions of the chromosomal chbR and nagC. These transformants also showed a Cel(+) phenotype on MacConkey cellobiose medium, suggesting that the wild-type permease and phospho-beta-glucosidase, upon induction, could recognize, transport and cleave cellobiose respectively. This was confirmed by expressing the wild-type genes encoding the permease and phospho-beta-glucosidase under a heterologous promoter. Biochemical characterization of one of the chbR mutants, chbRN238S, showed that the mutant regulator makes stronger contact with the target DNA sequence within the chb promoter and has enhanced recognition of cellobiose 6-phosphate as an inducer compared with the wild-type regulator.