Hybrid Mass Spectrometry Applied across the Production of Antibody Biotherapeutics.

Post expression from the host cells, biotherapeutics undergo downstream processing steps before final formulation. Mass spectrometry and biophysical characterization methods are valuable for examining conformational and stoichiometric changes at these stages, although typically not used in biomanufacturing, where stability is assessed via bulk property studies. Here we apply hybrid MS methods to understand how solution condition changes impact the structural integrity of a biopharmaceutical across the processing pipeline.

Second Generation G-Quadruplex Stabilizing Trimethine Cyanines.

G-Quadruplex DNA has been recognized as a highly appealing target for the development of new selective chemotherapeutics, which could result in markedly reduced toxicity toward normal cells. In particular, the cyanine dyes that bind selectively to G-quadruplex structures without targeting duplex DNA have attracted attention due to their high amenability to structural modifications that allows fine-tuning of their biomolecular interactions. We have previously reported pentamethine and symmetric trimethine cyanines designed to effectively bind G-quadruplexes through end stacking interactions.

Fe(II)/Fe(III) Redox Process Can Significantly Modulate the Conformational Dynamics and Electrostatics of Pirin in NF-κB Regulation.

Pirin is an iron (Fe)-dependent regulatory protein of nuclear factor κB (NF-κB) transcription factors. Binding studies have suggested that the oxidative state of iron plays a crucial role in modulating the binding of Pirin to NF-κB p65, in turn enhancing the binding of p65 to DNA. The Fe(III) form of Pirin is the active form and binds to NF-κB, whereas the Fe(II) form does not bind to NF-κB.

Mechanisms of peptide hydrolysis by aspartyl and metalloproteases.

Peptide hydrolysis has been involved in a wide range of biological, biotechnological, and industrial applications. In this perspective, the mechanisms of three distinct peptide bond cleaving enzymes, beta secretase (BACE1), insulin degrading enzyme (IDE), and bovine lens leucine aminopeptidase (BILAP), have been discussed. BACE1 is a catalytic Asp dyad [Asp, Asp] containing aspartyl protease, while IDE and BILAP are mononuclear [Zn(His, His, Glu)] and binuclear [Zn1(Asp, Glu, Asp)-Zn2(Lys, Glu, Asp, Asp)] core possessing metallopeptidases, respectively.

Coupled Dynamics and Entropic Contribution to the Allosteric Mechanism of Pin1.

Allosteric communication in proteins regulates a plethora of downstream processes in subcellular signaling pathways. Describing the effects of cooperative ligand binding on the atomic level is a key to understanding many regulatory processes involving biomolecules. Here, we use microsecond-long molecular dynamics simulations to investigate the allosteric mechanism of Pin1, a potential therapeutic target and a phosphorylated-Ser/Thr dependent peptidyl-prolyl cis-trans isomerase that regulates several subcellular processes and has been implicated in many diseases, including cancer and Alzheimer's.

Conserved Hydration Sites in Pin1 Reveal a Distinctive Water Recognition Motif in Proteins.

Structurally conserved water molecules are important for biomolecular stability, flexibility, and function. X-ray crystallographic studies of Pin1 have resolved a number of water molecules around the enzyme, including two highly conserved water molecules within the protein. The functional role of these localized water molecules remains unknown and unexplored.

Pushing the Limits of a Molecular Mechanics Force Field To Probe Weak CH···π Interactions in Proteins.

The relationship among biomolecular structure, dynamics, and function is far from being understood, and the role of subtle, weak interactions in stabilizing different conformational states is even less well-known. The cumulative effect of these interactions has broad implications for biomolecular stability and recognition and determines the equilibrium distribution of the ensemble of conformations that are critical for function. Here, we accurately capture the stabilizing effects of weak CH···π interaction using an empirical molecular mechanics force field in excellent agreement with experiments.

An Iron Reservoir to the Catalytic Metal: THE RUBREDOXIN IRON IN AN EXTRADIOL DIOXYGENASE.

The rubredoxin motif is present in over 74,000 protein sequences and 2,000 structures, but few have known functions. A secondary, non-catalytic, rubredoxin-like iron site is conserved in 3-hydroxyanthranilate 3,4-dioxygenase (HAO), from single cellular sources but not multicellular sources. Through the population of the two metal binding sites with various metals in bacterial HAO, the structural and functional relationship of the rubredoxin-like site was investigated using kinetic, spectroscopic, crystallographic, and computational approaches.

Theoretical insights into the functioning of metallopeptidases and their synthetic analogues.

CONSPECTUS: The selective hydrolysis of a peptide or amide bond (-(O═)C-NH-) by a synthetic metallopeptidase is required in a wide range of biological, biotechnological, and industrial applications. In nature, highly specialized enzymes known as proteases and peptidases are used to accomplish this daunting task. Currently, many peptide bond cleaving enzymes and synthetic reagents have been utilized to achieve efficient peptide hydrolysis.

Computational perspective and evaluation of plausible catalytic mechanisms of peptidyl-prolyl cis-trans isomerases.

Background: Peptidyl prolyl cis-trans isomerization of the protein backbone is involved in the regulation of many biological processes. Cis-trans isomerization is notoriously slow and is catalyzed by a family of cis-trans peptidyl prolyl isomerases (PPIases) that have been implicated in many diseases. A general consensus on how these enzymes speed up prolyl isomerization has not been reached after decades of both experimental and computational studies.

Loss of intramolecular electrostatic interactions and limited conformational ensemble may promote self-association of cis-tau peptide.

Self-association of proteins can be triggered by a change in the distribution of the conformational ensemble. Posttranslational modification, such as phosphorylation, can induce a shift in the ensemble of conformations. In the brain of Alzheimer's disease patients, the formation of intra-cellular neurofibrillary tangles deposition is a result of self-aggregation of hyper-phosphorylated tau protein.

Computational Insights into Substrate and Site Specificities, Catalytic Mechanism, and Protonation States of the Catalytic Asp Dyad of β -Secretase.

In this review, information regarding substrate and site specificities, catalytic mechanism, and protonation states of the catalytic Asp dyad of β-secretase (BACE1) derived from computational studies has been discussed. BACE1 catalyzes the rate-limiting step in the generation of Alzheimer amyloid beta peptide through the proteolytic cleavage of the amyloid precursor protein. Due to its biological functioning, this enzyme has been considered as one of the most important targets for finding the cure for Alzheimer's disease.

Cysteine-mediated dynamic hydrogen-bonding network in the active site of Pin1.

Enzymes catalyze a plethora of chemical reactions that are tightly regulated and intricately coupled in biology. Catalysis of phosphorylation-dependent cis-trans isomerization of peptidyl-prolyl bonds, which act as conformational switches in regulating many post-phosphorylation processes, is considered to be one of the most critical. Pin1 is a cis-trans isomerase of peptidyl-prolyl(ω-) bonds of phosphorylated-Ser/Thr-Pro motifs and has been implicated in many diseases.

Comparative molecular dynamics simulation studies for determining factors contributing to the thermostability of chemotaxis protein "CheY".

Comparative molecular dynamics simulations of chemotaxis protein "CheY" from thermophilic origin Thermotoga maritima and its mesophilic counterpart Salmonella enterica have been performed for 10 ns each at 300 and 350 K, and 20 ns each at 400 and 450 K. The trajectories were analyzed in terms of different factors like root-mean-square deviation, root-mean-square fluctuation, radius of gyration, solvent accessible surface area, H-bonds, salt bridge content, and protein-solvent interactions which indicate distinct differences between the two of them. The two proteins also follow dissimilar unfolding pathways.

Elucidating the catalytic mechanism of β-secretase (BACE1): a quantum mechanics/molecular mechanics (QM/MM) approach.

In this quantum mechanics/molecular mechanics (QM/MM) study, the mechanisms of the hydrolytic cleavage of the Met2-Asp3 and Leu2-Asp3 peptide bonds of the amyloid precursor protein (WT-substrate) and its Swedish mutant (SW) respectively catalyzed by β-secretase (BACE1) have been investigated by explicitly including the electrostatic and steric effects of the protein environment in the calculations. BACE1 catalyzes the rate-determining step in the generation of Alzheimer amyloid beta peptides and is widely acknowledged as a promising therapeutic target. The general acid-base mechanism followed by the enzyme proceeds through the following two steps: (1) formation of the gem-diol intermediate and (2) cleavage of the peptide bond.

Hydrocarbons depending on the chain length and head group adopt different conformations within a water-soluble nanocapsule: 1H NMR and molecular dynamics studies.

In this study we have examined the conformational preference of phenyl-substituted hydrocarbons (alkanes, alkenes, and alkynes) of different chain lengths included within a confined space provided by a molecular capsule made of two host cavitands known by the trivial name "octa acid" (OA). One- and two-dimensional (1)H NMR experiments and molecular dynamics (MD) simulations were employed to probe the location and conformation of hydrocarbons within the OA capsule. In general, small hydrocarbons adopted a linear conformation while longer ones preferred a folded conformation.

Computational Insights into Dynamics of Protein Aggregation and Enzyme-Substrate Interactions.

In this Perspective, the roles of protein dynamics have been discussed in the aggregation of amyloid beta (Aβ) peptides and formation of enzyme-substrate complexes of beta-secretase (BACE1) and insulin-degrading enzyme (IDE). The studies regarding the influence of individual amino acid residues and specific regions on the structures and oligomerization of early Aβ aggregates and computations of their translational and rotational diffusion coefficients and order parameters exhibited that even the short-time-scale molecular dynamics simulations can reproduce certain experimental parameters with reasonable accuracy. The simulations elucidating the enzyme-substrate interactions of BACE1 and IDE successfully showed that the chemical nature and length of the substrates influence the dynamics and plasticity of both the enzyme and substrate.

Protonation states of the catalytic dyad of β-secretase (BACE1) in the presence of chemically diverse inhibitors: a molecular docking study.

In this molecular docking study, the protonation states of the catalytic Asp dyad of the beta-secretase (BACE1) enzyme in the presence of eight chemically diverse inhibitors have been predicted. BACE1 catalyzes the rate-determining step in the generation of Alzheimer amyloid beta peptides and is widely considered as a promising therapeutic target. All the inhibitors were redocked into their corresponding X-ray structures using a combination of eight different protonation states of the Asp dyad for each inhibitor.

Dimerization of the full-length Alzheimer amyloid β-peptide (Aβ42) in explicit aqueous solution: a molecular dynamics study.

In this study, the mechanism of dimerization of the full-length Alzheimer amyloid beta (Aβ42) peptide and structural properties of the three most stable dimers have been elucidated through 0.8 μs classical molecular dynamics (MD) simulations. The Aβ42 dimer has been reported to be the smallest neurotoxic species that adversely affects both memory and synaptic plasticity.

Mechanism of peptide hydrolysis by co-catalytic metal centers containing leucine aminopeptidase enzyme: a DFT approach.

In this density functional theory study, reaction mechanisms of a co-catalytic binuclear metal center (Zn1-Zn2) containing enzyme leucine aminopeptidase for two different metal bridging nucleophiles (H(2)O and -OH) have been investigated. In addition, the effects of the substrate (L-leucine-p-nitroanilide → L-leucyl-p-anisidine) and metal (Zn1 → Mg and Zn2 → Co, i.e.

Loss of cleavage at β'-site contributes to apparent increase in β-amyloid peptide (Aβ) secretion by β-secretase (BACE1)-glycosylphosphatidylinositol (GPI) processing of amyloid precursor protein.

Several lines of evidence implicate lipid raft microdomains in Alzheimer disease-associated β-amyloid peptide (Aβ) production. Notably, targeting β-secretase (β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1)) exclusively to lipid rafts by the addition of a glycosylphosphatidylinositol (GPI) anchor to its ectodomain has been reported to elevate Aβ secretion. Paradoxically, Aβ secretion is not reduced by the expression of non-raft resident S-palmitoylation-deficient BACE1 (BACE1-4C/A (C474A/C478A/C482A/C485A)).

Computational modeling of substrate specificity and catalysis of the β-secretase (BACE1) enzyme.

In this combined MD simulation and DFT study, interactions of the wild-type (WT) amyloid precursor protein (APP) and its Swedish variant (SW), Lys670 → Asn and Met671 → Leu, with the beta-secretase (BACE1) enzyme and their cleavage mechanisms have been investigated. BACE1 catalyzes the rate-limiting step in the generation of 40-42 amino acid long Alzheimer amyloid beta (Aβ) peptides. All key structural parameters such as position of the flap, volume of the active site, electrostatic binding energy, structures, and positions of the inserts A, D, and F and 10s loop obtained from the MD simulations show that, in comparison to the WT-substrate, BACE1 exhibits greater affinity for the SW-substrate and orients it in a more reactive conformation.

Cotinine reduces amyloid-β aggregation and improves memory in Alzheimer's disease mice.

Alzheimer's disease (AD) affects millions of people world-wide and new effective and safe therapies are needed. Cotinine, the main metabolite of nicotine, has a long half-life and does not have cardiovascular or addictive side effects in humans. We studied the effect of cotinine on amyloid-β (Aβ) aggregation as well as addressed its impact on working and reference memories.

Which one among aspartyl protease, metallopeptidase, and artificial metallopeptidase is the most efficient catalyst in peptide hydrolysis?

In this comparative DFT study, the hydrolysis of a peptide bond (Phe1-Phe2) by the following three types of catalysts has been studied: (1) beta-secretase (BACE2), (2) matrix metalloproteinase (MMP) and insulin degrading enzyme (IDE), and (3) [Pd(H(2)O)(4)](2+) (I(MPC)) and [Pd(2)(mu-OH)([18]aneN(6))](3+) (I(DPC)). The computed energetics predict that among these catalysts, the Zn(2+) metal center containing MMP is the most efficient in catalyzing this reaction. The two active site aspartate residues containing BACE2 catalyze this reaction with 5.

Computational insights into aspartyl protease activity of presenilin 1 (PS1) generating Alzheimer amyloid beta-peptides (Abeta40 and Abeta42).

In this combined bioinformatics, molecular dynamics (MD), and density functional theory study, mechanisms for the hydrolytic cleavage of Val-Ile and Ala-Thr peptide bonds of amyloid precursor protein by the intramembrane aspartyl protease presenilin 1 (PS1) have been elucidated. These processes lead to the formation of 40-42 amino acids long Alzheimer amyloid beta (Abeta) peptides (Abeta40 and Abeta42, respectively). In the absence of an X-ray structure of PS1, based on the substrate specificity and structural characteristics of the active site, another aspartyl protease BACE1 was selected as a model for PS1.