Decoupling of catalysis and transition state analog binding from mutations throughout a phosphatase revealed by high-throughput enzymology.

Using high-throughput microfluidic enzyme kinetics (HT-MEK), we measured over 9,000 inhibition curves detailing impacts of 1,004 single-site mutations throughout the alkaline phosphatase PafA on binding affinity for two transition state analogs (TSAs), vanadate and tungstate. As predicted by catalytic models invoking transition state complementary, mutations to active site and active-site-contacting residues had highly similar impacts on catalysis and TSA binding. Unexpectedly, most mutations to more distal residues that reduced catalysis had little or no impact on TSA binding and many even increased tungstate affinity. These disparate effects can be accounted for by a model in which distal mutations alter the enzyme's conformational landscape, increasing the occupancy of microstates that are catalytically less effective but better able to accommodate larger transition state analogs. In support of this ensemble model, glycine substitutions (rather than valine) were more likely to increase tungstate affinity (but not more likely to impact catalysis), presumably due to increased conformational flexibility that allows previously disfavored microstates to increase in occupancy. These results indicate that residues throughout an enzyme provide specificity for the transition state and discriminate against analogs that are larger only by tenths of an Ångström. Thus, engineering enzymes that rival the most powerful natural enzymes will likely require consideration of distal residues that shape the enzyme's conformational landscape and fine-tune active-site residues. Biologically, the evolution of extensive communication between the active site and remote residues to aid catalysis may have provided the foundation for allostery to make it a highly evolvable trait.

Incorporation of automated buffer exchange empowers high-throughput protein and plasmid purification for downstream uses.

The continued acceleration of time-to-market product development and rising demand for biotherapeutics have hastened the need for higher throughput within the biopharmaceutical industry. Automated liquid handlers (ALH) are increasingly popular due to flexible programming that enables processing of multiple samples with an array of functions. This flexibility is useful in streamlining research that requires chromatographic procedures to achieve product purity for downstream analysis. However, purification of biologics often requires additional off-deck buffer exchange steps due to undesirable elution conditions such as high acid or high salt content. Expanding the capability of ALHs to perform purification in sequence with buffer exchange would, therefore, increase workflow efficiency by eliminating the need for manual intervention, thus expediting sample preparation. Here we demonstrate two different automated purifications using pipet-based dispersive solid-phase extraction (dSPE). The first is an affinity purification of His-tagged proteins from bacterial lysate. The second is an anion-exchange purification of plasmid DNA. Both methods are followed by buffer exchange performed by an ALH. Percent recoveries for the three purified recombinant proteins ranged from 51 ± 1.2 to 86 ± 10%. The yields were inversely correlated to starting sample load and protein molecular weight. Yields for plasmid purification ranged between 11.4 ± 0.8 and 13.7 ± 0.9 µg, with the largest plasmid providing the highest yield. Both programs were rapid, with protein purification taking <80 min and plasmid purification <60 min. Our results demonstrate that high-quality, ready-to-use biologics can be obtained rapidly from a crude sample after two separate chromatographic processes without manual intervention.

Energetically unfavorable protein angles: Exploration of a conserved dihedral angle in triosephosphate isomerase.

Over the past 3.5 billion years of evolution, enzymes have adopted a myriad of conformations to suit life on earth. However, torsional angles of proteins have settled into limited zones of energetically favorable dihedrals observed in Ramachandran plots. Areas outside said zones are believed to be disallowed to all amino acids, except glycine, due to steric hindrance. Triosephosphate isomerase (TIM), a homodimer with a catalytic rate approaching the diffusion limit, contains an active site lysine residue (K13) with dihedrals within the fourth quadrant (Φ = +51/Ψ = -143). Both the amino acid and the dihedral angles are conserved across all species of TIM and known crystal structures regardless of ligand. Only crystal structures of the engineered monomeric version (1MSS) show accepted ß-sheet dihedral values of Φ = -135/Ψ = +170 but experiments show a 1000-fold loss in activity. Based on these results, we hypothesized that adopting the unfavorable torsion angle for K13 contributes to catalysis. Using both, computational and experimental approaches, four residues that interact with K13 (N11, M14, E97, and Q64) were mutated to alanine. In silico molecular dynamics (MD) simulations were performed using 2JK2 unliganded human TIM as a starting structure. Ramachandran plots, containing K13 dihedral values reveal full or partial loss of disallowed zone angles. N11A showed no detectable catalytic activity and lost the unfavorable K13 dihedral angles across four separate force fields during simulation while all other mutants plus wild type retained activity and retained the conserved K13 dihedral angles.

Effect of Bench Press Load Knowledge on One Repetition Maximum Strength.

ABSTRACT: Snarr, RL, Adams, K, and Cook, J. Effect of bench press load knowledge on one repetition maximum strength. J Strength Cond Res 35(8): 2121-2126, 2021-Knowledge of a given load and visual feedback are crucial while performing a given task. Although previous literature has examined the consequences of lifting a submaximal weight, the removal of load knowledge has yet to be examined under maximal effort. Therefore, the purpose of this investigation was to examine the differences in 1 repetition maximum (1RM) bench press values between a known and unknown external load. Twenty resistance-trained subjects (10 male subjects and 10 female subjects) completed 2 trials of a 1RM bench press protocol separated by 48-72 hours. The two 1RM trials consisted of (a) a traditional 1RM lift in which subjects could see the external load and (b) a 1RM lift in which the external load was blocked from view of the subject. Trials were randomized for all subjects. Results indicated that no differences were observed when the load was known vs. unknown within all subjects (p = 0.094; Cohen's d = 0.03; intraclass correlation coefficient = 0.99). In addition, 40% decreased and 15% increased their 1RM, despite the unknown condition. A statistical difference was found between the initial 1RM estimation vs. measured 1RM during the known load trial (p = 0.034, Cohen's d = 0.07). Practitioners should note that load knowledge before a maximal lift does not appear to present any significant detrimental effects on performance. Therefore, training during a "blinded" condition may present a unique modality and additional balance component that would not be presented during a traditional maximal lift. Further examination of attentional focus and muscle activation differences are warranted.

The Relationship Between the National Football League Scouting Combine and Game Performance Over a 5-Year Period.

Cook, J, Ryan, GA, Snarr, RL, and Rossi, S. The relationship between the National Football League scouting Combine and game performance over a 5-year period. J Strength Cond Res 34(8): 2492-2499, 2020-There has been doubt on the ability of the National Football League (NFL) Scouting Combine to predict successful future game performance. This study analyzed data from athletes who participated in the Combine between 2013 and 2017 (n = 1,537) and their subsequent year's performance in the NFL. Data from 6 athletic measures were normalized for each athlete when compared with all other athletes (avgCZ) and athletes of the same position (avgPZ). Correlational analysis was used to ascertain whether the physical performance tests were associated with subsequent year's game performance (avgS). A multiple linear regression was performed to examine whether individual event Combine performance could predict the subsequent year's avgS in the NFL. Of the 35 correlations found when examining relationships, only 2 correlations were found to be moderately strong, avgCZ-avgS2 (r = 0.320), avgPZ-avgS2 (r = 0.332), whereas most were found to be weak (r < 0.3). Furthermore, data analysis suggests that Combine measures can only explain approximately 2.6% of the variance in avgS 1 year after the Combine when using 3 (vertical jump, bench press, and PRO) performance tests as predictors. The primary results of this study suggest that the NFL Combine lacks predictive ability when examining first year game performance. Furthermore, it also lacks correlational strength when examining relationships between performance and subsequent 5-year performance in the NFL. Caution should be used if coaches, general managers, and other front office staff are considering the use of Combine data as a possible selection for the upcoming NFL Draft.

BGP-15 Protects against Oxaliplatin-Induced Skeletal Myopathy and Mitochondrial Reactive Oxygen Species Production in Mice.

Chemotherapy is a leading intervention against cancer. Albeit highly effective, chemotherapy has a multitude of deleterious side-effects including skeletal muscle wasting and fatigue, which considerably reduces patient quality of life and survivability. As such, a defense against chemotherapy-induced skeletal muscle dysfunction is required. Here we investigate the effects of oxaliplatin (OXA) treatment in mice on the skeletal muscle and mitochondria, and the capacity for the Poly ADP-ribose polymerase (PARP) inhibitor, BGP-15, to ameliorate any pathological side-effects induced by OXA. To do so, we investigated the effects of 2 weeks of OXA (3 mg/kg) treatment with and without BGP-15 (15 mg/kg). OXA induced a 15% (p < 0.05) reduction in lean tissue mass without significant changes in food consumption or energy expenditure. OXA treatment also altered the muscle architecture, increasing collagen deposition, neutral lipid and Ca2+ accumulation; all of which were ameliorated with BGP-15 adjunct therapy. Here, we are the first to show that OXA penetrates the mitochondria, and, as a possible consequence of this, increases mtROS production. These data correspond with reduced diameter of isolated FDB fibers and shift in the fiber size distribution frequency of TA to the left. There was a tendency for reduction in intramuscular protein content, albeit apparently not via Murf1 (atrophy)- or p62 (autophagy)- dependent pathways. BGP-15 adjunct therapy protected against increased ROS production and improved mitochondrial viability 4-fold and preserved fiber diameter and number. Our study highlights BGP-15 as a potential adjunct therapy to address chemotherapy-induced skeletal muscle and mitochondrial pathology.

Ontogeny of Feeding Mechanics in Smoothhound Sharks: Morphology and Cartilage Stiffness.

The diet of dusky smoothhound sharks, Mustelus canis, shifts over ontogeny from soft foods to a diet dominated by crabs. This may be accompanied by changes in the skeletal system that facilitates the capture and processing of large and bulky prey. The hyoid arch, for example, braces the jaws against the cranium, and generates suction for prey capture and intraoral transport. In this study, ontogenetic changes in the hyoid arch were investigated by quantifying size, mineralization, and stiffness to determine whether increasingly stiffer cartilages are associated with the dietary switch. Total length and length of the hyomandibula and ceratohyal cartilages over ontogeny were the proxy for body size. Cross-sectional area, percent mineralization, and second moment of area were quantified in 28 individuals spanning most of the natural size range. Mechanical compression tests were conducted to compare flexural stiffness to size. Our results show that the morphological characters tested for the hyomandibular and ceratohyal cartilages scales isometrically with length. While stiffness of the hyomandibular and ceratohyal cartilages scales isometrically with length when assessed on morphological characters alone (second moment of area), this relationship becomes allometric when mechanical properties are included (flexural stiffness). Thus, while the hyoid arch elements grow isometrically, the mechanical properties dictate a scaling relationship that dwarfs morphological characteristics. The various combinations of morphologies and ontogenetic trajectories of chondrichthyan species illustrate the tremendous flexibility that they possess in the functional organization of the feeding apparatus.