The induced fit and conformational selection/population shift models are two extreme cases of a continuum aimed at understanding the mechanism by which the final key-lock or active enzyme conformation is achieved upon formation of the correctly ligated enzyme. Structures of complexes representing the Michaelis and enolate intermediate complexes of the reaction catalyzed by phosphoenolpyruvate carboxykinase provide direct structural evidence for the encounter complex that is intrinsic to the induced fit model and not required by the conformational selection model. In addition, the structural data demonstrate that the conformational selection model is not sufficient to explain the correlation between dynamics and catalysis in phosphoenolpyruvate carboxykinase and other enzymes in which the transition between the uninduced and the induced conformations occludes the active site from the solvent. The structural data are consistent with a model in that the energy input from substrate association results in changes in the free energy landscape for the protein, allowing for structural transitions along an induced fit pathway.enzyme dynamics ͉ population shift ͉ phosphoenolpyruvate carboxykinase I t has been 50 years since the original presentation of the induced fit hypothesis by Daniel Koshland (1) that built upon Emil Fisher's key-lock principle (2) and introduced the idea that the inherent dynamic properties of enzymes play an essential role in the processes of molecular recognition and catalysis. Over the last decade, with the advent of new instrumentation and novel experimental approaches there has been a resurgence in investigations focused upon understanding the specific ways in that these dynamic properties influence the ability of enzymes to achieve enormous levels of selectivity and catalytic power (refs. 3-6 and references therein).Presently there are two primary models used to explain the mechanism by which an enzyme can move toward adopting the final key-lock state ( Fig. 1): (i) the induced fit model and (ii) the conformational selection/population shift model (7-14). As defined originally by Koshland (1), the induced fit model states that the achievement of the final key-lock state, that is, the precise orientation of catalytic groups and residues necessary for catalysis, occurs only after changes in protein structure that are induced by the binding of a substrate. This pathway is represented by the equilibrium constants K 1 and K 2 in Fig. 1. Intrinsic to this model is the ability of the enzyme to form an ''encounter complex'' † (Fig. 1B). In this complex the enzyme is liganded identically to the eventual catalytically competent key-lock state; however, the conformational change leading to the active key-lock state has not yet occurred. In contrast, the conformational selection model states that the enzyme exists in multiple conformational states, of which the substrate has a high affinity for the active or key-lock state. In this model the ligand is suggested to bind to this lowly populated high-energy ...

Protein aggregation is a hallmark of many diseases, including amyotrophic lateral sclerosis (ALS), where aggregation of Cu/Zn superoxide dismutase (SOD1) is implicated in causing neurodegeneration. Recent studies have suggested that destabilization and aggregation of the most immature form of SOD1, the disulfide-reduced, unmetallated (apo) protein is particularly important in causing ALS. We report herein in depth analyses of the effects of chemically and structurally diverse ALS-associated mutations on the stability and aggregation of reduced apo SOD1. In contrast with previous studies, we find that various reduced apo SOD1 mutants undergo highly reversible thermal denaturation with little aggregation, enabling quantitative thermodynamic stability analyses. In the absence of ALS-associated mutations, reduced apo SOD1 is marginally stable but predominantly folded. Mutations generally result in slight decreases to substantial increases in the fraction of unfolded protein. Calorimetry, ultracentrifugation, and light scattering show that all mutations enhance aggregation propensity, with the effects varying widely, from subtle increases in most cases, to pronounced formation of 40–100 nm soluble aggregates by A4V, a mutation that is associated with particularly short disease duration. Interestingly, although there is a correlation between observed aggregation and stability, there is minimal to no correlation between observed aggregation, predicted aggregation propensity, and disease characteristics. These findings suggest that reduced apo SOD1 does not play a dominant role in modulating disease. Rather, additional and/or multiple forms of SOD1 and additional biophysical and biological factors are needed to account for the toxicity of mutant SOD1 in ALS.

A systematic correlation between finite element models (FEMs) and histopathology is needed to define deformation thresholds associated with traumatic brain injury (TBI). In this study, a FEM of a transected piglet brain was used to reverse engineer the range of optimal shear moduli for infant (5-day-old, 553-658 Pa) and 4-week old toddler piglet brain, (692-811 Pa) from comparisons with measured in situ tissue strains. The more mature brain modulus was found to have significant strain and strain rate dependencies not observed with the infant brain. Age-appropriate FEMs were then used to simulate experimental TBI in infant (n=36) and pre-adolescent (n=17) piglets undergoing a range of rotational head loads. The experimental animals were evaluated for the presence of clinically significant traumatic axonal injury (TAI), which was then correlated with FEM-calculated measures of overall and white matter tract-oriented tissue deformations, and used to identify the metric with the highest sensitivity and specificity for detecting TAI. The best predictors of TAI were the tract-oriented strain (6–7%), strain rate (38–40 s−1), and strain times strain rate (1.3–1.8 s−1) values exceeded by 90% of the brain. These tract-oriented strain and strain rate thresholds for TAI were comparable to those found in isolated axonal stretch studies. Furthermore, we proposed that the higher degree of agreement between tissue distortion aligned with white matter tracts and TAI may be the underlying mechanism responsible for more severe TAI after horizontal and sagittal head rotations in our porcine model of nonimpact TAI than coronal plane rotations.

Animal approach-avoidance conflict paradigms have been used extensively to characterize effects of anxiolytic agents and probe neural circuitry related to anxiety. However, there are few behavioral approaches to measure conflict in human populations, limiting the translation of findings from animal conflict tasks to human clinical research. We developed a novel approachavoidance conflict (AAC) paradigm involving situations in which the same decision is associated with "reward" (points) and "punishment" (negative affective stimuli). The AAC task was completed by 95 young adults (56 female) with varying levels of self-reported trait anxiety. As expected, conflict-related approach behavior correlated with self-reported motivation to approach reward and avoid punishment and greater reward level increased approach behavior. Additionally, females exhibited less approach behavior than males. Anxiety Sensitivity Index (Physical subscale) scores related negatively to approach behavior for males, while Behavioral Activation Scale (BAS, Fun Seeking subscale) scores related positively to approach behavior for females. Results support the utility of the AAC task as a behavioral test that has strong reverse translational features. Findings indicate that approach drives and anxiety sensitivity may be important in determining conflict behavior for females and males respectively. The approach-avoidance Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript conflict task offers a novel, translational measure to probe neural systems underlying conflict behavior, motivational processes, and anxiety disorders.

Brief measures of anxiety related severity and impairment that can be used across anxiety disorders and with subsyndromal anxiety are lacking. The Overall Anxiety Severity and Impairment Scale (OASIS) have shown strong psychometric properties with college students and primary care patients. This study examines sensitivity, specificity, and efficiency of an abbreviated version of the OASIS that takes only 2-3 minutes to complete using a non-clinical (college student) sample. 48 participants completed the OASIS and SCID for anxiety disorders, 21 had a diagnosis of ≥1 anxiety disorder, and 4 additional participants had a subthreshold diagnosis. A cut-score of 8 best discriminated those with anxiety disorders from those without, successfully classifying 78% of the sample with 69% sensitivity and 74% specificity. Results from a larger sample (n=171) showed a single factor structure and excellent convergent and divergent validity. The availability of cut-scores for a non-clinical sample furthers the utility of this measure for settings where screening or brief assessment of anxiety is needed.