Understanding the [NiFe] Hydrogenase Active Site Environment through Ultrafast Infrared and 2D-IR Spectroscopy of the Subsite Analogue K[CpFe(CO)(CN)2] in Polar and Protic Solvents.

The [CpFe(CO)(CN)2]- unit is an excellent structural model for the Fe(CO)(CN)2 moiety of the active site found in [NiFe] hydrogenases. Ultrafast infrared (IR) pump-probe and 2D-IR spectroscopy have been used to study K[CpFe(CO)(CN)2] (M1) in a range of protic and polar solvents and as a dry film. Measurements of anharmonicity, intermode vibrational coupling strength, vibrational relaxation time, and solvation dynamics of the CO and CN stretching modes of M1 in H2O, D2O, methanol, dimethyl sulfoxide, and acetonitrile reveal that H-bonding to the CN ligands plays an important role in defining the spectroscopic characteristics and relaxation dynamics of the Fe(CO)(CN)2 unit. Comparisons of the spectroscopic and dynamic data obtained for M1 in solution and in a dry film with those obtained for the enzyme led to the conclusion that the protein backbone forms an important part of the bimetallic active site environment via secondary coordination sphere interactions.

Social cognitive disruptions in multiple sclerosis: The role of executive (dys)function.

OBJECTIVE: Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system, resulting in a range of potential motor and cognitive impairments. The latter can affect both executive functions that orchestrate general goal-directed behavior and social cognitive processes that support our ability to interact with others and maintain healthy interpersonal relationships. Despite a long history of research into the cognitive symptoms of MS, it remains uncertain if social cognitive disruptions occur independently of, or reflect underlying disturbances to, more foundational executive functions. The present preregistered study investigated this directly. METHOD: Employing an experimental design, we administered a battery of computerized tasks online to a large sample comprising 134 individuals with MS and 134 age- and sex-matched healthy controls (HCs). Three tasks measured elements of executive function (working memory, response inhibition, and switching) and two assessed components of social cognition disrupted most commonly in MS (emotion perception and theory of mind). RESULTS: Individuals with MS exhibited poorer working memory (d = .31), response inhibition (d = -.26), emotion perception (d = .32), and theory of mind (d = .35) compared with matched HCs. Furthermore, exploratory mediation analyses revealed that working memory performance accounted for approximately 20% of the group differences in both measures of social cognition. CONCLUSIONS: Disruptions to working memory appear to serve as one of the mechanisms underpinning disturbances to social cognition in MS. Future research should examine if the benefits of cognitive rehabilitation programs that incorporate working memory training transfer to these social cognitive processes. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Relationships between the race implicit association test and other measures of implicit and explicit social cognition.

Background: The race-based Implicit Association Test (IAT) was proposed to measure individual differences in implicit racial bias subsumed within social cognition. In recent years, researchers have debated the theoretical tenets underpinning the IAT, questioning whether performance on this task: (1) measures implicit attitudes that operate automatically outside of conscious awareness; (2) reflects individual differences in social cognition; and (3) can predict social behavior. One way to better address these research questions is to assess whether the race-IAT correlates with other implicit processes that are subsumed within social cognition. Aims: The current study assessed whether the race-IAT was related to other commonly used individual difference measures of implicit (and explicit) social cognition. Experiment 1 assessed whether dissociable patterns of performance on the race-IAT were related to measures of implicit imitative tendencies, emotion recognition and perspective taking toward White task actors, as well as explicit measures of trait and state affective empathy and racial bias. Overcoming limitations of task conceptual correspondence, Experiment 2 assessed whether these latter tasks were sensitive in detecting racial biases by using both White and Black task actors and again examined their relationships with the race-IAT. Method: In two lab-based experiments, 226 and 237 participants completed the race-IAT followed by an extensive battery of social cognition measures. Results: Across both experiments, pro-White/anti-Black bias on the race-IAT was positively related to a pro-White bias on explicit measures of positive affective empathy. However, relationships between the race-IAT and implicit imitative tendencies, perspective taking, emotion recognition, and explicit trait and negative state affective empathy were statistically equivalent. Conclusion: The race-IAT was consistently related to explicit measures of positive state affective empathy but not to other individual difference measures of implicit social cognition. These findings are discussed with regards to the theoretical underpinnings of the race-IAT as an individual difference measure of implicit social cognition, as well as alternative explanations relating to the reliability of social cognition measures and the various combinations of general-purpose (social and non-social) executive processes that underpin performance on these tasks.

Modulation of IL-17 backbone dynamics reduces receptor affinity and reveals a new inhibitory mechanism.

Knowledge of protein dynamics is fundamental to the understanding of biological processes, with NMR and 2D-IR spectroscopy being two of the principal methods for studying protein dynamics. Here, we combine these two methods to gain a new understanding of the complex mechanism of a cytokine:receptor interaction. The dynamic nature of many cytokines is now being recognised as a key property in the signalling mechanism. Interleukin-17s (IL-17) are proinflammatory cytokines which, if unregulated, are associated with serious autoimmune diseases such as psoriasis, and although there are several therapeutics on the market for these conditions, small molecule therapeutics remain elusive. Previous studies, exploiting crystallographic methods alone, have been unable to explain the dramatic differences in affinity observed between IL-17 dimers and their receptors, suggesting there are factors that cannot be fully explained by the analysis of static structures alone. Here, we show that the IL-17 family of cytokines have varying degrees of flexibility which directly correlates to their receptor affinities. Small molecule inhibitors of the cytokine:receptor interaction are usually thought to function by either causing steric clashes or structural changes. However, our results, supported by other biophysical methods, provide evidence for an alternate mechanism of inhibition, in which the small molecule rigidifies the protein, causing a reduction in receptor affinity. The results presented here indicate an induced fit model of cytokine:receptor binding, with the more flexible cytokines having a higher affinity. Our approach could be applied to other systems where the inhibition of a protein-protein interaction has proved intractable, for example due to the flat, featureless nature of the interface. Targeting allosteric sites which modulate protein dynamics, opens up new avenues for novel therapeutic development.

It's not what you do, it's the way that you do it: An experimental task delineates among passive, reactive and interactive styles of behaviour on social networking sites.

Studies have produced vastly disparate findings when exploring relationships between social networking site (SNS) usage and psychosocial well-being. These inconsistencies might reflect a lack of consideration for how people use SNS; specifically, while meaningful interactions are suggested to foster positive feelings, the passive consumption of others' feeds is proposed to have negative effects on users' well-being. To facilitate the empirical evaluation of these claims, the present study developed a computerised task to measure styles of usage on a mock SNS platform. Administering this Social Network Site Behaviour Task (SNSBT) online to 526 individuals, we identified three dissociable usage styles that extend the active-passive dichotomy employed frequently in the literature: passive use (consuming content posted by others), reactive use (reacting to others' content), and interactive use (interacting with others through content sharing). Furthermore, our data reveal that these usage styles differ on several measures of psychosocial variables employed frequently in the disparate literature: more interactive users reported greater feelings of social connectedness and social capital than passive or reactive users. Importantly, however, our results also reveal the multi-dimensional nature of usage styles, with online network size and time spent on SNS platforms serving as potentially confounding influences on some psychosocial measures. These findings not only advance our understanding of SNS behaviour by providing empirical support for theoretic propositions, but also demonstrate the utility of the SNSBT for experimental investigations into the psychosocial outcomes of different SNS usage styles.

Metasurface-enhanced mid-infrared spectroscopy in the liquid phase.

Vibrational spectroscopy is an important tool in chemical and biological analysis. A key issue when applying vibrational spectroscopy to dilute liquid samples is the inherently low sensitivity caused by short interaction lengths and small extinction coefficients, combined with low target molecule concentrations. Here, we introduce a novel type of surface-enhanced infrared absorption spectroscopy based on the resonance of a dielectric metasurface. We demonstrate that the method is suitable for probing vibrational bands of dilute analytes with a range of spectral linewidths. We observe that the absorption signal is enhanced by 1-2 orders of magnitude and show that this enhancement leads to a lower limit of detection compared to attenuated total reflection (ATR). Overall, the technique provides an important addition to the spectroscopist's toolkit especially for probing dilute samples.

A Novel Statistical Model for Predicting the Efficacy of Vagal Nerve Stimulation in Patients With Epilepsy (Pre-X-Stim) Is Applicable to Different EEG Systems.

Background: Identifying patients with intractable epilepsy who would benefit from therapeutic chronic vagal nerve stimulation (VNS) preoperatively remains a major clinical challenge. We have developed a statistical model for predicting VNS efficacy using only routine preimplantation electroencephalogram (EEG) recorded with the TruScan EEG device (Brazdil et al., 2019). It remains to be seen, however, if this model can be applied in different clinical settings. Objective: To validate our model using EEG data acquired with a different recording system. Methods: We identified a validation cohort of eight patients implanted with VNS, whose preimplantation EEG was recorded on the BrainScope device and who underwent the EEG recording according to the protocol. The classifier developed in our earlier work, named Pre-X-Stim, was then employed to classify these patients as predicted responders or non-responders based on the dynamics in EEG power spectra. Predicted and real-world outcomes were compared to establish the applicability of this classifier. In total, two validation experiments were performed using two different validation approaches (single classifier or classifier voting). Results: The classifier achieved 75% accuracy, 67% sensitivity, and 100% specificity. Only two patients, both real-life responders, were classified incorrectly in both validation experiments. Conclusion: We have validated the Pre-X-Stim model on EEGs from a different recording system, which indicates its application under different technical conditions. Our approach, based on preoperative EEG, is easily applied and financially undemanding and presents great potential for real-world clinical use.

Raising the bar: improving methodological rigour in cognitive alcohol research.

BACKGROUND AND AIMS: A range of experimental paradigms claim to measure the cognitive processes underpinning alcohol use, suggesting that heightened attentional bias, greater approach tendencies and reduced cue-specific inhibitory control are important drivers of consumption. This paper identifies methodological shortcomings within this broad domain of research and exemplifies them in studies focused specifically on alcohol-related attentional bias. ARGUMENT AND ANALYSIS: We highlight five main methodological issues: (i) the use of inappropriately matched control stimuli; (ii) opacity of stimulus selection and validation procedures; (iii) a credence in noisy measures; (iv) a reliance on unreliable tasks; and (v) variability in design and analysis. This is evidenced through a review of alcohol-related attentional bias (64 empirical articles, 68 tasks), which reveals the following: only 53% of tasks use appropriately matched control stimuli; as few as 38% report their stimulus selection and 19% their validation procedures; less than 28% used indices capable of disambiguating attentional processes; 22% assess reliability; and under 2% of studies were pre-registered. CONCLUSIONS: Well-matched and validated experimental stimuli, the development of reliable cognitive tasks and explicit assessment of their psychometric properties, and careful consideration of behavioural indices and their analysis will improve the methodological rigour of cognitive alcohol research. Open science principles can facilitate replication and reproducibility in alcohol research.

High frequency oscillations in epileptic and non-epileptic human hippocampus during a cognitive task.

Hippocampal high-frequency electrographic activity (HFOs) represents one of the major discoveries not only in epilepsy research but also in cognitive science over the past few decades. A fundamental challenge, however, has been the fact that physiological HFOs associated with normal brain function overlap in frequency with pathological HFOs. We investigated the impact of a cognitive task on HFOs with the aim of improving differentiation between epileptic and non-epileptic hippocampi in humans. Hippocampal activity was recorded with depth electrodes in 15 patients with focal epilepsy during a resting period and subsequently during a cognitive task. HFOs in ripple and fast ripple frequency ranges were evaluated in both conditions, and their rate, spectral entropy, relative amplitude and duration were compared in epileptic and non-epileptic hippocampi. The similarity of HFOs properties recorded at rest in epileptic and non-epileptic hippocampi suggests that they cannot be used alone to distinguish between hippocampi. However, both ripples and fast ripples were observed with higher rates, higher relative amplitudes and longer durations at rest as well as during a cognitive task in epileptic compared with non-epileptic hippocampi. Moreover, during a cognitive task, significant reductions of HFOs rates were found in epileptic hippocampi. These reductions were not observed in non-epileptic hippocampi. Our results indicate that although both hippocampi generate HFOs with similar features that probably reflect non-pathological phenomena, it is possible to differentiate between epileptic and non-epileptic hippocampi using a simple odd-ball task.

A neuroscientific evaluation of driver rehabilitation: Functional neuroimaging demonstrates the effectiveness of empathy induction in altering brain responses during social information processing.

An alarming number of traffic-related deaths occur each year on European roads alone. Figures reveal that the vast majority of road-traffic accidents are caused by drivers themselves, and so further improvements in road safety require developments in driver training and rehabilitation. This study evaluated a novel approach to driver rehabilitation-specifically, empathy induction as a means of changing attitudes towards risky driving. To assess the effectiveness of this method, the present study employed functional magnetic resonance imaging (fMRI) to compare brain function before and after a short program of empathy induction in 27 drivers whose licenses had been revoked after serious traffic offences (rehabilitated drivers [RDs]). In an extension of our previous research, we first assessed whether neural responses to empathy-eliciting social stimuli changed in these RDs. In order to isolate the neurophysiological effects of empathy induction from any other potential influences, we compared these RDs to a sample of 27 age-, handedness- and driving experience-matched control drivers (CDs) who had no exposure to the program. We then performed dual-fMRI "hyperscanning" to evaluate whether empathy induction changed brain responses during real-world social interactions among drivers; namely, during co-operative and/or competitive exchanges. Our data reveal that RDs exhibited weaker brain responses to socio-emotional stimuli compared with CDs prior to the program, but this difference was reversed after empathy induction. Moreover, we observed differences between pre- and post-program assessments in patterns of brain responses in RDs elicited during competitive social exchanges, which we interpret to reflect a change in their proclivity to react to the perceived wrong-doing of other road users. Together, these findings suggest that empathy induction is an effective form of driver rehabilitation, and the utility of neuroscientific techniques for evaluating and improving rehabilitation programs.

Where's the wine? Heavy social drinkers show attentional bias towards alcohol in a visual conjunction search task.

BACKGROUND AND AIMS: Research indicates that high consumers of alcohol exhibit attentional bias (AB) towards alcohol-related cues, suggestive of a cognitive mechanism that might drive substance seeking. Many tasks that measure AB (e.g. visual probe, addiction Stroop), however, are limited by their reliance on non-appetitive control cues, the serial presentation of stimuli and their poor internal reliability. The current study employed a visual conjunction search (VCS) task capable of presenting multiple alcoholic and non-alcoholic appetitive cues simultaneously to assess whether social drinkers attend selectively to alcoholic stimuli. To assess the construct validity of this task, we examined whether alcohol consumption and related problems, subjective craving and drinking motives predict alcohol-specific AB. DESIGN AND SETTING: A VCS task was performed in a laboratory setting, which required participants to detect the presence of appetitive alcoholic (wine, beer) and non-alcoholic (cola, lemonade) targets within arrays of matching and non-matching distractors. PARTICIPANTS: Data from 99 participants were assessed [meanage  = 20.77, standard deviation (SD) = 2.98; 64 (65%) females], with 81.8% meeting the threshold for harmful alcohol consumption (meanAUDIT  = 12.89, SD = 5.79). MEASUREMENTS: Self-reports of alcohol consumption and related problems [Alcohol Use Disorders Identification Test (AUDIT)], subjective craving (Alcohol Craving Questionnaire Short Form) and drinking motives (Drinking Motives Questionnaire Short Form) were obtained, and the VCS task measured response times for the correct detection of alcoholic and non-alcoholic targets. FINDINGS: Participants were significantly quicker to detect alcoholic relative to non-alcoholic appetitive targets (P < 0.001, dz  = 0.41), which was predicted positively by AUDIT scores (P = 0.013, R2  = 0.06%). The VCS task achieved excellent reliability (α > 0.79), superior to other paradigms. CONCLUSIONS: The visual conjunction search task appears to be a highly reliable method for assessing alcohol-related attentional bias, and shows that heavy social drinkers prioritize alcoholic cues in their immediate environment.

Getting into sync: Data-driven analyses reveal patterns of neural coupling that distinguish among different social exchanges.

In social interactions, each individual's brain drives an action that, in turn, elicits systematic neural responses in their partner that drive a reaction. Consequently, the brain responses of both interactants become temporally contingent upon one another through the actions they generate, and different interaction dynamics will be underpinned by distinct forms of between-brain coupling. In this study, we investigated this by "performing functional magnetic resonance imaging on two individuals simultaneously (dual-fMRI) while they competed or cooperated with one another in a turn-based or concurrent fashion." To assess whether distinct patterns of neural coupling were associated with these different interactions, we combined two data-driven, model-free analytical techniques: group-independent component analysis and inter-subject correlation. This revealed four distinct patterns of brain responses that were temporally aligned between interactants: one emerged during co-operative exchanges and encompassed brain regions involved in social cognitive processing, such as the temporo-parietal cortex. The other three were associated with competitive exchanges and comprised brain systems implicated in visuo-motor processing and social decision-making, including the cerebellum and anterior cingulate cortex. Interestingly, neural coupling was significantly stronger in concurrent relative to turn-based exchanges. These results demonstrate the utility of data-driven approaches applied to "dual-fMRI" data in elucidating the interpersonal neural processes that give rise to the two-in-one dynamic characterizing social interaction.

Uncovering the Early Stages of Domain Melting in Calmodulin with Ultrafast Temperature-Jump Infrared Spectroscopy.

The signaling protein calmodulin (CaM) undergoes a well-known change in secondary structure upon binding Ca2+, but the structural plasticity of the Ca2+-free apo state is linked to CaM functionality. Variable temperature studies of apo-CaM indicate two structural transitions at 46 and 58 °C that are assigned to melting of the C- and N-terminal domains, respectively, but the molecular mechanism of domain unfolding is unknown. We report temperature-jump time-resolved infrared (IR) spectroscopy experiments designed to target the first steps in the C-terminal domain melting transition of human apo-CaM. A comparison of the nonequilibrium relaxation of apo-CaM with the more thermodynamically stable holo-CaM, with 4 equiv of Ca2+ bound, shows that domain melting of apo-CaM begins on microsecond time scales with α-helix destabilization. These observations enable the assignment of previously reported dynamics of CaM on hundreds of microsecond time scales to thermally activated melting, producing a complete mechanism for thermal unfolding of CaM.

EEG Reactivity Predicts Individual Efficacy of Vagal Nerve Stimulation in Intractable Epileptics.

Background: Chronic vagal nerve stimulation (VNS) is a well-established non-pharmacological treatment option for drug-resistant epilepsy. This study sought to develop a statistical model for prediction of VNS efficacy. We hypothesized that reactivity of the electroencephalogram (EEG) to external stimuli measured during routine preoperative evaluation differs between VNS responders and non-responders. Materials and Methods: Power spectral analyses were computed retrospectively on pre-operative EEG recordings from 60 epileptic patients with VNS. Thirty five responders and 25 non-responders were compared on the relative power values in four standard frequency bands and eight conditions of clinical assessment-eyes opening/closing, photic stimulation, and hyperventilation. Using logistic regression, groups of electrodes within anatomical areas identified as maximally discriminative by n leave-one-out iterations were used to classify patients. The reliability of the predictive model was verified with an independent data-set from 22 additional patients. Results: Power spectral analyses revealed significant differences in EEG reactivity between responders and non-responders; specifically, the dynamics of alpha and gamma activity strongly reflected VNS efficacy. Using individual EEG reactivity to develop and validate a predictive model, we discriminated between responders and non-responders with 86% accuracy, 83% sensitivity, and 90% specificity. Conclusion: We present a new statistical model with which EEG reactivity to external stimuli during routine presurgical evaluation can be seen as a promising avenue for the identification of patients with favorable VNS outcome. This novel method for the prediction of VNS efficacy might represent a breakthrough in the management of drug-resistant epilepsy, with wide-reaching medical and economic implications.

Dissecting social interaction: dual-fMRI reveals patterns of interpersonal brain-behavior relationships that dissociate among dimensions of social exchange.

During social interactions, each individual's actions are simultaneously a consequence of and an antecedent to their interaction partner's behavior. Capturing online the brain processes underlying such mutual dependency requires simultaneous measurements of all interactants' brains during real-world exchange ('hyperscanning'). This demands a precise characterization of the type of interaction under investigation, however, and analytical techniques capable of capturing interpersonal dependencies. We adapted an interactive task capable of dissociating between two dimensions of interdependent social exchange: goal structure (cooperation vs competition) and interaction structure [concurrent (CN) vs turn-based]. Performing dual-functional magnetic resonance imaging hyperscanning on pairs of individuals interacting on this task, and modeling brain responses in both interactants as systematic reactions to their partner's behavior, we investigated interpersonal brain-behavior dependencies (iBBDs) during each dimension. This revealed patterns of iBBDs that differentiated among exchanges; in players supporting the actions of another, greater brain responses to the co-player's actions were expressed in regions implicated in social cognition, such as the medial prefrontal cortex, precuneus and temporal cortices. Stronger iBBD during CN competitive exchanges was observed in brain systems involved in movement planning and updating, however, such as the supplementary motor area. This demonstrates the potential for hyperscanning to elucidate neural processes underlying different forms of social exchange.

A dual-fMRI investigation of the iterated Ultimatum Game reveals that reciprocal behaviour is associated with neural alignment.

Dyadic interactions often involve a dynamic process of mutual reciprocity; to steer a series of exchanges towards a desired outcome, both interactants must adapt their own behaviour according to that of their interaction partner. Understanding the brain processes behind such bidirectional reciprocity is therefore central to social neuroscience, but this requires measurement of both individuals' brains during real-world exchanges. We achieved this by performing functional magnetic resonance imaging (fMRI) on pairs of male individuals simultaneously while they interacted in a modified iterated Ultimatum Game (iUG). In this modification, both players could express their intent and maximise their own monetary gain by reciprocating their partner's behaviour - they could promote generosity through cooperation and/or discourage unfair play with retaliation. By developing a novel model of reciprocity adapted from behavioural economics, we then show that each player's choices can be predicted accurately by estimating expected utility (EU) not only in terms of immediate payoff, but also as a reaction to their opponent's prior behaviour. Finally, for the first time we reveal that brain signals implicated in social decision making are modulated by these estimates of EU, and become correlated more strongly between interacting players who reciprocate one another.

Hippocampal involvement in nonpathological déjà vu: Subfield vulnerability rather than temporal lobe epilepsy equivalent.

INTRODUCTION: Morphological correlates of nonpathological déjà vu (DV) have been identified recently within the human brain. Significantly reduced gray matter volume (GMV) within a set of cortical and subcortical regions reported in subjects experiencing DV seems to mirror the distribution of GMV reduction in mesial temporal lobe epilepsy (MTLE) patients but vary in terms of the hippocampus. Another condition associated with hippocampal GMV reduction and DV alike disturbance in memory processing is schizophrenia (SCH). Here, we tested the hypothesis that hippocampal involvement in nonpathological DV resembles more closely the pattern of GMV decrease observed in MTLE compared with that occurring in SCH. METHODS: Using automated segmentation of the MRI data we compared the medians of GMV within 12 specific hippocampal subfields in healthy individuals that do (DV+; N = 87) and do not report déjà vu experience (DV-; N = 26), and patients with MTLE (N = 47) and SCH (N = 29). By Pearson correlation, we then evaluated the similarity of MTLE and SCH groups to DV+ group with respect to spatial distribution of GMV deviation from DV- group. RESULTS: Significant GMV decrease was found in MTLE group in most of the subfields. There were just trends in the hippocampal GMV decrease found in DV+ or SCH groups. Concerning the spatial distribution of GMV decrease, we revealed statistically significant correlation for the left hippocampus for SCH vs DV+. Otherwise there was no statistically significant correlation. CONCLUSIONS: Our findings reveal structural features of hippocampal involvement in nonpathological DV, MTLE, and SCH. Despite our expectations, the pattern of GMV reduction in the DV+ relative to the DV- group does not resemble the pattern observed in MTLE any more than that observed in SCH. The highly similar patterns of the three clinical groups rather suggest an increased vulnerability of certain hippocampal subfields; namely, Cornu Ammonis (CA)4, CA3, dentate gyrus granular cell layer (GC-DG), hippocampal-amygdaloid transition area (HATA) and subiculum.

Quantifying Secondary Structure Changes in Calmodulin Using 2D-IR Spectroscopy.

Revealing the details of biomolecular processes in solution needs tools that can monitor structural dynamics over a range of time and length scales. We assess the ability of 2D-IR spectroscopy in combination with multivariate data analysis to quantify changes in secondary structure of the multifunctional calcium-binding messenger protein Calmodulin (CaM) as a function of temperature and Ca2+ concentration. Our approach produced quantitative agreement with circular dichroism (CD) spectroscopy in detecting the domain melting transitions of Ca2+-free (apo) CaM (reduction in α-helix structure by 13% (CD) and 15% (2D)). 2D-IR also allows accurate differentiation between melting transitions and generic heating effects observed in the more thermally stable Ca2+-bound (holo) CaM. The functionally relevant random-coil-α-helix transition associated with Ca2+ uptake that involves just 7-8 out of a total of 148 amino acid residues was clearly detected. Temperature-dependent Molecular Dynamics (MD) simulations show that apo-CaM exists in dynamic equilibrium with holo-like conformations, while Ca2+ uptake reduces conformational flexibility. The ability to combine quantitative structural insight from 2D-IR with MD simulations thus offers a powerful approach for measuring subtle protein conformational changes in solution.

Disruption of key NADH-binding pocket residues of the Mycobacterium tuberculosis InhA affects DD-CoA binding ability.

Tuberculosis (TB) is a global health problem that affects over 10 million people. There is an urgent need to develop novel antimicrobial therapies to combat TB. To achieve this, a thorough understanding of key validated drug targets is required. The enoyl reductase InhA, responsible for synthesis of essential mycolic acids in the mycobacterial cell wall, is the target for the frontline anti-TB drug isoniazid. To better understand the activity of this protein a series of mutants, targeted to the NADH co-factor binding pocket were created. Residues P193 and W222 comprise a series of hydrophobic residues surrounding the cofactor binding site and mutation of both residues negatively affect InhA function. Construction of an M155A mutant of InhA results in increased affinity for NADH and DD-CoA turnover but with a reduction in Vmax for DD-CoA, impairing overall activity. This suggests that NADH-binding geometry of InhA likely permits long-range interactions between residues in the NADH-binding pocket to facilitate substrate turnover in the DD-CoA binding region of the protein. Understanding the precise details of substrate binding and turnover in InhA and how this may affect protein-protein interactions may facilitate the development of improved inhibitors enabling the development of novel anti-TB drugs.