A Highly Ordered Nitroxide Side Chain for Distance Mapping and Monitoring Slow Structural Fluctuations in Proteins.

Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) is an established tool for exploring protein structure and dynamics. Although nitroxide side chains attached to a single cysteine via a disulfide linkage are commonly employed in SDSL-EPR, their internal flexibility complicates applications to monitor slow internal motions in proteins and to structure determination by distance mapping. Moreover, the labile disulfide linkage prohibits the use of reducing agents often needed for protein stability. To enable the application of SDSL-EPR to the measurement of slow internal dynamics, new spin labels with hindered internal motion are desired. Here, we introduce a highly ordered nitroxide side chain, designated R9, attached at a single cysteine residue via a non-reducible thioether linkage. The reaction to introduce R9 is highly selective for solvent-exposed cysteine residues. Structures of R9 at two helical sites in T4 Lysozyme were determined by X-ray crystallography and the mobility in helical sequences was characterized by EPR spectral lineshape analysis, Saturation Transfer EPR, and Saturation Recovery EPR. In addition, interspin distance measurements between pairs of R9 residues are reported. Collectively, all data indicate that R9 will be useful for monitoring slow internal structural fluctuations, and applications to distance mapping via dipolar spectroscopy and relaxation enhancement methods are anticipated. Supplementary Information: The online version contains supplementary material available at 10.1007/s00723-023-01618-8.

Subacute Combined Degeneration of the Spinal Cord Induced by Nitrous Oxide Abuse: A Rare Patient Presentation to a Spine Surgery Clinic: Illustrative Case.

Subacute combined degeneration (SCD) of the spinal cord is a disease involving the lateral and posterior columns of the spinal cord that can manifest in patients with vitamin B12 deficiency. Nitrous oxide (N2O)-induced SCD of the spinal cord is a result of N2O interfering with the metabolism of vitamin B12 and results in nervous system demyelination. This is an infrequent complication of N2O anesthesia; however, cases are rising with recreational N2O use. This case report describes a patient with SCD of the spinal cord induced by recreational N2O abuse. The patient presented to a spine surgery clinic with a 3-week history of progressive global weakness and paresthesias. After a detailed history and physical examination, the diagnosis was made and supported by various tests and imaging findings. Despite marked neurologic deficits, the patient's symptoms improved markedly with therapy and vitamin B12 supplementation. Spine surgery clinicians may be confronted with these cases and should be aware of this atypical presentation of SCD. As in our case, patients may present with neurologic deficits of unclear etiology. Neurologic dysfunction may be irreversible; therefore, accurate diagnosis, medical treatment, and complete neurologic evaluation are of the utmost importance to prevent additional progression.

Conformational flexibility of HIV-1 envelope glycoproteins modulates transmitted / founder sensitivity to broadly neutralizing antibodies.

HIV-1 envelope glycoproteins (Envs) mediate viral entry and are the sole target of neutralizing antibodies. Envs of most primary HIV-1 strains exist in a closed conformation and occasionally sample more open states. Thus, current knowledge guides immunogen design to mimic the closed Env conformation as the preferred target for eliciting broadly neutralizing antibodies (bnAbs) to block HIV-1 entry. Here we show that Env-preferred conformations of 6 out of 13 (46%) transmitted/founder (T/F) strains tested are incompletely closed. As a result, entry of these T/Fs into target cells is sensitive to antibodies that recognize internal epitopes exposed on open Env conformations. A cryo-electron microscopy structure of unliganded, incompletely closed T/F Envs (1059-SOSIP) at 3.6 Å resolution exhibits an asymmetric configuration of Env protomers with increased sampling of states with incompletely closed trimer apex. Double electron-electron resonance spectroscopy provided further evidence for enriched occupancy of more open Env conformations. Consistent with conformational flexibility, 1059 Envs were associated with resistance to most bnAbs that exhibit reduced potency against functional Env intermediates. To follow the fate of incompletely closed Env in patients, we reconstructed de novo the post-transmission evolutionary pathway of a second T/F Env (CH040), which is sensitive to the V3-targeting antibody 19b and highly resistant to most bnAbs. Evolved viruses exhibited increased resistance to cold, soluble CD4 and 19b, all of which correlate with closing of the adapted Env trimer. Lastly, we show a correlation between efficient neutralization of multiple Env conformations and increased antiviral breadth of CD4-binding site (CD4bs) bnAbs. In particular, N6 bnAb, which uniquely recognizes different Env conformations, efficiently neutralizes 50% of the HIV-1 strains that were resistant to VRC01 and transmitted during the first-in-humans antibody-mediated prevention trial (HVTN 704). VRC01-resistant Envs are incompletely closed based on their sensitivity to cold and on partial sensitivity to antibodies targeting internal, typically occluded, epitopes. Most VRC01-resistant Envs retain the VRC01 epitope according to VRC01 binding to their gp120 subunit at concentrations that have no significant effect on virus entry, and they exhibit cross resistance to other CD4bs bnAbs that poorly recognize functional Env intermediates. Our findings refine current knowledge of Env conformational states and provide guidance for developing new strategies for bnAb immunotherapy and Env-based immunogen design.

Neutralizing antibodies induced in immunized macaques recognize the CD4-binding site on an occluded-open HIV-1 envelope trimer.

Broadly-neutralizing antibodies (bNAbs) against HIV-1 Env can protect from infection. We characterize Ab1303 and Ab1573, heterologously-neutralizing CD4-binding site (CD4bs) antibodies, isolated from sequentially-immunized macaques. Ab1303/Ab1573 binding is observed only when Env trimers are not constrained in the closed, prefusion conformation. Fab-Env cryo-EM structures show that both antibodies recognize the CD4bs on Env trimer with an 'occluded-open' conformation between closed, as targeted by bNAbs, and fully-open, as recognized by CD4. The occluded-open Env trimer conformation includes outwardly-rotated gp120 subunits, but unlike CD4-bound Envs, does not exhibit V1V2 displacement, 4-stranded gp120 bridging sheet, or co-receptor binding site exposure. Inter-protomer distances within trimers measured by double electron-electron resonance spectroscopy suggest an equilibrium between occluded-open and closed Env conformations, consistent with Ab1303/Ab1573 binding stabilizing an existing conformation. Studies of Ab1303/Ab1573 demonstrate that CD4bs neutralizing antibodies that bind open Env trimers can be raised by immunization, thereby informing immunogen design and antibody therapeutic efforts.

Lipid bilayer induces contraction of the denatured state ensemble of a helical-bundle membrane protein.

Defining the denatured state ensemble (DSE) and disordered proteins is essential to understanding folding, chaperone action, degradation, and translocation. As compared with water-soluble proteins, the DSE of membrane proteins is much less characterized. Here, we measure the DSE of the helical membrane protein GlpG of Escherichia coli (E. coli) in native-like lipid bilayers. The DSE was obtained using our steric trapping method, which couples denaturation of doubly biotinylated GlpG to binding of two streptavidin molecules. The helices and loops are probed using limited proteolysis and mass spectrometry, while the dimensions are determined using our paramagnetic biotin derivative and double electron-electron resonance spectroscopy. These data, along with our Upside simulations, identify the DSE as being highly dynamic, involving the topology changes and unfolding of some of the transmembrane (TM) helices. The DSE is expanded relative to the native state but only to 15 to 75% of the fully expanded condition. The degree of expansion depends on the local protein packing and the lipid composition. E. coli's lipid bilayer promotes the association of TM helices in the DSE and, probably in general, facilitates interhelical interactions. This tendency may be the outcome of a general lipophobic effect of proteins within the cell membranes.

Structural basis for activation of voltage sensor domains in an ion channel TPC1.

Voltage-sensing domains (VSDs) couple changes in transmembrane electrical potential to conformational changes that regulate ion conductance through a central channel. Positively charged amino acids inside each sensor cooperatively respond to changes in voltage. Our previous structure of a TPC1 channel captured an example of a resting-state VSD in an intact ion channel. To generate an activated-state VSD in the same channel we removed the luminal inhibitory Ca2+-binding site (Cai2+), which shifts voltage-dependent opening to more negative voltage and activation at 0 mV. Cryo-EM reveals two coexisting structures of the VSD, an intermediate state 1 that partially closes access to the cytoplasmic side but remains occluded on the luminal side and an intermediate activated state 2 in which the cytoplasmic solvent access to the gating charges closes, while luminal access partially opens. Activation can be thought of as moving a hydrophobic insulating region of the VSD from the external side to an alternate grouping on the internal side. This effectively moves the gating charges from the inside potential to that of the outside. Activation also requires binding of Ca2+ to a cytoplasmic site (Caa2+). An X-ray structure with Caa2+ removed and a near-atomic resolution cryo-EM structure with Cai2+ removed define how dramatic conformational changes in the cytoplasmic domains may communicate with the VSD during activation. Together four structures provide a basis for understanding the voltage-dependent transition from resting to activated state, the tuning of VSD by thermodynamic stability, and this channel's requirement of cytoplasmic Ca2+ ions for activation.

Conformational heterogeneity of the allosteric drug and metabolite (ADaM) site in AMP-activated protein kinase (AMPK).

AMP-activated protein kinase (AMPK) is a master regulator of energy homeostasis and a promising drug target for managing metabolic diseases such as type 2 diabetes. Many pharmacological AMPK activators, and possibly unidentified physiological metabolites, bind to the allosteric drug and metabolite (ADaM) site at the interface between the kinase domain (KD) in the α-subunit and the carbohydrate-binding module (CBM) in the ß-subunit. Here, using double electron-electron resonance (DEER) spectroscopy, we demonstrate that the CBM-KD interaction is partially dissociated and the interface highly disordered in the absence of pharmacological ADaM site activators as inferred from a low depth of modulation and broad DEER distance distributions. ADaM site ligands such as 991, and to a lesser degree phosphorylation, stabilize the KD-CBM association and strikingly reduce conformational heterogeneity in the ADaM site. Our findings that the ADaM site, formed by the KD-CBM interaction, can be modulated by diverse ligands and by phosphorylation suggest that it may function as a hub for integrating regulatory signals.

DEER Spectroscopy Measurements Reveal Multiple Conformations of HIV-1 SOSIP Envelopes that Show Similarities with Envelopes on Native Virions.

HIV-1 Envelope (Env) mediates viral-host membrane fusion after binding host-receptor CD4 and coreceptor. Soluble envelopes (SOSIPs), designed to mimic prefusion conformational states of virion-bound envelopes, are proposed immunogens for eliciting neutralizing antibodies, yet only static structures are available. To evaluate conformational landscapes of ligand-free, CD4-bound, inhibitor-bound, and antibody-bound SOSIPs, we measured inter-subunit distances throughout spin-labeled SOSIPs using double electron-electron resonance (DEER) spectroscopy and compared results to soluble and virion-bound Env structures, and single-molecule fluorescence resonance energy transfer (smFRET)-derived dynamics of virion-bound Envs. Unliganded SOSIP measurements were consistent with closed, neutralizing antibody-bound structures and shielding of non-neutralizing epitopes, demonstrating homogeneity at Env apex, increased flexibility near Env base, and no evidence for the intra-subunit flexibility near Env apex suggested by smFRET. CD4 binding increased inter-subunit distances and heterogeneity, consistent with rearrangements required for coreceptor binding. Results suggest similarities between SOSIPs and virion-bound Envs and demonstrate DEER's relevance for immunogen design.

Probing the structural basis and adsorption mechanism of an enzyme on nano-sized protein carriers.

Silica nanoparticles (SiNPs) are important nano-sized, solid-state carriers/hosts to load, store, and deliver biological or pharmaceutical cargoes. They are also good potential solid supports to immobilize proteins for fundamental protein structure and dynamics studies. However, precaution is necessary when using SiNPs in these areas because adsorption might alter the activity of the cargoes, especially when enzymes are loaded. Therefore, it becomes important to understand the structural basis of the cargo enzyme activity changes, if there is any. The high complexity and dynamics of the nano-bio interface present many challenges. Reported here is a comprehensive study of the structure, dynamics, and activity of a model enzyme, T4 lysozyme, upon adsorption to a few surface-modified SiNPs using several experimental techniques. Not surprisingly, a significant activity loss on each studied SiNP was found. The structural basis of the activity loss was identified based on results from a unique technique, the Electron Paramagnetic Resonance (EPR) spectroscopy, which probes structural information regardless of the complexity. Several docking models of the enzyme on SiNPs with different surfaces, at different enzyme-to-SiNP ratios are proposed. Interestingly, we found that the adsorbed enzyme can be desorbed via pH adjustment, which highlighted the potential to use SiNPs for enzyme/protein delivery or storage due to the high capacity. In order to use SiNPs as enzyme hosts, minimizing the enzymatic activity loss upon adsorption is needed. Lastly, the work outlined here demonstrate the use of EPR in probing structural information on the complex (inorganic)nano-bio interface.

A triarylmethyl spin label for long-range distance measurement at physiological temperatures using T1 relaxation enhancement.

Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy has become an important tool for measuring distances in proteins on the order of a few nm. For this purpose pairs of spin labels, most commonly nitroxides, are site-selectively introduced into the protein. Recent efforts to develop new spin labels are focused on tailoring the intrinsic properties of the label to either extend the upper limit of measurable distances at physiological temperature, or to provide a unique spectral lineshape so that selective pairwise distances can be measured in a protein or complex containing multiple spin label species. Triarylmethyl (TAM) radicals are the foundation for a new class of spin labels that promise to provide both capabilities. Here we report a new methanethiosulfonate derivative of a TAM radical that reacts rapidly and selectively with an engineered cysteine residue to generate a TAM containing side chain (TAM1) in high yield. With a TAM1 residue and Cu(2+) bound to an engineered Cu(2+) binding site, enhanced T1 relaxation of TAM should enable measurement of interspin distances up to 50Å at physiological temperature. To achieve favorable TAM1-labeled protein concentrations without aggregation, proteins are tethered to a solid support either site-selectively using an unnatural amino acid or via native lysine residues. The methodology is general and readily extendable to complex systems, including membrane proteins.

Saturation Recovery EPR and Nitroxide Spin Labeling for Exploring Structure and Dynamics in Proteins.

Experimental techniques capable of determining the structure and dynamics of proteins are continuously being developed in order to understand protein function. Among existing methods, site-directed spin labeling in combination with saturation recovery (SR) electron paramagnetic resonance spectroscopy contributes uniquely to the determination of secondary and tertiary protein structure under physiological conditions, independent of molecular weight and complexity. In addition, SR of spin labeled proteins was recently demonstrated to be sensitive to conformational exchange events with characteristic lifetimes on the order of µs, a time domain that presents a significant challenge to other spectroscopic techniques. In this chapter, we present the theoretical background necessary to understand the capabilities of SR as applied to spin labeled proteins, the instrumental requirements, and practical experimental considerations necessary to obtain interpretable data, and the use of SR to obtain information on protein: (1) secondary structure via solvent accessibility measurements, (2) tertiary structure using interspin distance measurements, and (3) conformational exchange.

Multiple structural states exist throughout the helical nucleation sequence of the intrinsically disordered protein stathmin, as reported by electron paramagnetic resonance spectroscopy.

The intrinsically disordered protein (IDP) stathmin plays an important regulatory role in cytoskeletal maintenance through its helical binding to tubulin and microtubules. However, it lacks a stable fold in the absence of its binding partner. Although stathmin has been a focus of research over the past two decades, the solution-phase conformational dynamics of this IDP are poorly understood. It has been reported that stathmin is purely monomeric in solution and that it bears a short helical region of persistent foldedness, which may act to nucleate helical folding in the C-terminal direction. Here we report a comprehensive study of the structural equilibria local to this region in stathmin that contradicts these two claims. Using the technique of electron paramagnetic resonance (EPR) spectroscopy on spin-labeled stathmin mutants in the solution-phase and when immobilized on Sepharose solid support, we show that all sites in the helical nucleation region of stathmin exhibit multiple spectral components that correspond to dynamic states of differing mobilities and stabilities. Importantly, a state with relatively low mobility dominates each spectrum with an average population greater than 50%, which we suggest corresponds to an oligomerized state of the protein. This is in contrast to a less populated, more mobile state, which likely represents a helically folded monomeric state of stathmin, and a highly mobile state, which we propose is the random coil conformer of the protein. Our interpretation of the EPR data is confirmed by further characterization of the protein using the techniques of native and SDS PAGE, gel filtration chromatography, and multiangle and dynamic light scattering, all of which show the presence of oligomeric stathmin in solution. Collectively, these data suggest that stathmin exists in a diverse equilibrium of states throughout the purported helical nucleation region and that this IDP exhibits a propensity toward oligomerization.

Long-range distance measurements in proteins at physiological temperatures using saturation recovery EPR spectroscopy.

Site-directed spin labeling in combination with EPR is a powerful method for providing distances on the nm scale in biological systems. The most popular strategy, double electron-electron resonance (DEER), is carried out at cryogenic temperatures (50-80 K) to increase the short spin-spin relaxation time (T2) upon which the technique relies. A challenge is to measure long-range distances (20-60 Å) in proteins near physiological temperatures. Toward this goal we are investigating an alternative approach based on the distance-dependent enhancement of spin-lattice relaxation rate (T1(-1)) of a nitroxide spin label by a paramagnetic metal. With a commonly used nitroxide side chain (R1) and Cu(2+), it has been found that interspin distances ≤25 Å can be determined in this way (Jun et al. Biochemistry 2006, 45, 11666). Here, the upper limit of the accessible distance is extended to ≈40 Å using spin labels with long T1, a high-affinity 5-residue Cu(2+) binding loop inserted into the protein sequence, and pulsed saturation recovery to measure relaxation enhancement. Time-domain Cu(2+) electron paramagnetic resonance, quantum mechanical calculations, and molecular dynamics simulations provide information on the structure and geometry of the Cu(2+) loop and indicate that the metal ion is well-localized in the protein. An important aspect of these studies is that both Cu(2+)/nitroxide DEER at cryogenic temperatures and T1 relaxation measurements at room temperature can be carried out on the same sample, allowing both validation of the relaxation method and assessment of the effect of freezing on protein structure.

The effect of medical student feedback about resident teaching on resident teaching identity: a randomized controlled trial.

BACKGROUND AND OBJECTIVES: Residents often teach medical students, other residents, and patients. However, few residents get feedback about their teaching. This study's objective was to determine if feedback from medical students increases resident teaching identity. METHODS: This was a stratified, single blinded, randomized controlled trial of an educational intervention. Residents were stratified based on year of residency and then randomized to receive feedback by medical students or not. Medical students evaluated resident teaching effectiveness by ranking resident ability to apply the five microskills for clinical teaching and to role model being an effective clinician. Residents were surveyed to determine their level of teacher identity before and after the intervention. Allocation concealment and intention to treat principles were used. RESULTS: All residents (n=32) that met inclusion criteria participated with complete response rate to both pre-intervention and post-intervention surveys. There was no difference in teaching identity between residents who received feedback and those who did not, except in one subscale of the Teacher Identity Scale-global teaching identity, where residents who received medical student feedback scored lower. CONCLUSIONS: There was no difference between intervention and control group in resident teaching identity over time. The residents found feedback important. This was a randomized controlled trial with strong methodology that helps advance understanding of the importance of medical student feedback on resident teaching.

Fitwits: preparing residency-based physicians to discuss childhood obesity with preteens.

OBJECTIVE: To evaluate the Fitwits MD office tool and games for obesity discussions with 9- to 12-year-olds. METHODS: A nonrandomized intervention study using pre- and posttest assessments in 2 residency programs compared 31 control group and 55 intervention physicians (34 previously trained, 21 newly trained to use Fitwits). Surveys addressed comfort and competence regarding: obesity prevention and treatment, nutrition, exercise, portion size, body mass index (BMI), and the term "obesity." We surveyed all groups at baseline and 5 months (post 1) and new trainees 3 months later (post 2). RESULTS: In post 1, prior trainees reported significantly increased comfort and competence for discussing obesity prevention, portion size, BMI, and "obesity." In post 2, new trainees reported significantly increased comfort and competence discussing obesity prevention and treatment, portion size, and BMI. CONCLUSIONS: Experience using Fitwits improved residency-based physician comfort and competence in obesity prevention and treatment, portion size, BMI, and "obesity" discussions with preadolescents.

A FRET-based assay for the discovery of West Nile Virus NS2B-NS3 protease inhibitors.

The West Nile Virus (WNV) has been a worldwide epidemic since the early 1990s. Currently there are no therapeutic treatments for WNV infections. One particular avenue of treatment is inhibition of the NS2B-NS3 protease, an enzyme that is crucial for WNV replication. In our effort to increase the number of NS2B-NS3 protease inhibitors, we report a novel FRET-based high throughput assay for the discovery of WNV NS2B-NS3 protease inhibitors. For this assay, a FRET-based peptide substrate was synthesized and kinetically characterized with the NS2B-NS3 protease. The new substrate exhibits a K(m) of 3.35 ± 0.31 µM, a k(cat) of 0.0717 ± 0.0016 s(-1) and a k(cat)/K(m) of 21,400 ± 2000 M(-1) s(-1).

Reversal of glial and neurovascular markers of unhealthy brain aging by exercise in middle-aged female mice.

Healthy brain aging and cognitive function are promoted by exercise. The benefits of exercise are attributed to several mechanisms, many which highlight its neuroprotective role via actions that enhance neurogenesis, neuronal morphology and/or neurotrophin release. However, the brain is also composed of glial and vascular elements, and comparatively less is known regarding the effects of exercise on these components in the aging brain. Here, we show that aerobic exercise at mid-age decreased markers of unhealthy brain aging including astrocyte hypertrophy, a hallmark of brain aging. Middle-aged female mice were assigned to a sedentary group or provided a running wheel for six weeks. Exercise decreased hippocampal astrocyte and myelin markers of aging but increased VEGF, a marker of angiogenesis. Brain vascular casts revealed exercise-induced structural modifications associated with improved endothelial function in the periphery. Our results suggest that age-related astrocyte hypertrophy/reactivity and myelin dysregulation are aggravated by a sedentary lifestyle and accompanying reductions in vascular function. However, these effects appear reversible with exercise initiated at mid-age. As this period of the lifespan coincides with the appearance of multiple markers of brain aging, including initial signs of cognitive decline, it may represent a window of opportunity for intervention as the brain appears to still possess significant vascular plasticity. These results may also have particular implications for aging females who are more susceptible than males to certain risk factors which contribute to vascular aging.

Structure and dynamics of a conformationally constrained nitroxide side chain and applications in EPR spectroscopy.

A disulfide-linked nitroxide side chain (R1) is the most widely used spin label for determining protein topology, mapping structural changes, and characterizing nanosecond backbone motions by site-directed spin labeling. Although the internal motion of R1 and the number of preferred rotamers are limited, translating interspin distance measurements and spatial orientation information into structural constraints is challenging. Here, we introduce a highly constrained nitroxide side chain designated RX as an alternative to R1 for these applications. RX is formed by a facile cross-linking reaction of a bifunctional methanethiosulfonate reagent with pairs of cysteine residues at i and i + 3 or i and i + 4 in an α-helix, at i and i + 2 in a ß-strand, or with cysteine residues in adjacent strands in a ß-sheet. Analysis of EPR spectra, a crystal structure of RX in T4 lysozyme, and pulsed electron-electron double resonance (ELDOR) spectroscopy on an immobilized protein containing RX all reveal a highly constrained internal motion of the side chain. Consistent with the constrained geometry, interspin distance distributions between pairs of RX side chains are narrower than those from analogous R1 pairs. As an important consequence of the constrained internal motion of RX, spectral diffusion detected with ELDOR reveals microsecond internal motions of the protein. Collectively, the data suggest that the RX side chain will be useful for distance mapping by EPR spectroscopy, determining spatial orientation of helical segments in oriented specimens, and measuring structural fluctuations on the microsecond time scale.

Genetic classification of populations using supervised learning.

There are many instances in genetics in which we wish to determine whether two candidate populations are distinguishable on the basis of their genetic structure. Examples include populations which are geographically separated, case-control studies and quality control (when participants in a study have been genotyped at different laboratories). This latter application is of particular importance in the era of large scale genome wide association studies, when collections of individuals genotyped at different locations are being merged to provide increased power. The traditional method for detecting structure within a population is some form of exploratory technique such as principal components analysis. Such methods, which do not utilise our prior knowledge of the membership of the candidate populations. are termed unsupervised. Supervised methods, on the other hand are able to utilise this prior knowledge when it is available.In this paper we demonstrate that in such cases modern supervised approaches are a more appropriate tool for detecting genetic differences between populations. We apply two such methods, (neural networks and support vector machines) to the classification of three populations (two from Scotland and one from Bulgaria). The sensitivity exhibited by both these methods is considerably higher than that attained by principal components analysis and in fact comfortably exceeds a recently conjectured theoretical limit on the sensitivity of unsupervised methods. In particular, our methods can distinguish between the two Scottish populations, where principal components analysis cannot. We suggest, on the basis of our results that a supervised learning approach should be the method of choice when classifying individuals into pre-defined populations, particularly in quality control for large scale genome wide association studies.

Isotope effects and the temperature dependences of the hyperfine coupling constants of muoniated sec-butyl radicals in condensed phases.

Reported here is the first µSR study of the muon (A(µ)) and proton (A(p)) ß-hyperfine coupling constants (Hfcc) of muoniated sec-butyl radicals, formed by muonium (Mu) addition to 1-butene and to cis- and trans-2-butene. The data are compared with in vacuo spin-unrestricted MP2 and hybrid DFT/B3YLP calculations reported in the previous paper (I), which played an important part in the interpretation of the data. The T-dependences of both the (reduced) muon, A(µ)'(T), and proton, A(p)(T), Hfcc are surprisingly well explained by a simple model, in which the calculated Hfcc from paper I at energy minima of 0 and near ±120° are thermally averaged, assuming an energy dependence given by a basic 2-fold torsional potential. Fitted torsional barriers to A(µ)'(T) from this model are similar (~3 kJ/mol) for all muoniated butyl radicals, suggesting that these are dominated by ZPE effects arising from the C−Mu bond, but for A(p)(T) exhibit wide variations depending on environment. For the cis- and trans-2-butyl radicals formed from 2-butene, A(µ)'(T) exhibits clear discontinuities at bulk butene melting points, evidence for molecular interactions enhancing these muon Hfcc in the environment of the solid state, similar to that found in earlier reports for muoniated tert-butyl. In contrast, for Mu−sec-butyl formed from 1-butene, there is no such discontinuity. The muon hfcc for the trans-2-butyl radical are seemingly very well predicted by B3LYP calculations in the solid phase, but for sec-butyl from 1-butene, showing the absence of further interactions, much better agreement is found with the MP2 calculations across the whole temperature range. Examples of large proton Hfcc near 0 K are also reported, due to eclipsed C−H bonds, in like manner to C−Mu, which then also exhibit clear discontinuities in A(p)(T) at bulk melting points. The data suggest that the good agreement found between theory and experiment from the B3LYP calculations for eclipsed bonds in the solid phase may be fortuitous. For the staggered protons of the sec-butyl radicals formed, no discontinuities are seen at all in A(p)(T), also demonstrating no further effects of molecular interactions on these particular proton Hfcc.