Human apo-metallothionein 1a is not a random coil: Evidence from guanidinium chloride, high temperature, and acidic pH unfolding studies.

The structures of apo-metallothioneins (apo-MTs) have been relatively elusive due to their fluxional, disordered state which has been difficult to characterize. However, intrinsically disordered protein (IDP) structures are rather diverse, which raises questions about where the structure of apo-MTs fit into the protein structural spectrum. In this paper, the unfolding transitions of apo-MT1a are discussed with respect to the effect of the chemical denaturant GdmCl, temperature conditions, and pH environment. Cysteine modification in combination with electrospray ionization mass spectrometry was used to probe the unfolding transition of apo-MT1a in terms of cysteine exposure. Circular dichroism spectroscopy was also used to monitor the change in secondary structure as a function of GdmCl concentration. For both of these techniques, cooperative unfolding was observed, suggesting that apo-MT1a is not a random coil. More GdmCl was required to unfold the protein backbone than to expose the cysteines, indicating that cysteine exposure is likely an early step in the unfolding of apo-MT1a. MD simulations complement the experimental results, suggesting that apo-MT1a adopts a more compact structure than expected for a random coil. Overall, these results provide further insight into the intrinsically disordered structure of apo-MT.

ESI-MS analysis of Cu(I) binding to apo and Zn7 human metallothionein 1A, 2, and 3 identifies the formation of a similar series of metallated species with no individual isoform optimization for Cu(I).

Metallothioneins (MTs) are cysteine-rich proteins involved in metal homeostasis, heavy metal detoxification, and protection against oxidative stress. Whether the four mammalian MT isoforms exhibit different metal binding properties is not clear. In this paper, the Cu(I) binding properties of the apo MT1A, apo MT2, and apo MT3 are compared and the relative Cu(I) binding affinities are reported. In all three isoforms, Cu4, Cu6, and Cu10 species form cooperatively, and MT1A and MT2 also form a Cu13 species. The Cu(I) binding properties of Zn7-MT1A, Zn7-MT2, and Zn7-MT3 are compared systematically using isotopically pure 63Cu(I) and 68Zn(II). The species formed in each MT isoform were detected through electrospray ionization-mass spectrometry and further characterized using room temperature phosphorescence spectroscopy. The mixed metal Cu, Zn species forming in MT1A, MT2, and MT3 have similar stoichiometries and their emission spectral properties indicate that analogous clusters form in the three isoforms. Three parallel metallation pathways have been proposed through analysis of the detailed Cu, Zn speciation in MT1A, MT2, and MT3. Pathway ① results in Cu5Zn5-MT and Cu9Zn3-MT. Pathway ② involves Cu6Zn4-MT and Cu10Zn2-MT. Pathway ③ includes Cu8Zn4-MT. Speciation analysis indicates that Pathway ② is the preferred pathway for MT2. This is also evident in the phosphorescence spectra with the 750 nm emission from Cu6Zn4-MT being most prominent in MT2. We see no evidence for different MT isoforms being optimized or exhibiting preferences for certain metals. We discuss the probable stoichiometry for MTs in vivo based on the in vitro determined binding constants.

Physician and Advanced Practice Clinician Burnout in Rural and Urban Settings.

INTRODUCTION: Recruiting rural-practicing clinicians is a high priority. In this study, we explored burnout and contributing work conditions among rural, urban, and family practice physicians and advanced practice clinicians (APCs) in an Upper Midwestern health care system. METHODS: The Mini Z burnout reduction measure was administered by anonymous electronic survey in March 2022. We conducted bivariate analyses of study variables, then assessed relationships of study variables to burnout with multivariate binary logistic regression. RESULTS: Of 1118 clinicians (63% response rate), 589 physicians and 496 APCs were included in this study (n = 1085). Most were female (56%), physicians (54%), and White (86%), while 21% were in family practice, 46% reported burnout, and 349 practiced rurally. Rural and urban clinician burnout rates were comparable (45% vs 47%). Part-time work protected against burnout for family practice and rural clinicians, but not urban clinicians. In multivariate models for rural clinicians, stress (OR: 8.53, 95% CI: 4.09 to 17.78, P < .001), lack of workload control (OR: 3.06, 95% CI: 1.47-6.36, P = .003), busy/chaotic environments (OR: 2.53, 95% CI: 1.29-4.99, P = .007), and intent to leave (OR: 2.18, 95% CI: 1.06-4.45, P = .033) increased burnout odds. In family practice clinicians, stress (OR: 13.43 95% CI: 4.90-36.79, P < .001) also significantly increased burnout odds. CONCLUSIONS: Burnout was comparable between rural and urban physicians and APCs. Part-time work was associated with decreased burnout in rural and family practice clinicians. Addressing burnout drivers (stress, workload control, chaos) may improve rural work environments, reduce turnover, and aid rural clinician recruitment. Addressing stress may be particularly impactful in family practice.

Bi(III) Binding Stoichiometry and Domain-Specificity Differences Between Apo and Zn(II)-bound Human Metallothionein 1a.

Bismuth is a xenobiotic metal with a high affinity to sulfur that is used in a variety of therapeutic applications. Bi(III) induces the cysteine-rich metallothionein (MT), a protein known to form two-domain cluster structures with certain metals such as Zn(II), Cd(II), or Cu(I). The binding of Bi(III) to MTs has been previously studied, but there are conflicting reports on the stoichiometry and binding pathway, which appear to be highly dependent on pH and initial metal-loading status of the MT. Additionally, domain specificity has not been thoroughly investigated. In this paper, ESI-MS was used to determine the binding constants of [Bi(EDTA)]- binding to apo-MT1a and its individual αMT fragment. The results were compared to previous experiments using ßMT1a and ßαMT3. Domain specificity was investigated using proteolysis methods and the initial cooperatively formed Bi2MT was found to bind to cysteines that spanned across the traditional metal binding domain regions. Titrations of [Bi(EDTA)]- into Zn7MT were performed and were found to result in a maximum stoichiometry of Bi7MT, contrasting the Bi6MT formed when [Bi(EDTA)]- was added to apo-MT. These results show that the initial structure of the apo-MT determines the stoichiometry of new incoming metals and explains the previously observed differences in stoichiometry.

Structural motifs in the early metallation steps of Zn(II) and Cd(II) binding to apo-metallothionein 1a.

Many proteins require a metal cofactor to function and these metals are often involved in the protein folding process. The protein metallothionein (MT) has a dynamic structure capable of binding to a variety of metals with different stoichiometries. The most well-understood structure is the seven-metal, two domain structure formed upon metallation using Zn(II) or Cd(II). However, the partially metallated states and the pathways to form these clusters are less well-understood, although it is known that the pathways are pH dependent. Using stopped flow methods, it is shown that the metallation rates of the less cooperative Zn(II) binding pathway is much more impacted by low pH conditions that that of the more cooperative Cd(II) binding pathway. Electrospray ionization mass spectrometry (ESI-MS) methods reveal specific mixtures of bridging and terminally bound MxSy structures form in the first few metallation steps. Using a combination of methods, the data show that the result of unfolding this intrinsically disordered apo-MT structure using guanidinium chloride is that the formation of preliminary bridging structures that form in the first few metallation steps is impeded. The data show that more terminally bound structures form. Our conclusion is that the compact conformation of the native apo-MT at physiological pH allows for rapid formation of complex metal-thiolate structures with high affinity that provides protection from oxidation, a function that is suppressed upon unfolding. Overall, these results highlight both the importance of the apo-MT structure in the metallation pathway, but also the differences in Zn(II) and Cd(II) binding under different conditions.

Supermetalation of Cd-MT3 beyond the two-domain model.

The flexibility of mammalian metallothioneins (MTs) has contributed to the difficulty in obtaining structural information for this family of metalloproteins that bind divalent metals with its twenty cysteines. While the two-domain structure for Cd7MT is well-established as a Cd4S11 and Cd3S9, a third structure has been reported when 8 Cd(II) ions bind to MT1. Isoform 3 of the MT family, MT3, has been of interest to the research community since its isolation as a growth inhibitory factor isolated in brain tissue, and has since been noted as a prominent participant in the mediation of neurodegenerative diseases and regular brain development. The differences between MT3 and the other isoforms of MT include an additional hexapeptide insertion of acidic residues in the α domain as well as the introduction of two prolines in the ß domain. It is unclear whether these changes impact the metalation properties of MT3. We report the formation of a Cd8MT3 species is characterized by electrospray ionization mass spectrometry and UV-visible absorption spectroscopy. We report that the spectroscopic properties of this supermetalated Cd8MT3 are similar to those of the supermetalated Cd8MT1, with a clear indication of changes in structure from "fully-metalated" Cd7MT3 to supermetalated Cd8MT3 from circular dichroism spectra and both 1D 113Cd and 2D 1H-113Cd HSQC NMR spectra. We conclude that the metalation properties are not impacted significantly due to the amino acid changes in MT3, and that the cysteinyl thiols are the key players in determining the capacity of metal-binding and the structure of metal-thiolate clusters.

Metallothionein-3 and carbonic anhydrase metalation properties with Zn(II) and Cd(II) change as a result of protein-protein interactions.

Metallothioneins (MT) are regulators of the metals Zn(II) and Cu(I) and act as antioxidants in many organisms, including in humans. Isoform 3 (MT3) is expressed constitutively in central nervous tissue and has been shown to have additional biological functions, including the inhibition of neuronal growth, the regulation of apoptosis, and cytoskeleton modulation. To facilitate these functions, protein-protein interactions likely occur. These interactions may then impact the metalation status of the MT and the recipient metalloprotein. Using electrospray ionization mass spectrometry and circular dichroism spectroscopy, we report that the interaction between the zinc metalloenzyme, carbonic anhydrase (CA), and MT3, impacts the metalation profiles of both apo-MT3 and apo-CA with Cd(II) and Zn(II). We observe two phases in the metalation of the apo-CA, the first of which is associated with an increased binding affinity of apo-CA for Cd/Zn(II) and the second pathway is associated with apo-CA metalated without a change in binding affinity. The weak interactions that result in this change of binding affinity are not detectable as a protein complex in the ESI-mass spectral data or in the circular dichroism spectra. These unusual metalation properties of apo-CA in the presence of apo-MT3 are evidence of the effects of protein-protein interactions. With adjustment to take into account the interaction of both proteins, we report the complete Cd(II) and Zn(II) binding constants of MT3 under physiological conditions, as well as the pH dependence of these binding pathways.

Cu(I) binds to Zn7-MT2 via two parallel pathways.

Metallothionein proteins are essential for Cu(I) and Zn(II) homeostasis as well as heavy metal detoxification. The metallation properties of MT2 are of great interest due to their wide patterns of expression and correlation with multiple diseases including cancers, neurological disorders, and respiratory diseases. Use of isotopically pure 63Cu(I) and 68Zn(II) eliminates the complexity of the Cu, Zn-MT2 mass spectral peaks due to significant overlap of naturally abundant isotopes. This allows for the resolution of the precise Cu(I) and Zn(II) stoichiometries when both Cu(I) and Zn(II) are bound to MT2 at physiological pH as expected in vivo. Exact Cu: Zn ratios were determined from mass spectral simulations carried out for every point in the titration. We report that Cu(I) metallation of Zn7-MT2 can only be understood in terms of two pathways occurring in parallel with pathway ① resulting in Cu5Zn5-MT2 and Cu9Zn3-MT2. Pathway ② results in Cu6Zn4-MT2 and Cu10Zn2-MT2, which are the major products of the reaction. From the electrospray ionization (ESI)-mass spectral data we report a series of formation constants (KF) for species starting from Zn7-MT2 up to Cu11Zn2-MT2. Room temperature phosphorescence and circular dichroism (CD) spectra were measured in parallel with the ESI-mass spectrometry data allowing for the assignment of specific species to specific spectral bands. Through analysis of the CD spectral bands, we propose that Cu(I) binds to the ß domain first to form a Cu5Zn1 cluster or Cu6 cluster with emission at 670 and 750 nm, respectively, leaving the Zn4 cluster in the α domain.

Xenobiotic Bi3+ Coordination by Cysteine-Rich Metallothionein-3 Reveals a Cooperatively Formed Thiolate-Sharing Bi2S5 Cluster.

The field of designing artificial metalloproteins has yet to effectively tackle the incorporation of multimetal clusters, which is a key component of natural metalloproteins, such as metallothioneins (MTs) and calmodulin. MT is a physiological, essential, cysteine-rich metalloprotein that binds to a variety of metals but is only known to form metal-thiolate clusters with Cd2+, Zn2+, and Cu+. Bismuth is a xenobiotic metal and a component of metallodrugs used to treat gastric ulcers and cancer, as well as an emerging metal used in industrial practices. Electrospray ionization mass spectrometry, UV-visible spectroscopy, and extended X-ray absorption fine structure spectroscopy were used to probe the Bi3+ binding site structures in apo-MT3 (brain-located MT) at pH 7.4 and 2 and provide the complete set of binding affinities. We discovered the highly cooperative formation of a novel Bi3+ species, Bi2MT3, under physiological conditions, where each Bi3+ ion is coordinated by three cysteinyl thiolates, with one of the thiolates bridging between the two Bi3+ ions. This cluster structure was associated with a strong visible region absorption band, which was disrupted by the addition of Zn2+ and reversibly disrupted by acidification and increased temperature. This is the first reported presence of bridging cysteines for a xenobiotic metal in MT3 and the Bi2MT structure is the first Bi cluster found in a metalloprotein.

Arsenic binding to human metallothionein-3.

Arsenic poisoning is of great concern with respect to its neurological toxicity, which is especially significant for young children. Human exposure to arsenic occurs worldwide from contaminated drinking water. In human physiology, one response to toxic metals is through coordination with the metallochaperone metallothionein (MT). Central nervous system expression of MT isoform 3 (MT3) is thought to be neuroprotective. We report for the first time on the metalation pathways of As3+ binding to apo-MT3 under physiological conditions, yielding the absolute binding constants (log Kn, n = 1-6) for each sequential As3+ binding event: 10.20, 10.02, 9.79, 9.48, 9.06, and 8.31 M-1. We report on the rate of the reaction of As3+ with apo-MT3 at pH 3.5 with rate constants (kn, n = 1-6) determined for each sequential As3+ binding event: 116.9, 101.2, 85.6, 64.0, 43.9, and 21.0 M-1 s-1. We further characterize the As3+ binding pathway to fully metalated Zn7MT3 and partially metalated Zn-MT3. As3+ binds rapidly with high binding constants under physiological conditions in a noncooperative manner, but is unable to replace the Zn2+ in fully-metalated Zn-MT3. As3+ binding to partially metalated Zn-MT3 takes place with a rearrangement of the Zn-binding profile. Our work shows that As 3+ rapidly and efficiently binds to both apo-MT3 and partially metalated Zn-MT3 at physiological pH.

Metallothionein-3: 63 Cu(I) binds to human 68 Zn7 -ßα MT3 with no preference for Cu4 -ß cluster formation.

Human metallothioneins (MTs) are involved in binding the essential elements, Cu(I) and Zn(II), and the toxic element, Cd(II), in metal-thiolate clusters using 20 reduced cysteines. The brain-specific MT3 binds a mixture of Cu(I) and Zn(II) in vivo. Its metallation properties are critically important because of potential connections between Cu, Zn and neurodegenerative diseases. We report that the use of isotopically pure 63 Cu(I) and 68 Zn(II) greatly enhances the element resolution in the ESI-mass spectral data revealing species with differing Cu:Zn ratios but the same total number of metals. Room temperature phosphorescence and circular dichroism spectral data measured in parallel with ESI-mass spectral data identified the presence of specific Cu(I)-thiolate clusters in the presence of Zn(II). A series of Cu(I)-thiolate clusters form following Cu(I) addition to apo MT3: the two main clusters that form are a Cu6 cluster in the ß domain followed by a Cu4 cluster in the α domain. 63 Cu(I) addition to 68 Zn7 -MT3 results in multiple species, including clustered Cu5 Zn5 -MT3 and Cu9 Zn3 -MT3. We assign the domain location of the metals for Cu5 Zn5 -MT3 as a Cu5 Zn1 -ß cluster and a Zn4 -α cluster and for Cu9 Zn3 -MT3 as a Cu6 -ß cluster and a Cu3 Zn3 -α cluster. While many reports of the average MT3 metal content exist, determining the exact Cu,Zn stoichiometry has proven very difficult even with native ESI-MS. The work in this paper solves the ambiguity introduced by the overlap of the naturally abundant Cu(I) and Zn(II) isotopes. Contrary to other reports, there is no indication of a major fraction of Cu4 -ß-Znn -α-MT3 forming.

Structural Role of Cadmium and Zinc in Metallothionein Oxidation by Hydrogen Peroxide: The Resilience of Metal-Thiolate Clusters.

Oxidative stress is a state involving an imbalance of reactive oxygen species in a cell and is linked to a variety of diseases. The metal-binding protein metallothionein (MT) may play a role in protection due to its high cysteine content. Many studies have shown that oxidative stress will cause MT to both form disulfide bonds and release bound metals. However, studies on the more biologically relevant partially metalated MTs have been largely neglected. Additionally, most studies to date have used spectroscopic methods that cannot detect specific intermediate species. In this paper, we describe the oxidation and the subsequent metal displacement pathway of fully and partially metalated MTs with hydrogen peroxide. The rates of the reactions were monitored using electrospray ionization mass spectrometry (ESI-MS) techniques, which resolved and characterized the individual intermediate Mx(SH)yMT species. The rate constants were calculated for each species formation. Through ESI-MS and circular dichroism spectroscopy, it was found that the three metals in the ß-domain were the first to be released from the fully metalated MTs. The Cd(II) in the partially metalated Cd(II)-bound MTs rearranged to form a protective Cd4MT cluster structure upon exposure to oxidation. The partially metalated Zn(II)-bound MTs oxidized at a faster rate as the Zn(II) did not rearrange in response to oxidation. Additionally, density functional theory calculations showed that the terminally bound cysteines were more negative and thus more susceptible to oxidation than the bridging cysteines. The results of this study highlight the importance of metal-thiolate structures and metal identity in MT's response to oxidation.

Apo-metallothionein-3 cooperatively forms tightly compact structures under physiological conditions.

Metallothioneins (MTs) are essential mammalian metal chaperones. MT isoform 1 (MT1) is expressed in the kidneys and isoform 3 (MT3) is expressed in nervous tissue. For MTs, the solution-based NMR structure was determined for metal-bound MT1 and MT2, and only one X-ray diffraction structure on a crystallized mixed metal-bound MT2 has been reported. The structure of solution-based metalated MT3 is partially known using NMR methods; however, little is known about the fluxional de novo apo-MT3 because the structure cannot be determined by traditional methods. Here, we used cysteine modification coupled with electrospray ionization mass spectrometry, denaturing reactions with guanidinium chloride, stopped-flow methods measuring cysteine modification and metalation, and ion mobility mass spectrometry to reveal that apo-MT3 adopts a compact structure under physiological conditions and an extended structure under denaturing conditions, with no intermediates. Compared with apo-MT1, we found that this compact apo-MT3 binds to a cysteine modifier more cooperatively at equilibrium and 0.5 times the rate, providing quantitative evidence that many of the 20 cysteines of apo-MT3 are less accessible than those of apo-MT1. In addition, this compact apo-MT3 can be identified as a distinct population using ion mobility mass spectrometry. Furthermore, proposed structural models can be calculated using molecular dynamics methods. Collectively, these findings provide support for MT3 acting as a noninducible regulator of the nervous system compared with MT1 as an inducible scavenger of trace metals and toxic metals in the kidneys.

63Cu(I) binding to human kidney 68Zn7-ßα MT1A: determination of Cu(I)-thiolate cluster domain specificity from ESI-MS and room temperature phosphorescence spectroscopy.

Mammalian metallothioneins (MTs) are important proteins in Zn(II) and Cu(I) homeostasis with the Zn(II) and Cu(I) binding to the 20 cysteines in metal-thiolate clusters. Previous electrospray ionization (ESI) mass spectrometric (MS) analyses of Cu(I) binding to Zn7-MT were complicated by significant overlap of the natural abundance isotopic patterns for Zn(II) and Cu(I) leading to impossibly ambiguous stoichiometries. In this paper, isotopically pure 63Cu(I) and 68Zn(II) allowed determination of the specific stoichiometries in the 68 Zn,63Cu-ßα MT1A species formed following the stepwise addition of 63Cu(I) to 68Zn7-ßα MT1A. These species were characterized by ESI-MS and room temperature emission spectroscopy. The key species that form and their emission band centres are Zn5Cu5-ßα MT1A (λ = 684 nm), Zn4Cu6-ßα MT1A (λ = 750 nm), Zn3Cu9-ßα MT1A (λ = 750 nm), Zn2Cu10-ßα MT1A (λ = 750 nm), and Zn1Cu14-ßα MT1A (λ = 634 nm). The specific domain stoichiometry of each species was determined by assessing the species forming following 63Cu(I) addition to the 68Zn3-ß MT1A and 68Zn4-α MT1A domain fragments. The domain fragment emission suggests that Zn5Cu5-ßα MT1A contains a Zn1Cu5-ß cluster and the Zn4Cu6-ßα MT1A, Zn3Cu9-ßα MT1A, and Zn2Cu10-ßα MT1A each contain a Cu6-ß cluster. The species forming with >10 mol. eq. of 63Cu(I) in ßα-MT1A exhibit emission from the Cu6-ß cluster and an α domain cluster. This high emission intensity is seen at the end of the titrations of 68Zn7-ßα MT1A and the 68Zn4-α MT1A domain fragment suggesting that the initial presence of the Zn(II) results in clustered Cu(I) binding in the α domain.

COVID-Related Stress and Work Intentions in a Sample of US Health Care Workers.

OBJECTIVE: To evaluate relationships between coronavirus disease 2019 (COVID-19)-related stress and work intentions in a sample of US health care workers. PATIENTS AND METHODS: Between July 1 and December 31, 2020, health care workers were surveyed for fear of viral exposure or transmission, COVID-19-related anxiety or depression, work overload, burnout, and intentions to reduce hours or leave their jobs. RESULTS: Among 20,665 respondents at 124 institutions (median organizational response rate, 34%), intention to reduce hours was highest among nurses (33.7%; n=776), physicians (31.4%; n=2914), and advanced practice providers (APPs; 28.9%; n=608) while lowest among clerical staff (13.6%; n=242) and administrators (6.8%; n=50; all P<.001). Burnout (odds ratio [OR], 2.15; 95% CI, 1.93 to 2.38), fear of exposure, COVID-19-related anxiety/depression, and workload were independently related to intent to reduce work hours within 12 months (all P<.01). Intention to leave one's practice within 2 years was highest among nurses (40.0%; n=921), APPs (33.0%; n=694), other clinical staff (29.4%; n=718), and physicians (23.8%; n=2204) while lowest among administrators (12.6%; n=93; all P<.001). Burnout (OR, 2.57; 95% CI, 2.29 to 2.88), fear of exposure, COVID-19-related anxiety/depression, and workload were predictors of intent to leave. Feeling valued by one's organization was protective of reducing hours (OR, 0.65; 95% CI, 0.59 to 0.72) and intending to leave (OR, 0.40; 95% CI, 0.36 to 0.45; all P<.01). CONCLUSION: Approximately 1 in 3 physicians, APPs, and nurses surveyed intend to reduce work hours. One in 5 physicians and 2 in 5 nurses intend to leave their practice altogether. Reducing burnout and improving a sense of feeling valued may allow health care organizations to better maintain their workforces postpandemic.

A di-Copper Peptidyl Complex Mimics the Activity of Catalase, a Key Antioxidant Metalloenzyme.

Catalases (CAT) are antioxidant metalloenzymes necessary for life in oxygen-metabolizing cells to regulate H2O2 concentration by accelerating its dismutation. Many physiopathological situations are associated with oxidative stress resulting from H2O2 overproduction, during which antioxidant defenses are overwhelmed. We have used a combinatorial approach associated with an activity-based screening to discover a first peptidyl di-copper complex mimicking CAT. The complex was studied in detail and characterized for its CAT activity both in solutions and in cells using different analytical methods. The complex exhibited CAT activity in solutions and, more interestingly, on HyPer HeLa cells that possess a genetically encoded ratiometric fluorescent sensors of H2O2. These results highlight the efficiency of a combinatorial approach for the discovery of peptidyl complexes that exhibit catalytic activity.

Structurally restricted Bi(III) metallation of apo-ßMT1a: metal-induced tangling.

Non-toxic bismuth salts are used in anti-ulcer medications and to protect against nephrotoxicity from anticancer drugs. Bismuth salts also induce metallothionein (MT), a metal-binding protein that lacks a formal secondary structure. We report the impact on the metallation properties of Bi(III) to the 9-cysteine ß fragment of MT as a function of cysteine accessibility using electrospray ionization mass spectrometry. At pH 7.4, Bi2ßMT formed cooperatively. Cysteine modification shows that each Bi(III) was terminally bound to three cysteinyl thiolates. Non-cooperative Bi(III) binding was observed at pH 2.3, where cysteine accessibility is increased. However, competition from H4EDTA inhibited Bi(III) binding. When GdmCl, a well-known denaturing agent, was used to increase cysteine accessibility of the apoßMT at pH 7.4, a greater fraction of Bi3ßMT formed using all nine cysteines. The change in binding profile and equilibrium of Bi2ßMT was determined as a function of acidification, which changed as a result of competition with H4EDTA. There was no Bi(III) transfer between Bi2ßMT, Cd3ßMT, and Zn3ßMT. This lack of metal exchange and the resistance towards binding the third Bi(III) suggest a rigidity in the Bi2ßMT binding sites that inhibits Bi(III) mobility. These experiments emphasize the conformational control of metallation that results in substantially different metallated products: at pH 7.4 (many cysteines buried) Bi2ßMT, whereas at pH 7.4 (all cysteines accessible) enhanced formation of Bi3ßMT. These data suggest that the addition of the first two Bi(III) crosslinks the protein, blocking access to the remaining three cysteines for the third Bi(III), as a result of tangle formation.

Association of Physician Burnout With Suicidal Ideation and Medical Errors.

Importance: Addressing physician suicide requires understanding its association with possible risk factors such as burnout and depression. Objective: To assess the association between burnout and suicidal ideation after adjusting for depression and the association of burnout and depression with self-reported medical errors. Design, Setting, and Participants: This cross-sectional study was conducted from November 12, 2018, to February 15, 2019. Attending and postgraduate trainee physicians randomly sampled from the American Medical Association Physician Masterfile were emailed invitations to complete an online survey in waves until a convenience sample of more than 1200 practicing physicians agreed to participate. Main Outcomes and Measures: The primary outcome was the association of burnout with suicidal ideation after adjustment for depression. The secondary outcome was the association of burnout and depression with self-reported medical errors. Burnout, depression, suicidal ideation, and medical errors were measured using subscales of the Stanford Professional Fulfillment Index, Maslach Burnout Inventory-Human Services Survey for Medical Personnel, and Mini-Z burnout survey and the Patient-Reported Outcomes Measurement Information System depression Short Form. Associations were evaluated using multivariable regression models. Results: Of the 1354 respondents, 893 (66.0%) were White, 1268 (93.6%) were non-Hispanic, 762 (56.3%) were men, 912 (67.4%) were non-primary care physicians, 934 (69.0%) were attending physicians, and 824 (60.9%) were younger than 45 years. Each SD-unit increase in burnout was associated with 85% increased odds of suicidal ideation (odds ratio [OR], 1.85; 95% CI, 1.47-2.31). After adjusting for depression, there was no longer an association (OR, 0.85; 95% CI, 0.63-1.17). In the adjusted model, each SD-unit increase in depression was associated with 202% increased odds of suicidal ideation (OR, 3.02; 95% CI, 2.30-3.95). In the adjusted model for self-reported medical errors, each SD-unit increase in burnout was associated with an increase in self-reported medical errors (OR, 1.48; 95% CI, 1.28-1.71), whereas depression was not associated with self-reported medical errors (OR, 1.01; 95% CI, 0.88-1.16). Conclusions and Relevance: The results of this cross-sectional study suggest that depression but not physician burnout is directly associated with suicidal ideation. Burnout was associated with self-reported medical errors. Future investigation might examine whether burnout represents an upstream intervention target to prevent suicidal ideation by preventing depression.

The pathways and domain specificity of Cu(I) binding to human metallothionein 1A.

Copper is an essential element, but as a result of numerous adverse reactions, it is also a cellular toxin. Nature protects itself from these toxic reactions by binding cuprous copper to chaperones and other metalloproteins. Metallothionein has been proposed as a storage location for Cu(i) and potentially as the donor of Cu(i) to copper-dependent enzymes. We report that the addition of Cu(i) to apo recombinant human metallothionein 1a cooperatively forms a sequential series of Cu(i)-cysteinyl thiolate complexes that have specific Cu(i) : MT stoichiometries of 6 : 1, 10 : 1, and finally 13 : 1. The individual domain Cu : SCys stoichiometries were determined as Cu6S9 (for 6 : 1), Cu6S9 + Cu4S6 (for 10 : 1), and Cu6S9 + Cu7S9 (for 13 : 1) based on the number of modified free cysteines not involved in Cu(i) binding. The stoichiometries are associated with Cu-SCys cluster formation involving bridging thiols in the manner similar to the clusters formed with Cd(ii) and Zn(ii). The locations of these clustered species within the 20 cysteine full protein were determined from the unique speciation profiles of Cu(i) binding to the ß and α domain fragments of recombinant human metallothionein 1a with 9 and 11 cysteines, respectively. Competition reactions using these domain fragments challenged Cu(i) metallation of the ßα protein, allowing the sequence of cluster formation in the full protein to be determined. Relative binding constants for each Cu(i) bound are reported. The emission spectra of the Cu4S6, Cu6S9, and Cu7S9 clusters have unique λmax and phosphorescent lifetime properties. These phosphorescent data provide unambiguous supporting evidence for the presence of solvent shielded clusters reported concurrently by ESI-MS. Simulated emission spectra based on the cluster specific emission profiles matched the experimental spectra and are used to confirm that the relative concentrations seen by ESI-MS are representative of the solution. Our results suggest that the availability of a series of sequential Cu(i)-thiolate clusters provides flexibility as a means of protecting the cell from toxicity while still allowing for homeostatic control of the total copper content in the cell. This mechanism provides a dynamic and reactive method of reducing the cellular free copper concentrations.

Interplay between Carbonic Anhydrases and Metallothioneins: Structural Control of Metalation.

Carbonic anhydrases (CAs) and metallothioneins (MTs) are both families of zinc metalloproteins central to life, however, they coordinate and interact with their Zn2+ ion cofactors in completely different ways. CAs and MTs are highly sensitive to the cellular environment and play key roles in maintaining cellular homeostasis. In addition, CAs and MTs have multiple isoforms with differentiated regulation. This review discusses current literature regarding these two families of metalloproteins in carcinogenesis, with a dialogue on the association of these two ubiquitous proteins in vitro in the context of metalation. Metalation of CA by Zn-MT and Cd-MT is described. Evidence for protein-protein interactions is introduced from changes in metalation profiles of MT from electrospray ionization mass spectrometry and the metalation rate from stopped-flow kinetics. The implications on cellular control of pH and metal donation is also discussed in the context of diseased states.