Filling up the heme pocket stabilizes apomyoglobin and speeds up its folding.
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ABSTRACT:
SUBMITTER: Goodman JS
PROVIDER: S-EPMC4065233 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Apomyoglobin folds via sequential helical intermediates that are formed by rapid collapse of the A, B, G, and H helix regions. An equilibrium molten globule with a similar structure is formed near pH 4. Previous studies suggested that the folding intermediates are kinetically trapped states in which folding is impeded by non-native packing of the G and H helices. Fluorescence spectra of mutant proteins in which cysteine residues were introduced at several positions in the G and H helices show differential quenching of W14 fluorescence, providing direct evidence of translocation of the H helix relative to helices A and G in both the kinetic and equilibrium intermediates. Förster resonance energy transfer measurements show that a 5-({2-[(acetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid acceptor coupled to K140C (helix H) is closer to Trp14 (helix A) in the equilibrium molten globule than in the native state, by a distance that is consistent with sliding of the H helix in an N-terminal direction by approximately one helical turn. Formation of an S108C-L135C disulfide prevents H helix translocation in the equilibrium molten globule by locking the G and H helices into their native register. By enforcing nativelike packing of the A, G, and H helices, the disulfide resolves local energetic frustration and facilitates transient docking of the E helix region onto the hydrophobic core but has only a small effect on the refolding rate. The apomyoglobin folding landscape is highly rugged, with several energetic bottlenecks that frustrate folding; relief of any one of the major identified bottlenecks is insufficient to speed progression to the transition state.
| S-EPMC4067146 | biostudies-literature
Project description:The F helix region of sperm whale apomyoglobin is disordered, undergoing conformational fluctuations between a folded helical conformation and one or more locally unfolded states. To examine the effects of F helix stabilization on the folding pathway of apomyoglobin, we have introduced mutations to augment intrinsic helical structure in the F helix of the kinetic folding intermediate and to increase its propensity to fold early in the pathway, using predictions based on plots of the average area buried upon folding (AABUF) derived from the primary sequence. Two mutant proteins were prepared: a double mutant, P88K/S92K (F2), and a quadruple mutant, P88K/A90L/S92K/A94L (F4). Whereas the AABUF for F2 predicts that the F helix will not fold early in the pathway, the F helix in F4 shows a significantly increased AABUF and is therefore predicted to fold early. Protection of amide protons by formation of hydrogen-bonded helical structure during the early folding events has been analyzed by pH-pulse labeling. Consistent with the AABUF prediction, many of the F helix residues for F4 are significantly protected in the kinetic intermediate but are not protected in the F2 mutant. F4 folds via a kinetically trapped burst-phase intermediate that contains stabilized secondary structure in the A, B, F, G, and H helix regions. Rapid folding of the F helix stabilizes the central core of the misfolded intermediate and inhibits translocation of the H helix back to its native position, thereby decreasing the overall folding rate.
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Project description:Kinetic investigation on the wild-type apomyoglobin and its 12 mutants with substitutions of hydrophobic residues by Ala was performed using stopped-flow fluorescence. Characteristics of the kinetic intermediate I and the folding nucleus were derived solely from kinetic data, namely, the slow-phase folding rate constants and the burst-phase amplitudes of Trp fluorescence intensity. This allowed us to pioneer the phi-analysis for apomyoglobin. As shown, these mutations drastically destabilized the native state N and produced minor (for conserved residues of G, H helices) or even negligible (for nonconserved residues of B, C, D, E helices) destabilizing effect on the state I. On the other hand, conserved residues of A, G, H helices made a smaller contribution to stability of the folding nucleus at the rate-limiting I-->N transition than nonconserved residues of B, D, E helices. Thus, conserved side chains of the A-, G-, H-residues become involved in the folding nucleus before crossing the main barrier, whereas nonconserved side chains of the B-, D-, E-residues join the nucleus in the course of the I-->N transition.
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Project description:Bacterial chemotaxis is a canonical system for the study of signal transduction. One of the hallmarks of this system is its robust adaptive behavior. However, how fast the system adapts remains controversial. The adaptation time measured at the level of the kinase activity was tens of seconds, whereas that measured at the level of the flagellar motor was <10 s. The flagellar motor was recently shown to exhibit adaptive remodeling, its main physiological function being to provide a robust match between the chemoreceptor output and the motor input, whereas its adaptation timescale was thought to be too slow to contribute much to the overall adaptation timescale of the chemotaxis system. Here, through theoretical modeling of the motor adaptive remodeling and experimental tests, we show that this motor adaptation contributes significantly to speeding up the overall chemotactic adaptation, thereby resolving the previous inconsistency.
| S-EPMC5883565 | biostudies-literature
Project description:Under mildly acidic conditions (pH 4-4.5) apomyoglobin (apoMb) adopts a partially structured equilibrium state ( M-state) that structurally resembles a kinetic intermediate encountered at a late stage of folding to the native structure at neutral pH. We have previously reported that the M-state is formed rapidly (<1 ms) via a multistate process and thus offers a unique opportunity for exploring early stages of folding by both experimental and computational techniques. In order to gain structural insight into intermediates and barriers at the residue level, we studied the folding/unfolding kinetics of 12 apoMb mutants at pH 4.2 using fluorescence-detected ultrafast mixing techniques. Global analysis of the submillisecond folding/unfolding kinetics vs urea concentration for each variant, based on a sequential four-state mechanism ( U ? I ? L ? M), allowed us to determine elementary rate constants and their dependence on urea concentration for most transitions. Comparison of the free energy diagrams constructed from the kinetic data of the mutants with that of wild-type apoMb yielded quantitative information on the effects of mutations on the free energy (?? G) of both intermediates and the first two kinetic barriers encountered during folding. Truncation of conserved aliphatic side chains on helices A, G, and H gives rise to a stepwise increase in ?? G as the protein advances from U toward M, consistent with progressive stabilization of native-like contacts within the primary core of apoMb. Helix-helix contacts in the primary core contribute little to the first folding barrier ( U ? I) and thus are not required for folding initiation but are critical for the stability of the late intermediate, L, and the M-state. Alanine substitution of hydrophobic residues at more peripheral helix-helix contact sites of the native structure, which are still absent or unstable in the M-state, shows both positive (destabilizing) and negative (stabilizing) ?? G, indicating that non-native contacts are formed initially and weakened or lost as a result of subsequent structural rearrangement steps.
| S-EPMC6395567 | biostudies-literature
Project description:The earliest steps in the folding of proteins are complete on an extremely rapid time scale that is difficult to access experimentally. We have used rapid-mixing quench-flow methods to extend the time resolution of folding studies on apomyoglobin and elucidate the structural and dynamic features of members of the ensemble of intermediate states that are populated on a submillisecond time scale during this process. The picture that emerges is of a continuum of rapidly interconverting states. Even after only 0.4 ms of refolding time a compact state is formed that contains major parts of the A, G, and H helices, which are sufficiently well folded to protect amides from exchange. The B, C, and E helix regions fold more slowly and fluctuate rapidly between open and closed states as they search docking sites on this core; the secondary structure in these regions becomes stabilized as the refolding time is increased from 0.4 to 6 ms. No further stabilization occurs in the A, G, H core at 6 ms of folding time. These studies begin to time-resolve a progression of compact states between the fully unfolded and native folded states and confirm the presence an ensemble of intermediates that interconvert in a hierarchical sequence as the protein searches conformational space on its folding trajectory.
| S-EPMC2544544 | biostudies-literature
Project description:BACKGROUND: How do we estimate time when watching an action? The idea that events are timed by a centralized clock has recently been called into question in favour of distributed, specialized mechanisms. Here we provide evidence for a critical specialization: animate and inanimate events are separately timed by humans. METHODOLOGY/PRINCIPAL FINDINGS: In different experiments, observers were asked to intercept a moving target or to discriminate the duration of a stationary flash while viewing different scenes. Time estimates were systematically shorter in the sessions involving human characters moving in the scene than in those involving inanimate moving characters. Remarkably, the animate/inanimate context also affected randomly intermingled trials which always depicted the same still character. CONCLUSIONS/SIGNIFICANCE: The existence of distinct time bases for animate and inanimate events might be related to the partial segregation of the neural networks processing these two categories of objects, and could enhance our ability to predict critically timed actions.
| S-EPMC3012081 | biostudies-literature
Project description:The structures of apomyoglobin folding intermediates have been widely analyzed using physical chemistry methods including fluorescence, circular dichroism, small angle X-ray scattering, NMR, mass spectrometry, and rapid mixing. So far, at least two intermediates (on sub-millisecond- and millisecond-scales) have been demonstrated for apomyoglobin folding. The combination of pH-pulse labeling and NMR is a useful tool for analyzing the kinetic intermediates at the atomic level. Its use has revealed that the latter-phase kinetic intermediate of apomyoglobin (6 ms) was composed of helices A, B, G and H, whereas the equilibrium intermediate, called the pH 4 molten-globule intermediate, was composed mainly of helices A, G and H. The improved strategy for the analysis of the kinetic intermediate was developed to include (1) the dimethyl sulfoxide method, (2) data processing with the various labeling times, and (3) a new in-house mixer. Particularly, the rapid mixing revealed that helices A and G were significantly more protected at the earlier stage (400 µs) of the intermediate (former-phase intermediate) than the other helices. Mutation studies, where each hydrophobic residue was replaced with an alanine in helices A, B, E, F, G and H, indicated that both non-native and native-like structures exist in the latter-phase folding intermediate. The N-terminal part of helix B is a weak point in the intermediate, and the docking of helix E residues to the core of the A, B, G and H helices was interrupted by a premature helix B, resulting in the accumulation of the intermediate composed of helices A, B, G and H. The prediction-based protein engineering produced important mutants: Helix F in a P88K/A90L/S92K/A94L mutant folded in the latter-phase intermediate, although helix F in the wild type does not fold even at the native state. Furthermore, in the L11G/W14G/A70L/G73W mutant, helix A did not fold but helix E did, which is similar to what was observed in the kinetic intermediate of apoleghemoglobin. Thus, this protein engineering resulted in a changed structure for the apomyoglobin folding intermediate.
| S-EPMC5406622 | biostudies-literature
Project description:The characterization of protein folding dynamics in terms of secondary and tertiary structures is important in elucidating the features of intraprotein interactions that lead to specific folded structures. Apomyoglobin (apoMb), possessing seven helices termed A-E, G, and H in the native state, has a folding intermediate composed of the A, G, and H helices, whose formation in the submillisecond time domain has not been clearly characterized. In this study, we used a rapid-mixing device combined with circular dichroism and small-angle x-ray scattering to observe the submillisecond folding dynamics of apoMb in terms of helical content (f(H)) and radius of gyration (R(g)), respectively. The folding of apoMb from the acid-unfolded state at pH 2.2 was initiated by a pH jump to 6.0. A significant collapse, corresponding to approximately 50% of the overall change in R(g) from the unfolded to native conformation, was observed within 300 micros after the pH jump. The collapsed intermediate has a f(H) of 33% and a globular shape that involves >80% of all its atoms. Subsequently, a stepwise helix formation was detected, which was interpreted to be associated with a conformational search for the correct tertiary contacts. The characterized folding dynamics of apoMb indicates the importance of the initial collapse event, which is suggested to facilitate the subsequent conformational search and the helix formation leading to the native structure.
| S-EPMC337025 | biostudies-literature
Project description:ImportanceOne-third of US residents have trouble paying their medical bills. They often turn to their physicians for help navigating health costs and insurance coverage.ObjectiveTo determine whether physicians can accurately estimate out-of-pocket expenses when they are given all of the necessary information about a drug's price and a patient's insurance plan.Design, setting, and participantsThis national mail-in survey used a random sample of US physicians. The survey was sent to 900 outpatient physicians (300 each of primary care, gastroenterology, and rheumatology). Physicians were excluded if they were in training, worked primarily for the Veterans Administration or Indian Health Service, were retired, or reported 0% outpatient clinical effort. Analyses were performed from July to December 2020.Main outcomes and measuresIn a hypothetical vignette, a patient was prescribed a new drug costing $1000/month without insurance. A summary of her private insurance information was provided, including the plan's deductible, coinsurance rates, copays, and out-of-pocket maximum. Physicians were asked to estimate the drug's out-of-pocket cost at 4 time points between January and December, using the plan's 4 types of cost-sharing: (1) deductibles, (2) coinsurance, (3) copays, and (4) out-of-pocket maximums. Multivariate linear regression was used to assess differences in performance by specialty, adjusting for attitudes toward cost conversations, demographics, and clinical characteristics.ResultsThe response rate was 45% (405 of 900) and 371 respondents met inclusion criteria. Among the respondents included in this study, 59% (n = 220) identified as male, 23% (n = 84) as Asian, 3% (n = 12) as Black, 6% (n = 24) as Hispanic, and 58% (n = 216) as White; 30% (n = 112) were primary care physicians, 35% (n = 128) were gastroenterologists, and 35% (n = 131) were rheumatologists; and the mean (SD) age was 49 (10) years. Overall, 52% of physicians (n = 192) accurately estimated costs before the deductible was met, 62% (n = 228) accurately used coinsurance information, 61% (n = 224) accurately used copay information, and 57% (n = 210) accurately estimated costs once the out-of-pocket maximum was met. Only 21% (n = 78) of physicians answered all 4 questions correctly. Ability to estimate out-of-pocket costs was not associated with specialty, attitudes toward cost conversations, or clinic characteristics.Conclusions and relevanceThis survey study found that many US physicians have difficulty estimating out-of-pocket costs, even when they have access to their patients' insurance plans. The mechanics involved in calculating real-time out-of-pocket costs are complex. These findings suggest that increased price transparency and simpler insurance cost-sharing mechanisms are needed to enable informed cost conversations at the point of prescribing.
| S-EPMC8571653 | biostudies-literature