Three-state analysis of sperm whale apomyoglobin folding

Barrick, Doug; Baldwin, Robert L.

doi:10.1021/bi00065a035

Cited by 232 publications

(237 citation statements)

References 37 publications

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“…Although it cannot be ruled out that the reversible formation of β-sheet is the result of intermolecular interactions in small soluble aggregates, largescale aggregation would cause scattering not detected in the CD data. Thus, the population existing at 2 kbar is not unfolded in the literal sense, and certainly retains a higher fraction of secondary structure than that produced by chemical perturbation techniques, including GdnHCl (∼5%), urea (∼7%), and pH (∼13%) (67,68). Although EPR spectral changes at high pressure confirm the appearance of a dynamically disordered population in the noncore helices C, D, E, and F, the spectra at all sites retain two components, indicating the retention of a tertiary fold.…”

Section: Discussionmentioning

confidence: 99%

Circular dichroism and site-directed spin labeling reveal structural and dynamical features of high-pressure states of myoglobin

Lerch

Horwitz²,

McCoy³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Excited states of proteins may play important roles in function, yet are difficult to study spectroscopically because of their sparse population. High hydrostatic pressure increases the equilibrium population of excited states, enabling their characterization [Akasaka K (2003) Biochemistry 42:10875-85]. High-pressure site-directed spin-labeling EPR (SDSL-EPR) was developed recently to map the site-specific structure and dynamics of excited states populated by pressure. To monitor global secondary structure content by circular dichroism (CD) at high pressure, a modified optical cell using a custom MgF 2 window with a reduced aperture is introduced. Here, a combination of SDSL-EPR and CD is used to map reversible structural transitions in holomyoglobin and apomyoglobin (apoMb) as a function of applied pressure up to 2 kbar. CD shows that the high-pressure excited state of apoMb at pH 6 has helical content identical to that of native apoMb, but reversible changes reflecting the appearance of a conformational ensemble are observed by SDSL-EPR, suggesting a helical topology that fluctuates slowly on the EPR time scale. Although the high-pressure state of apoMb at pH 6 has been referred to as a molten globule, the data presented here reveal significant differences from the well-characterized pH 4.1 molten globule of apoMb. Pressure-populated states of both holomyoglobin and apoMb at pH 4.1 have significantly less helical structure, and for the latter, that may correspond to a transient folding intermediate.P roteins in solution are dynamic molecules, exhibiting conformational flexibility across a range of time and length scales (1). In addition to a well-ordered native state, conformational excursions to low-lying "excited states" may be required in protein function (2). For example, on a funnel-shaped energy landscape (3, 4), excited states have increased configurational entropy that may give rise to the promiscuous protein-protein interactions that define a protein interactome (5, 6). Structural changes involved in formation of the excited state may take a variety of forms, from rigid body motions of helices (7) to local unfolding of secondary structural elements (8). Despite their functional relevance, excited states are sparsely populated and may escape detection by standard spectroscopic techniques. Hydrostatic pressure apparently offers a solution to this problem by reversibly populating excited states of proteins, allowing for spectroscopic characterization (9-14). Pressure application is particularly useful because it shifts the relative population of preexisting conformational states while minimally affecting the conformational landscape itself (15).Assuming that pressure can populate excited states for study, the increased configurational entropy of such states presents a challenge to spectroscopic methods that aim to describe structure; depending on the intrinsic time scale of the method, either a population-weighted average structure or a heterogeneous ensemble is observed. The intrinsic time scale of con...

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Section: Discussionmentioning

confidence: 99%

Circular dichroism and site-directed spin labeling reveal structural and dynamical features of high-pressure states of myoglobin

Lerch

Horwitz²,

McCoy³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Data were fit according to the Henderson-Hasselbalch equation assuming a two-state protonation equilibrium. 34 Far UV CD Circular dichroism measurements were carried out using a Jasco-J810 spectropolarimeter. Spectra of domain 4 (12.1 lM) in 5 mM each of Bis-Tris/Hepes/cacodylic acid at pH 8 or 5, 67 mM NaCl were recorded between 180 and 260 nm in a 0.5 mm thermostatted cylindrical cell at 5 C for eight accumulations at a scan speed of 10 nm min À1 and a response time of 4 s. For variable temperature experiments, the molar ellipticity was monitored at 201 nm with a response time of 16 s, and temperature adjusted from 5 to 60 C and back from 60 to 5 C using a Julabo F25/HD circulating water bath.…”

Section: Fluorescencementioning

confidence: 99%

Domain 4 of the anthrax protective antigen maintains structure and binding to the host receptor CMG2 at low pH

et al. 2009

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Domain 4 of the anthrax protective antigen (PA) plays a key role in cellular receptor recognition as well as in pH-dependent pore formation. We present here the 1.95 Å crystal structure of domain 4, which adopts a fold that is identical to that observed in the full-length protein. We have also investigated the structural properties of the isolated domain 4 as a function of pH, as well as the pH-dependence on binding to the von Willebrand factor A domain of capillary morphogenesis protein 2 (CMG2). Our results provide evidence that the isolated domain 4 maintains structure and interactions with CMG2 at pH 5, a pH that is known to cause release of the receptor on conversion of the heptameric prepore (PA 63 ) 7 to a membrane-spanning pore. Our results suggest that receptor release is not driven solely by a pH-induced unfolding of domain 4.

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“…The following equation was used that is an adaptation of the three-state equazIu = e -( A G~~-m f u D ) / R T (3) tion of Barrick and Baldwin (1993).…”

Section: Equilibrium Urea Denaturation Of Apoflavodoxin At Neutral P Hmentioning

confidence: 99%

Conformational stability of apoflavodoxin

et al. 1996

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Flavodoxins are ar/p proteins that mediate electron transfer reactions. The conformational stability of apoflavodoxin from A n a b~n a PCC 71 19 has been studied by calorimetry and urea denaturation as a function of pH and ionic strength. At pH > 12, the protein is unfolded. Between pH 1 1 and pH 6, the apoprotein is folded properly as judged from near-ultraviolet (UV) circular dichroism (CD) and high-field 'H NMR spectra. In this pH interval, apoflavodoxin is a monomer and its unfolding by urea or temperature follows a simple two-state mechanism. The specific heat capacity of unfolding for this native conformation is unusually low. Near its isoelectric point (3.9), the protein is highly insoluble. At lower pH values (pH 3.5-2.0), apoflavodoxin adopts a conformation with the properties of a molten globule. Although apoflavodoxin at pH 2 unfolds cooperatively with urea in a reversible fashion and the fluorescence and far-UV CD unfolding curves coincide, the transition midpoint depends on the concentration of protein, ruling out a simple two-state process at acidic pH. Apoflavodoxin constitutes a prornising system for the analysis of the stability and folding of cr/0 proteins and for the study of the interaction between apoflavoproteins and their corresponding redox cofactors.

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Three-state analysis of sperm whale apomyoglobin folding

Cited by 232 publications

References 37 publications

Circular dichroism and site-directed spin labeling reveal structural and dynamical features of high-pressure states of myoglobin

Circular dichroism and site-directed spin labeling reveal structural and dynamical features of high-pressure states of myoglobin

Domain 4 of the anthrax protective antigen maintains structure and binding to the host receptor CMG2 at low pH

Conformational stability of apoflavodoxin

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