Characterization of transient intermediates in lysozyme folding with time-resolved small-angle X-ray scattering

Segel, Daniel J.; Bachmann, Annett; Hofrichter, James; Hodgson, Keith O.; Doniach, Sebastian; Kiefhaber, Thomas

doi:10.1006/jmbi.1999.2703

Cited by 159 publications

(142 citation statements)

References 28 publications

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“…14,38,41 The Kratky plot exhibits a plateau at the high Q region for particles with extended conformation, since the Debye function for scattering from a Gaussian coil has a limiting behavior of Q −2 at high Q. However, for globular/compact particles I(Q).Q 2 values will decrease at high Q since I(Q) should vary as Q −4 for them.…”

Section: Resultsmentioning

confidence: 99%

Surfactant Copolymers Prevent Aggregation of Heat Denatured Lysozyme

et al. 2006

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We investigated the ability of certain triblock copolymer surfactant poloxamers of the form polyethylene oxide-polypropylene oxide-polyethylene oxide (PEO-PPO-PEO), to prevent formation of stable aggregates of heat denatured hen egg lysozyme. Differential scanning calorimetry (DSC) and synchrotron small angle x-ray scattering (SAXS) experiments were performed to study the thermodynamics and solution structures of lysozyme at temperatures between 20 and 90°C in the presence and absence of poloxamers with various molecular weights (8.4-14.3 kDa), but similar hydrophile/hydrophobe (PEO:PPO) ratio of 80%. Poloxmer 188 was found to be very effective in preventing aggregation of heat denatured lysozyme and those functioned as a synthetic surfactant, thus enabling them to refold when the conditions become optimal. For comparison, we measured the ability of 8 kDa polyethylene glycol (PEG) to prevent lysozyme aggregation under same conditions. The results of these studies suggest that poloxamers are more efficient than PEG in preventing aggregation of heat denaturated lysozyme, To achieve equivalence, more than an order of magnitude higher concentration of PEG concentration was needed. Apparently, the presence of a hydrophobic segment in the poloxamers increases their ability to target the hydrophobic region of the unfolded proteins and protect them from self association. Given their biocompatibility and the low concentrations at which they effectively facilitate refolding of denatured proteins, they may be useful in the treatment of burns and other conditions resulting in the denaturation of proteins.

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Section: Resultsmentioning

confidence: 99%

Surfactant Copolymers Prevent Aggregation of Heat Denatured Lysozyme

et al. 2006

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“…The rather harsh and nonphysiological conditions required for populating unfolded states in equilibrium pose a major problem in the characterization of unfolded proteins under physiological conditions. For many proteins, rapid chain compaction in combination with major changes in spectroscopic properties was observed in refolding experiments starting from the denaturant-unfolded state (1)(2)(3)(4)(5). It has been discussed whether these rapidly formed compact structures represent the unfolded state under physiological conditions or whether the fast processes are due to the formation of specific folding intermediates separated from the unfolded state by energy barriers (3,5,6).…”

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confidence: 99%

End-to-end distance distributions and intrachain diffusion constants in unfolded polypeptide chains indicate intramolecular hydrogen bond formation

Möglich

Joder

Kiefhaber

2006

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

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234

335

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Characterization of the unfolded state is essential for the understanding of the protein folding reaction. We performed time-resolved FRET measurements to gain information on the dimensions and the internal dynamics of unfolded polypeptide chains. Using an approach based on global analysis of data obtained from two different donor–acceptor pairs allowed for the determination of distance distribution functions and diffusion constants between the chromophores. Results on a polypeptide chain consisting of 16 Gly-Ser repeats between the FRET chromophores reveal an increase in the average end-to-end distance from 18.9 to 39.2 Å between 0 and 8 M GdmCl. The increase in chain dimensions is accompanied by an increase in the end-to-end diffusion constant from (3.6 ± 1.0) × 10 −7 cm 2 s −1 in water to (14.8 ± 2.5) × 10 −7 cm 2 s −1 in 8 M GdmCl. This finding suggests that intrachain interactions in water exist even in very flexible chains lacking hydrophobic groups, which indicates intramolecular hydrogen bond formation. The interactions are broken upon denaturant binding, which leads to increased chain flexibility and longer average end-to-end distances. This finding implies that rapid collapse of polypeptide chains during refolding of denaturant-unfolded proteins is an intrinsic property of polypeptide chains and can, at least in part, be ascribed to nonspecific intramolecular hydrogen bonding. Despite decreased intrachain diffusion constants, the conformational search is accelerated in the collapsed state because of shorter diffusion distances. The measured distance distribution functions and diffusion constants in combination with Szabo–Schulten–Schulten theory were able to reproduce experimentally determined rate constants for end-to-end loop formation.

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“…The Kratky plot is considered a standard to determine the unfolding process. 28,49,50 The characteristic peak for a native globular protein decreases when partially folded states are present and tends to a typical flat shape for a fully denaturated structure (random coil). The spectra analysis permits to clearly characterize the stabilizing action of the ibuprofen.…”

Section: Saxs Datamentioning

confidence: 99%

Human serum albumin binding ibuprofen: A 3D description of the unfolding pathway in urea

Galantini¹,

Leggio²,

Konarev³

et al. 2010

Biophysical Chemistry

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT2 AbstractSmall angle X-ray scattering (SAXS) technique, supported by light scattering measurements and spectroscopic data (circular dichroism and fluorescence) allowed us to restore the 3D structure at low resolution of defatted human serum albumin (HSA) in interaction with ibuprofen. The data were carried out on a set of HSA solutions with urea concentrations between 0.00 and 9.00 M. The SingularValue Decomposition method, applied to the complete SAXS data set allowed us to distinguish three different states in solution. In particular a native conformation N (at 0.00 M urea), an intermediate I1(at 6.05 M urea) and an unfolded structure U (at 9.00 M urea) were recognized. The low resolution structures of these states were obtained by exploiting both ab initio and rigid body fitting methods. In particular, for the protein without denaturant, a conformation recently described (Leggio & al., PCCP, 2008, 10, 6741-6750), very similar to the crystallographic heart shape, with only a slight reciprocal movement of the three domains, was confirmed. The I1 structure was instead characterized by only a closed domain (domain III) and finally, the recovered structure of the U state revealed the characteristic feature of a completely open state. A direct comparison with the free HSA pointed out that the presence of the ibuprofen provokes a shift of the equilibrium towards higher urea concentrations without changing the unfolding sequence. The work represents a type of analysis which could be exploited in future investigations on proteins in solution, in the binding of drugs or endogenous compounds and in the pharmacokinetic properties as well as in the study of allosteric effects, cooperation or anticooperation mechanisms.

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Characterization of transient intermediates in lysozyme folding with time-resolved small-angle X-ray scattering

Cited by 159 publications

References 28 publications

Surfactant Copolymers Prevent Aggregation of Heat Denatured Lysozyme

Surfactant Copolymers Prevent Aggregation of Heat Denatured Lysozyme

End-to-end distance distributions and intrachain diffusion constants in unfolded polypeptide chains indicate intramolecular hydrogen bond formation

Human serum albumin binding ibuprofen: A 3D description of the unfolding pathway in urea

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