Rapid collapse and slow structural reorganisation during the refolding of bovine α-lactalbumin

Forge, Vincent; Wijesinha, Ramani; Balbach, Jochen; Brew, Keith; Robinson, Carol V.; Redfield, Christina; Dobson, Christopher M.

doi:10.1006/jmbi.1999.2687

Cited by 151 publications

(188 citation statements)

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“…consistent with experimental studies of the kinetics and the thermodynamics of HLA folding (27,28). The rmsd between the native state and the molten globule state is only Ϸ5 Å, and such a barrier is likely to originate at least in part from the fact that the entropic gain of the molten globule state due to the greater freedom of the side chains is lost in the transition to the native state (13), which is stabilized by strong interactions in a tightly packed low-energy structure.…”

Section: Ensembles Of Structures (Seesupporting

confidence: 83%

Structures and relative free energies of partially folded states of proteins

Vendruscolo

Paci

Karplus

et al. 2003

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

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The ability of proteins to fold to well defined compact structures is one of the most remarkable examples of the effect of natural selection on biological molecules. To understand their properties, including the stability, the mechanism of folding, and the possibilities of misfolding and association, it is necessary to know the protein free energy landscape. We use NMR data as restraints in a Monte Carlo sampling procedure to determine the ensemble of structures populated by human ␣-lactalbumin in the presence of increasing concentrations of urea. The ensembles of structures that represent the partially folded states of the protein show that two structural cores, corresponding to portions of the ␣ and ␤ domains of the native protein, are preserved even when the native-like interactions that define their existence are substantially weakened. Analysis of the network of residual contacts reveals the presence of a complex interface region between the two structural cores and indicates that the development of specific interactions within this interface is the key step in achieving the native structure. The relative probabilities of the conformations determined from the NMR data are used to construct a coarse-grained free energy landscape for ␣-lactalbumin in the absence of urea. The form of the landscape, together with the existence of distinct cores, supports the concept that robustness and modularity are the properties that make possible the folding of complex proteins.T he folding of small single-domain proteins is now relatively well understood as a result of recent progress in describing the structural properties of the transition state ensembles of a range of representative proteins that fold with two-state kinetics (1-3). A particularly important conclusion of these studies is that the protein fold is encoded by the sequence through key nucleation sites (1,4,5). By contrast, the folding mechanism of larger proteins, in which the population of one or more partially folded states can be significant, still remains to be resolved. We show here that it is possible to use experimental data to determine the structures and relative free energies of partially folded states for such more complex systems, which include the vast majority of proteins. These results provide the information necessary for the construction of the accessible portions of the free energy surface (3, 6) or ''landscape'' (7) from which the thermodynamic description of the folding process can be determined. The present approach incorporates experimental restraints into the energy function used in Monte Carlo simulations to bias the conformations that are sampled to the portions of space compatible with the measurements (5). In this way the conformation space accessible to rather complex proteins can be fully characterized. The work described here thus extends the theoretical analyses of energy landscapes for smaller proteins (see, for example, ref. 8).To illustrate the approach we apply it to the extensively studied protein human ␣-lactalbumin (HLA)...

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Section: Ensembles Of Structures (Seesupporting

confidence: 83%

Structures and relative free energies of partially folded states of proteins

Vendruscolo

Paci

Karplus

et al. 2003

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

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“…All NMR signals observed during the folding process can be assigned either to the MG or the native state. No additional peaks indicative of a significantly populated folding intermediate were detected, in agreement with previous findings (23,26,27). The refolding kinetics could be quantified for 92 of 121 backbone amide sites in the protein from intensity measurements of well resolved cross-peaks in the 1 H-15 N correlation spectra (Fig.…”

Section: Conformational Transition Kinetics Of ␣-Lactalbumin From An supporting

confidence: 76%

“…NMR assignments were taken from ref. 27. The reproducibility of the real-time folding data has been evaluated by repeating the experiment twice.…”

Section: Methodsmentioning

confidence: 99%

“…2b). In addition, and in contrast to the studies by Balbach et al (23,26,27), the intensity decay of five cross-peaks characteristic for the MG state could be quantified. Under the experimental conditions chosen (15°C, pH 8, Ca 2ϩ -free), the NMR intensity decay and buildup curves can be fitted to monoexponential functions.…”

Section: Conformational Transition Kinetics Of ␣-Lactalbumin From An mentioning

confidence: 96%

“…One-dimensional 1 H NMR spectra recorded during the refolding reaction of apo-␣-lactalbumin provided evidence that the MG state, accumulated during the early stages of the folding reaction, shows similar spectral characteristics (poorly dispersed and broad resonances) and therefore similar dynamic properties of the conformational ensemble to those observed for the MG state stabilized at acidic pH (25). Additional diffusion-edited and NOE transfer NMR measurements allowed monitoring the compactness of the protein and the establishing of native tertiary contacts, respectively (23,27). The folding kinetics of apo-␣-lactalbumin have also been studied previously at a residue level by line shape analysis of individual cross-peaks detected in a single 2D NMR spectrum recorded during the refolding event (26).…”

Section: Conformational Transition Kinetics Of ␣-Lactalbumin From An mentioning

confidence: 99%

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Protein folding and unfolding studied at atomic resolution by fast two-dimensional NMR spectroscopy

Schanda

Forge

Brutscher

2007

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

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152

151

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Atom-resolved real-time studies of kinetic processes in proteins have been hampered in the past by the lack of experimental techniques that yield sufficient temporal and atomic resolution. Here we present band-selective optimized flip-angle short transient (SOFAST) real-time 2D NMR spectroscopy, a method that allows simultaneous observation of reaction kinetics for a large number of nuclear sites along the polypeptide chain of a protein with an unprecedented time resolution of a few seconds. SOFAST real-time 2D NMR spectroscopy combines fast NMR data acquisition techniques with rapid sample mixing inside the NMR magnet to initiate the kinetic event. We demonstrate the use of SOFAST real-time 2D NMR to monitor the conformational transition of ␣-lactalbumin from a molten globular to the native state for a large number of amide sites along the polypeptide chain. The kinetic behavior observed for the disappearance of the molten globule and the appearance of the native state is monoexponential and uniform along the polypeptide chain. This observation confirms previous findings that a single transition state ensemble controls folding of ␣-lactalbumin from the molten globule to the native state. In a second application, the spontaneous unfolding of native ubiquitin under nondenaturing conditions is characterized by amide hydrogen exchange rate constants measured at high pH by using SOFAST real-time 2D NMR. Our data reveal that ubiquitin unfolds in a gradual manner with distinct unfolding regimes.hydrogen/deuterium exchange ͉ molecular kinetics ͉ ␣-lactalbumin ͉ ubiquitin A detailed description of the structural changes occurring during the folding and unfolding of proteins still remains a challenging objective in biophysics. The understanding of the fundamental mechanisms of the folding process will also shed light on the factors leading to protein misfolding responsible for neurodegenerative diseases such as Alzheimer's and Parkinson's diseases (1). The ideal method to study protein folding/unfolding provides structural information at atomic resolution and is sensitive to changes occurring on time scales ranging from microseconds to minutes, a task that no single technique is able to fulfill. NMR spectroscopy is especially well adapted to obtain detailed atomistic information about the mechanisms, kinetics, and energetics of the folding/unfolding process for virtually every nuclear site in the protein. NMR methods are sensitive to molecular dynamics occurring over a wide range of time scales (Fig. 1a). Although steady-state NMR methods are well suited to characterize equilibrium molecular dynamics occurring on a subsecond time scale (2, 3), unidirectional processes are best studied by real-time NMR (4, 5), where a series of NMR spectra is recorded during the reaction (Fig. 1b). Two difficulties have so far hampered the widespread application of real-time NMR to the study of protein folding. The first problem is the low intrinsic sensitivity of NMR at ambient temperature, a consequence of the small transition energies ...

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Hydrogen exchange study of canine milk lysozyme: Stabilization mechanism of the molten globule

Kobashigawa¹,

Demura²,

Koshiba³

et al. 2000

Proteins

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The native state 1H, 15N resonance assignment of 123 of the 128 nonproline residues of canine milk lysozyme has enabled measurements of the amide hydrogen exchange of over 70 amide hydrogens in the molten globule state. To elucidate the mechanism of protein folding, the molten globule state has been studied as a model of the folding intermediate state. Lysozyme and α‐lactalbumin are homologous to each other, but their equilibrium unfolding mechanisms differ. Generally, the folding mechanism of lysozyme obeys a two‐state model, whereas that of α‐lactalbumin follows a three‐state model. Exceptions to this rule are equine and canine milk lysozymes, which exhibit a partially unfolded state during the equilibrium unfolding; this state resembles the molten globule state of α‐lactalbumin but with extreme stability. Study of the molten globules of α‐lactalbumin and equine milk lysozyme showed that the stabilities of their α‐helices are similar, despite the differences in the thermodynamic stability of their molten globule states. On the other hand, our hydrogen exchange study of the molten globule of canine milk lysozyme showed that the α‐helices are more stabilized than in α‐lactalbumin or equine milk lysozyme and that this enhanced stability is caused by the strengthened cooperative interaction between secondary structure elements. Thus, our results underscore the importance of the cooperative interaction in the stability of the molten globule state. Proteins 2000;40:579–589. © 2000 Wiley‐Liss, Inc.

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Rapid collapse and slow structural reorganisation during the refolding of bovine α-lactalbumin

Cited by 151 publications

References 56 publications

Structures and relative free energies of partially folded states of proteins

Structures and relative free energies of partially folded states of proteins

Protein folding and unfolding studied at atomic resolution by fast two-dimensional NMR spectroscopy

Hydrogen exchange study of canine milk lysozyme: Stabilization mechanism of the molten globule

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