Multiple intermediates and transition states during protein unfolding

Zaidi, Faisal N.; Nath, Utpal; Udgaonkar, Jayant B.

doi:10.1038/nsb1297-1016

Cited by 110 publications

(126 citation statements)

References 45 publications

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“…As mentioned above, curved unfolding limbs have also been observed in connection with unfolding intermediates (38). In this case, mutants with reduced m u are those that increase the partial disrupture of N-i.e., where ‡ and the refolding ground state move closer together.…”

Section: Discussionmentioning

confidence: 98%

“…Further, the curvature of U1A's unfolding limb is seen also with the noncharged denaturant urea (data not shown). A third explanation is unfolding intermediates (37)-i.e., the curvatures arise from partial disrupture of N prior to global unfolding (38). In the case of U1A, however, we disfavor this scenario also, because the structure in question shows high H͞D protection factors (see below).…”

Section: Curvedmentioning

confidence: 99%

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From snapshot to movie: φ analysis of protein folding transition states taken one step further

Ternström

Mayor

Akke

et al. 1999

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

147

172

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Kinetic anomalies in protein folding can result from changes of the kinetic ground states (D, I, and N), changes of the protein folding transition state, or both. The 102-residue protein U1A has a symmetrically curved chevron plot which seems to result mainly from changes of the transition state. At low concentrations of denaturant the transition state occurs early in the folding reaction, whereas at high denaturant concentration it moves close to the native structure. In this study we use this movement to follow continuously the formation and growth of U1A's folding nucleus by analysis. Although U1A's transition state structure is generally delocalized and displays a typical nucleation-condensation pattern, we can still resolve a sequence of folding events. However, these events are sufficiently coupled to start almost simultaneously throughout the transition state structure.

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Section: Discussionmentioning

confidence: 98%

Section: Curvedmentioning

confidence: 99%

From snapshot to movie: φ analysis of protein folding transition states taken one step further

Ternström

Mayor

Akke

et al. 1999

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

147

172

View full text Add to dashboard Cite

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“…Because of the roughness of the energy landscape, unfolded molecules follow both direct and indirect pathways, and the folding flux undergoes kinetic partitioning (45). Most studies have focused on relatively small proteins such as lysozyme, barstar, and titin I27 (3,32,33). It is only in the case of T4 lysozyme that parallel pathways have been revealed using mechanical unfolding.…”

Section: Resultsmentioning

confidence: 99%

Ligand-modulated Parallel Mechanical Unfolding Pathways of Maltose-binding Proteins

Aggarwal

Kulothungan

Balamurali

et al. 2011

Journal of Biological Chemistry

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Protein folding and unfolding are complex phenomena, and it is accepted that multidomain proteins generally follow multiple pathways. Maltose-binding protein (MBP) is a large (a two-domain, 370-amino acid residue) bacterial periplasmic protein involved in maltose uptake. Despite the large size, it has been shown to exhibit an apparent two-state equilibrium unfolding in bulk experiments. Single-molecule studies can uncover rare events that are masked by averaging in bulk studies. Here, we use single-molecule force spectroscopy to study the mechanical unfolding pathways of MBP and its precursor protein (preMBP) in the presence and absence of ligands. Our results show that MBP exhibits kinetic partitioning on mechanical stretching and unfolds via two parallel pathways: one of them involves a mechanically stable intermediate (path I) whereas the other is devoid of it (path II). The apoMBP unfolds via path I in 62% of the mechanical unfolding events, and the remaining 38% follow path II. In the case of maltose-bound MBP, the protein unfolds via the intermediate in 79% of the cases, the remaining 21% via path II. Similarly, on binding to maltotriose, a ligand whose binding strength with the polyprotein is similar to that of maltose, the occurrence of the intermediate is comparable (82% via path I) with that of maltose. The precursor protein preMBP also shows a similar behavior upon mechanical unfolding. The percentages of molecules unfolding via path I are 53% in the apo form and 68% and 72% upon binding to maltose and maltotriose, respectively, for preMBP. These observations demonstrate that ligand binding can modulate the mechanical unfolding pathways of proteins by a kinetic partitioning mechanism. This could be a general mechanism in the unfolding of other large two-domain ligand-binding proteins of the bacterial periplasmic space.Small proteins (with fewer than 100 amino acids) are still under the scrutiny of having smooth versus the rough energy landscape; however, for proteins larger than 100 amino acid residues it is generally believed that they follow multiple pathways involving one or many intermediates during protein unfolding (1-4). Contrary to this notion, there exist few exceptions among large proteins, for example, Borrelia burgdorferi VlsE (5), Alteromonas haloplanctis ␣-amylase (6), and maltosebinding protein (MBP) 2 (7), which are shown to exhibit an apparent two-state equilibrium unfolding pathway in bulk solution. Very recent single-molecule pulling studies showed that out-of-equilibrium mechanical unfolding of MBP is very complex and consists of on-pathway sequential intermediates (8, 9). Our earlier studies on MBP have shown the existence of intermediates during the folding pathway of MBP (10, 11). Traditional bulk biophysical techniques like fluorescence, time-resolved unfolding and folding probe ensembles of molecules and hence suffer many drawbacks compared with methods that probe one molecule at a time. Therefore, single-molecule studies on protein folding/unfolding have proved vital in givin...

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“…However, the wild-type folding pathway is readily perturbed by single mutations, revealing an alternative folding mechanism in which the transition-state structure is polarized at the other end of the molecule. The concept of energy landscapes suggests that a protein can have multiple folding pathways, but to date, there has been very little experimental evidence to support this view (10)(11)(12)(13)(14). By contrast, the accessibility of multiple routes to the native state may be a general feature of repeat proteins that arises from the symmetry inherent in their structures.…”

mentioning

confidence: 98%

Rational redesign of the folding pathway of a modular protein

Lowe

Itzhaki

2007

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

100

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The modular structures of repeat proteins afford them distinct properties compared with globular proteins, enabling them to function in a large and diverse range of cellular processes. Here, we show that they can also have different folding mechanisms. Myotrophin comprises four ankyrin repeats stacked linearly to form an elongated structure. Using site-directed mutagenesis, we find that folding of wild-type myotrophin is initiated at the C-terminal repeats. However, close examination of the mutant chevron plots reveals that simple models are insufficient to describe all of the data, and double mutant analysis subsequently confirms that there are parallel folding pathways. Destabilizing mutations in the C-terminal repeats reduce flux through the wild-type pathway, making a new route accessible in which folding is initiated at the N-terminal repeats. Thus, the folding mechanism of the repeat protein is poised on a fulcrum: When one end of the molecule is perturbed, the balance shifts between the different nucleation sites. The vast majority of studies on small globular proteins indicate a single, well defined route between the denatured and native states. By contrast, the potential to initiate folding at more than one site may be a general feature of repeat proteins arising from the symmetry inherent in their structures. We show that this simple architecture makes it straightforward to direct the folding pathway of a repeat protein by design.ankyrin repeat ͉ myotrophin ͉ ⌽ value ͉ protein engineering ͉ parallel pathways R epeat proteins, such as ankyrin repeats, leucine-rich repeats, and tetratricopeptide repeats, consist of tandem arrays of a structural motif of 20-40 aa that stack in a roughly linear fashion, creating elongated and superhelical architectures (1-3). Repeat protein structures are composed entirely of short-range interactions, either within a repeat or in adjacent repeats, in striking contrast to the more complex topologies of globular proteins that are stabilized by contacts between residues distant in sequence (4). They are ubiquitous, representing Ͼ5% of eukaryotic proteins in the Swiss-Prot database, and highly versatile, mediating molecular recognition in many different biological processes, but little is known about their folding mechanisms. The simple, modular nature of these structures has made them uniquely successful as scaffolds for engineering novel binding specificities (5-7). Does the repeat architecture likewise give rise to distinct folding properties, and are their folding pathways also amenable to design?In this study, we have addressed this question using the small, well behaved ankyrin (ANK) repeat protein myotrophin. The folding and unfolding properties of ANK repeats are of particular interest because these motifs are thought to play a role in mechanical signal transduction by virtue of their putative elastic properties (8, 9). The ANK repeat is a 33-residue motif consisting of a pair of antiparallel ␣-helices linked to the neighboring repeat via an antiparallel ␤-loop. The 11...

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Multiple intermediates and transition states during protein unfolding

Cited by 110 publications

References 45 publications

From snapshot to movie: φ analysis of protein folding transition states taken one step further

From snapshot to movie: φ analysis of protein folding transition states taken one step further

Ligand-modulated Parallel Mechanical Unfolding Pathways of Maltose-binding Proteins

Rational redesign of the folding pathway of a modular protein

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