Conformational strain in the hydrophobic core and its implications for protein folding and design

Ventura, Salvador; Vega, M. Cristina; Lacroix, Emmanuel; Angrand, Isabelle; Spagnolo, Laura; Serrano, Luís

doi:10.1038/nsb799

Cited by 101 publications

(91 citation statements)

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“…For a given fold, a higher stability usually correlates with faster folding rates and slower unfolding speed (36). This is the case of TCI domains, because the folding of Ct TCI is faster and more efficient than that of the Nt domain, especially in the presence of redox agents, and also the reductive unfolding of Ct TCI is much slower than the one of its Nt counterpart.…”

Section: Discussionmentioning

confidence: 99%

Characterizing the Tick Carboxypeptidase Inhibitor

Arolas

Bronsoms

Ventura

et al. 2006

Journal of Biological Chemistry

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Tick carboxypeptidase inhibitor (TCI) is a small, disulfiderich protein that selectively inhibits metallocarboxypeptidases and strongly accelerates the fibrinolysis of blood clots. TCI consists of two domains that are structurally very similar, each containing three disulfide bonds arranged in an almost identical fashion. The oxidative folding and reductive unfolding pathways of TCI and its separated domains have been characterized by kinetic and structural analysis of the acid-trapped folding intermediates. TCI folding proceeds through a sequential formation of 1-, 2-, 3-, 4-, 5-, and 6-disulfide species to reach the native form. Folding intermediates of TCI comprise two predominant 3-disulfide species (named IIIa and IIIb) and a major 6-disulfide scrambled isomer (Xa) that consecutively accumulate along the reaction and are strongly prevented by the presence of protein disulfide isomerase. This study demonstrates that IIIa and IIIb are 3-disulfide species containing the native disulfide pairings of the N-and C-terminal domains of TCI, respectively, and explains why the two domains of TCI fold sequentially and independently. Also, we show that the reductive unfolding of TCI undergoes two main independent unfolding events through the formation of IIIa and IIIb intermediates. Together, the comparison of the folding, stability, and inhibitory activity of TCI with those of the isolated domains reveals the reasons behind the two-domain nature of this protein: both domains contribute to the specificity and high affinity of its double-headed binding to carboxypeptidases. The results obtained herein provide valuable information for the design of more potent and selective TCI molecules.The mechanism by which an unfolded protein achieves its native state is one of the most complex problems in structural biology. Understanding the sequence of folding events in proteins may not only help to predict protein structures from amino acid sequences but also provide invaluable information to a variety of related fields, such as protein design or protein misfolding associated with pathological diseases (1, 2). A number of studies concerning folding have been focused on small, disulfide-rich proteins, given that the chemistry of disulfide bond formation allows the trapping and subsequent characterization of the folding intermediates that accumulate, something difficult to attain with disulfide-free proteins (3, 4). In oxidative folding, reduced and denatured proteins are allowed to recover both its native disulfide bonds and native structures in the absence or presence of redox agents. Bovine pancreatic trypsin inhibitor, ribonuclease A, lysozyme, ␣-lactalbumin, and hirudin have been extensively investigated using this methodology (5-9). The heterogeneity and structures of the intermediates that occur during the process define their respective folding landscapes (10).Although most of the analyzed proteins comprise a small, single-domain fold containing three or four disulfide bonds, a great diversity of folding mechanisms is observ...

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Section: Discussionmentioning

confidence: 99%

Characterizing the Tick Carboxypeptidase Inhibitor

Arolas

Bronsoms

Ventura

et al. 2006

Journal of Biological Chemistry

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“…Typically, unstable mutant proteins display decreased folding rates and͞or increased unfolding rates. Although destabilized mutants that fold considerably faster than WT have been previously isolated (7,29), the Gly-48 mutants present by far the most extreme examples of such behavior, especially considering that they are only single-site substitutions. The use of NMR spin relaxation dispersion experiments to provide accurate measurements of k f H2O and k u H2O for the Gly-48 mutants in water without denaturant allowed us to eliminate the possibility that the k f H2O and k u H2O values extrapolated from stopped-flow measurements in urea were aberrantly high due to undetected chevron plot curvature at low denaturant concentrations.…”

Section: Discussionmentioning

confidence: 99%

Dramatic acceleration of protein folding by stabilization of a nonnative backbone conformation

Nardo

Korzhnev

Stogios

et al. 2004

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

100

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Through a mutagenic investigation of Gly-48, a highly conserved position in the Src homology 3 domain, we have discovered a series of amino acid substitutions that are highly destabilizing, yet dramatically accelerate protein folding, some up to 10-fold compared with the wild-type rate. The unique folding properties of these mutants allowed for accurate measurement of their folding and unfolding rates in water with no denaturant by using an NMR spin relaxation dispersion technique. A strong correlation was found between ␤-sheet propensity and the folding rates of the Gly-48 mutants, even though Gly-48 lies in an unusual non-␤-strand backbone conformation in the native state. This finding indicates that the accelerated folding rates of the Gly-48 mutants are the result of stabilization of a nonnative ␤-strand conformation in the transition-state structure at this position, thus providing the first, to our knowledge, experimentally elucidated example of a mechanism by which folding can occur fastest through a nonnative conformation. We also demonstrate that residues that are most stabilizing in the transition-state structure are most destabilizing in the native state, and also cause the greatest reductions in in vitro functional activity. These data indicate that the unusual native conformation of the Gly-48 position is important for function, and that evolutionary selection for function can result in a domain that folds at a rate far below the maximum possible. T he energetics involved as proteins progress from a broad ensemble of unfolded conformations to a single native structure are still not thoroughly understood. A widely used approach to study folding is the protein engineering method, which measures the thermodynamic and kinetic effects of single amino acid substitutions (usually with Ala) at many different sites in a protein as a means to define the most structured regions of the folding transition state (1, 2). Mapping the transition state identifies regions that fold more quickly than others, and such knowledge has led to important theoretical advances in our understanding of the folding process (3, 4). Although this method provides a general picture of folding pathways, it does not define the mechanisms by which individual residues can facilitate or retard the folding process. Our approach to study these mechanisms is to investigate the effects of multiple amino acid substitutions at single positions (5-7). In the present study, this approach is used to investigate the role in folding and function of position 48 of the Src homology 3 (SH3) domain, which is one of most conserved positions in this domain (8).The SH3 domain is well suited for protein-folding studies due to its small size and amenability to biophysical analysis (9-11). Composed of two three-stranded ␤-sheets packed orthogonally against one another (Fig. 1A), SH3 domains function as proteinprotein interaction modules in a wide variety of eukaryotic proteins. The structure of the SH3 domain transition state has been extensively characterized...

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“…Whereas the relative importance, selectivity, and context dependence of these forces remain imperfectly understood, the burial of hydrophobic surfaces is thought to play a major role in governing protein architecture and folding, at least in relatively small protein folds (3,(33)(34)(35)(41)(42)(43)(44). Ala-14 provides a striking example of how a native structure can still form once this localized driving force has been severely attenuated.…”

Section: For the Intensity (I) Of I Observations Of Reflection Hmentioning

confidence: 99%

An Alanine-Zipper Structure Determined by Long Range Intermolecular Interactions

Liu¹,

2002

Journal of Biological Chemistry

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A major challenge in protein folding is to identify and quantify specific structural determinants that allow native proteins to acquire their unique folded structures. Here we report the engineering of a 52-residue protein (Ala-14) that contains exclusively alanine residues at the hydrophobic a and d positions of a natural heptad-repeat sequence. Ala-14 is unfolded under normal solution conditions yet forms a parallel three-stranded ␣-helical coiled coil in crystals. Ala-14 trimers in the solid state associate with each other through the pairing of polar side chains and formation of an extended network of water-mediated hydrogen bonds. In contrast to the classical view that local intramolecular tertiary interactions dictate the three-dimensional structure of small single-domain proteins, Ala-14 shows that long range intermolecular interactions can be essential in determining the metastable alanine-zipper structure. A similar interplay between short range local and longer range global forces may underlie the conformational properties of the growing class of natively unstructured proteins in biological processes.The classical view of protein folding posits that local secondary and tertiary interactions promote the association of cooperative structural elements in a process of sequential stabilization to form a limited set of transient intermediates and finally the native state (1-12). The current theory emphasizes the role of the free energy landscape of a polypeptide chain in driving rapid and efficient folding into a unique well defined structure (13)(14)(15)(16)(17). In any description of protein folding, a major challenge is to identify specific structural determinants that guide the folding process. Although considerable progress has been achieved toward understanding how compact single-domain proteins fold (1-12), the factors that specify and stabilize larger and more extended protein complexes remain largely undefined despite their critical importance in the complex environment inside a living cell. We report here a new alaninezipper structure that undergoes a native folding transition only during crystallization. A network of long range solvent-mediated interactions is observed in the 1.3-Å x-ray crystal structure of this alanine-zipper trimer that appears to directly control its folding into a helical trimer structure. EXPERIMENTAL PROCEDURESProtein Production-Plasmid pAla14 encoding Ala-14 was derived from pLpp56 (Ref. 18). The sequence Cys-Gly-Gly was added to the N terminus of Ala-14 to produce Ala-14 N . Similarly, Gly-Gly-Cys was added to the C terminus of Ala-14 to produce Ala-14 C . The sequences Cys-Gly-Gly and Gly-Gly-Cys were added at the N and C termini, respectively, of Ala-14 to generate Ala-14 NC . The proteins were expressed in Escherichia coli strain BL21(DE3)/pLysS (Novagen). Cells were grown at 20°C in LB media to an optical density of 0.8 at 600 nm and induced with isopropylthio-␤-D-galactoside for 10 h. Cells were lysed by glacial acetic acid on ice. The bacterial lysate was centrifuge...

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Conformational strain in the hydrophobic core and its implications for protein folding and design

Cited by 101 publications

References 50 publications

Characterizing the Tick Carboxypeptidase Inhibitor

Characterizing the Tick Carboxypeptidase Inhibitor

Dramatic acceleration of protein folding by stabilization of a nonnative backbone conformation

An Alanine-Zipper Structure Determined by Long Range Intermolecular Interactions

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