Folding and Misfolding in a Naturally Occurring Circularly Permuted PDZ Domain

One of the most extreme and fascinating examples of naturally occurring mutagenesis is represented by circular permutation. Circular permutations involve the linking of two chain ends and cleavage at another site. Here we report the first description of the folding mechanism of a naturally occurring circularly permuted protein, a PDZ domain from the green alga Scenedesmus obliquus. Data reveal that the folding of the permuted protein is characterized by the presence of a low energy off-pathway kinetic trap. This finding contrasts with what was previously observed for canonical PDZ domains that, although displaying a similar primary structure when structurally re-aligned, fold via an on-pathway productive intermediate. Although circular permutation of PDZ domains may be necessary for a correct orientation of their functional sites in multi-domain protein scaffolds, such structural rearrangement may compromise their folding pathway. This study provides a straightforward example of the divergent demands of folding and function

Unveiling the folding mechanism of the bromodomains

Bromodomains (BRDs) are small protein domains often present in large multidomain proteins involved in transcriptional regulation in eukaryotic cells. They currently represent valuable targets for the development of inhibitors of aberrant transcriptional processes in a variety of human diseases. Here we report urea-induced equilibrium unfolding experiments monitored by circular dichroism (CD) and fluorescence on two structurally similar BRDs: BRD2(2) and BRD4(1), showing that BRD4(1) is more stable than BRD2(2). Moreover, we report a description of their kinetic folding mechanism, as obtained by careful analysis of stopped-flow and temperature-jump data. The presence of a high energy intermediate for both proteins, suggested by the non-linear dependence of the folding rate on denaturant concentration in the millisec time regime, has been experimentally observed by temperature-jump experiments. Quantitative global analysis of all the rate constants obtained over a wide range of urea concentrations, allowed us to propose a common, three-state, folding mechanism for these two BRDs. Interestingly, the intermediate of BRD4(1) appears to be more stable and structurally native-like than that populated by BRD2(2). Our results underscore the role played by structural topology and sequence in determining and tuning the folding mechanism

Exploring the cytochrome c folding mechanism: cytochrome c552 from thermus thermophilus folds through an on-pathway intermediate.

Understanding the role of partially folded intermediate states in the folding mechanism of a protein is a crucial yet very difficult problem. We exploited a kinetic approach to demonstrate that a transient intermediate of a thermostable member of the widely studied cytochrome c family (cytochrome c552 from Thermus thermophilus) is indeed on-pathway. This is the first clear indication of an obligatory intermediate in the folding mechanism of a cytochrome c. The fluorescence properties of this intermediate demonstrate that the relative position of the heme and of the only tryptophan residue cannot correspond to their native orientation. Based on an analysis of the three-dimensional structure of cytochrome c552, we propose an interpretation of the data which explains the residual fluorescence of the intermediate and is consistent with the established role played by some conserved interhelical interactions in the folding of other members of this family. A limited set of topologically conserved contacts may guide the folding of evolutionary distant cytochromes c through the same partially structured state, which, however, can play different kinetic roles, acting either as an intermediate or a transition state

Role of myristoylation in modulating PCaP1 interaction with calmodulin

Plasma membrane-associated Cation-binding Protein 1 (PCaP1) belongs to the plant-unique DREPP protein family with largely unknown biological functions but ascertained roles in plant development and calcium (Ca2+) signaling. PCaP1 is anchored to the plasma membrane via N-myristoylation and a polybasic cluster, and its N-terminal region can bind Ca2+/calmodulin (CaM). However, the molecular determinants of PCaP1-Ca2+-CaM interaction and the functional impact of myristoylation in the complex formation and Ca2+ sensitivity of CaM remained to be elucidated. Herein, we investigated the direct interaction between Arabidopsis PCaP1 (AtPCaP1) and CaM1 (AtCaM1) using both myristoylated and non-myristoylated peptides corresponding to the N-terminal region of AtPCaP1. ITC analysis showed that AtCaM1 forms a high affinity 1:1 complex with AtPCaP1 peptides and the interaction is strictly Ca2+-dependent. Spectroscopic and kinetic Ca2+ binding studies showed that the myristoylated peptide dramatically increased the Ca2+-binding affinity of AtCaM1 and slowed the Ca2+ dissociation rates from both the C- and N-lobes, thus suggesting that the myristoylation modulates the mechanism of AtPCaP1 recognition by AtCaM1. Furthermore, NMR and CD spectroscopy revealed that the structure of both the N- and C-lobes of Ca2+-AtCaM1 changes markedly in the presence of the myristoylated AtPCaP1 peptide, which assumes a helical structure in the final complex. Overall, our results indicate that AtPCaP1 biological function is strictly related to the presence of multiple ligands, i.e., the myristoyl moiety, Ca2+ ions and AtCaM1 and only a full characterization of their equilibria will allow for a complete molecular understanding of the putative role of PCaP1 as signal protein

Protein Machineries Involved in the Attachment of Heme to Cytochrome c: Protein Structures and Molecular Mechanisms

Cytochromes c (Cyt c) are ubiquitous heme-containing proteins, mainly involved in electron transfer processes, whose structure and functions have been and still are intensely studied. Surprisingly, our understanding of the molecular mechanism whereby the heme group is covalently attached to the apoprotein (apoCyt) in the cell is still largely unknown. This posttranslational process, known as Cyt c biogenesis or Cyt c maturation, ensures the stereospecific formation of the thioether bonds between the heme vinyl groups and the cysteine thiols of the apoCyt heme binding motif. To accomplish this task, prokaryotic and eukaryotic cells have evolved distinctive protein machineries composed of different proteins. In this review, the structural and functional properties of the main maturation apparatuses found in gram-negative and gram-positive bacteria and in the mitochondria of eukaryotic cells will be presented, dissecting the Cyt c maturation process into three functional steps: (i) heme translocation and delivery, (ii) apoCyt thioreductive pathway, and (iii) apoCyt chaperoning and heme ligation. Moreover, current hypotheses and open questions about the molecular mechanisms of each of the three steps will be discussed, with special attention to System I, the maturation apparatus found in gram-negative bacteria

The folding mechanism of c-type cytochromes

The protein folding field has undoubtedly benefited from studies on the folding mechanism of c-type cytochromes. Many different structural aspects of these small heme-containing proteins led this protein family to be considered a well established model system for such studies. In this chapter, we shall briefly describe some of the major results that have been obtained on the folding mechanism of this protein family and highlight unanswered questions. 1. Introduction Cytochromes c (cyt c) are small monomeric proteins of 80-120 residues involved in different and crucial aspects of cellular life, from electron transport processes to apoptosis. These heme-proteins show a typical α-helical fold that is recognized as a structural superfamily in protein classification tools such as SCOP (Andreeva et al. 2008) or CATH (Orengo et al. 1997). The three major α-helices (generally referred to as N-terminal helix, 60’-helix and C-terminal helix, following numbering of aminoacidic residues of horse heart cyt c) wrap around the heme group that is covalently linked to the protein via two thioether bonds between its vinyl groups and two cysteine residues in the conserved CysXaaXaaCysHis motif (Figure 1). Attachment of the heme group to the apoprotein in vivo is a complex post-translational process that involves different enzymatic activities (Ferguson et al. 2008). It is therefore not surprising that attempts to obtain properly folded recombinant holo cytochromes c by heterologous expression in E. coli, was unsuccessful for a long time; efficient production of recombinant prokaryotic holo cyt c in E. coli, is now generally accomplished under control of the E. coli enzymatic apparatus for heme attachment (Thöny-Meyer et al. 1995). The heme iron is always axially coordinated to the His residue of the CysXaaXaaCysHis motif on the proximal side. While the His ligand is maintained even under denaturing conditions, the distal ligand, generally Met, is inherently labile and readily displaced by other side-chains, such as a deprotonated His or Lys, which can become trapped during folding (Babul and Stellwagen 1971). The presence of a covalently bound heme can be considered as the privilege and disgrace of these small single-domain proteins: on one hand, it is an ideal natural quencher of the intrinsic fluorescence of the protein, it enables the breakage or formation of some individual bonds to be monitored (Gianni et al. 2003), and it represents a perfect tool for the design of photo-induced experiments to follow ultra-fast conformational transitions (Jones et al. 1993), (Hagen et al. 1996), (Yeh and Rousseau 1998), (Chang 2003). On the other hand, the presence of this large prosthetic group has often hindered a generalization of the rules emerging from in vitro folding studies on this system. In this chapter we report some of the key findings on the folding pathway of c-type cytochromes. We first describe the equilibrium studies on this protein family and then extend our discussion to kinetic measurements. Finally, we present the hypothesis of a common folding mechanism for all c-type cytochromes, and discuss the possibility to tune their folding pathways along different parallel routes by mutagenesis. Each section briefly outlines experimental methodologies and analytical approaches classically employed in protein folding studies

Plasticity of the protein folding landscape: Switching between on- and off-pathway intermediates

Proteins may fold via parallel routes partitioned by the relative effect of solvent conditions on the relevant transition states. Thus, intermediates may or may not necessarily be obligatory species accumulated during the folding process, but rather kinetic traps due to the ruggedness of the folding landscape. Implicit in this view is the notion of plasticity of the folding pathway: proteins can be rerouted through the energy landscape by mutational, topological or solvent perturbations. Our work was specifically aimed to the experimental identification of a switch in the folding mechanism of a c-type cytochrome from the thermophilic bacterium Hydrogenobacwter themophilus (HT cyt c(552)) induced by acidic conditions. We present evidence that, by destabilizing the relevant transition state, the native state of FIT cyt c(552) can be reached along alternative folding routes, which may involve an off-pathway intermediate. 2007 Elsevier Inc. All rights reserved

Refolding kinetics of cyt c551 from Pseudomonas aeruginosa reveals a mechanistic difference between guanidine and urea.

The energetic parameters for the folding of small globular proteins can be very different if derived from guanidine hydrochloride (GdnHCl) or urea denaturation experiments. A study of the equilibrium and kinetics of the refolding of wild-type (wt) cytochrome c(551) (cyt c(551)) from Pseudomonas aeruginosa and of two site-directed mutants (E70Q and E70V) shows that the nonionic nature of urea reveals the role of a salt bridge between residues E70 and K10 on the transition state, which is otherwise completely masked in GdnHCl experiments. Mixed denaturant refolding experiments allow us to conclude that the masking effect of GdnHCl is complete at fairly low GdnHCl concentrations (congruent to0.1 M). The fact that potassium chloride is unable to reproduce this quenching effect, together with the results obtained on the mutants, suggests a specific binding of the Gdn(+) cation, which involves the E70-K10 ion pair in wt cyt c(551).We propose, therefore, a simple kinetic test to obtain a mechanistic interpretation of nonlinear dependences of DeltaG(w) on GdnHCl concentration on the basis of kinetic refolding experiments in the presence of both denaturants

A common folding mechanism in the cytochrome c family

Of the globular proteins, cytochrome c (cyt c) has been used extensively as a model system for folding studies. Here we analyse the folding pathway of different cyt c proteins from prokaryotes and eukaryotes, and attempt to single out general correlations between structural determinants and folding mechanisms. Recent studies provide evidence that the folding pathway of several cyt c proteins involves the formation of a partially structured intermediate. Using state-of-the-art kinetic analysis on published data, we show that such a folding intermediate is an obligatory on-pathway species that might represent either a defined local minimum in the reaction coordinate or an unstable high-energy state. Available data also indicate that some essential structural features of the folding intermediate and transition states are highly conserved across this protein family. Thus, cyt c proteins share a consensus folding mechanism in spite of large differences in physico-chemical properties and thermodynamic stability. This novel outlook on the folding of cyt c can shed light on much published data and might offer a general scheme by which to plan new experiments