Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an essential adaptor protein in the formation of a multiprotein complex that activates procaspase-1. ASC is also known as a modulator of NF-B activation pathways. ASC has a bipartite domain structure, consisting of an N-terminal pyrin domain (PYD) and a C-terminal caspaserecruitment domain. The PYD of ASC (ASC_PYD) is known to interact with various PYD-containing intracellular danger signal sensors and PYD-only proteins. Using purified proteins, we characterized the in vitro interaction of ASC_PYD with PYDonly protein 1 (POP1). POP1 specifically interacts with ASC_PYD with a dissociation constant of 4.08 ؎ 0.52 M but does not interact with Cryopyrin. NMR and mutagenesis experiments show that a negative electrostatic potential surface patch (EPSP) on ASC_PYD, consisting of the first (H1) and fourth (H4) helices, is essential in the interaction with POP1. A positive EPSP on POP1, consisting of the second (H2) and third (H3) helices, is a counterpart of this interaction. The interaction between ASC_PYD and POP1 is similar to the interaction between caspase recruitment domains of Apaf-1 and procaspase-9. In addition, we present evidence that conformational changes at the long loop of ASC_PYD between the H2 and H3 helices can affect its interaction with POP1. Based on our observations, we propose that the positive EPSP of ASC_PYD, including the H2 and H3 helices, may be the binding site for Cryopyrin, and the interaction with Cryopyrin may induce the dissociation of POP1 from ASC_PYD.Apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC; also known as Pycard and TMS1) is a bipartite adaptor protein that plays a role in proinflammatory cytokine production as well as NF-B activation (1, 2). ASC consists of an N-terminal pyrin domain (ASC_PYD) 3 and a C-terminal caspase-recruitment domain (ASC_CARD).Accumulating evidence indicates that ASC_CARD is involved in the recruitment and activation of procaspase-1 (3, 4). ASC_PYD interacts with PYD-containing NALP proteins to initiate a multiprotein platform known as an inflammasome, where procaspase-1 is activated by the induced proximity mechanism (3, 4). The NALP proteins are characterized by a common structure, consisting of N-terminal PYD, an intermediate NATCH domain, and C-terminal leucine-rich repeats. The NALP proteins are expected to function as intracellular danger signal sensors (5, 6). Cryopyrin (also known as NALP3, PYPAF1, and CIAS1) is a relatively well studied member of the NALP proteins. Recent studies using Cryopyrin Ϫ/Ϫ mice indicate that this protein is pivotal to the interleukin-1␤ production by macrophages in response to bacterial RNA (7), certain Gram-positive bacteria (8), uric acid, calcium pyrophosphate crystals (9), cellular toxins (10), and some Toll-like receptor agonists (8). In addition, Cryopyrin and other NALP proteins, in particular NALP6 (also known as PYPAF5) and NALP12 (also known as Monarch and PYPAF7), induce NF-B activat...

Nod1 is an essential cytoplasmic sensor for bacterial peptidoglycans in the innate immune system. The caspase-recruitment domain of Nod1 (Nod1_CARD) is indispensable for recruiting a downstream kinase, receptor-interacting protein 2 (RIP2), that activates nuclear factor-kappaB (NF-kappaB). The crystal structure of human Nod1_CARD at 1.9 A resolution reveals a novel homodimeric conformation. Our structural and biochemical analysis shows that the homodimerization of Nod1_CARD is achieved by swapping the H6 helices at the carboxy termini and stabilized by forming an interchain disulfide bond between the Cys39 residues of the two monomers in solution and in the crystal. In addition, we present experimental evidence for a pH-sensitive conformational change of Nod1_CARD. Our results suggest that the pH-sensitive monomer/dimer transition is a unique molecular property of Nod1_CARD.

Proline effectively inhibits protein aggregation during the refolding of bovine carbonic anhydrase. Other osmolytes used such as glycine and ethylene glycol fail to exhibit the 'aggregation-blockade' role shown by proline. Results of viscosity and ANS fluorescence (1-anilino-8-naphthalene sulphonic acid) experiments suggest that proline at high concentrations forms an ordered supramolecular assembly. Based on these results, it is proposed that proline behaves as a protein folding chaperone due to the formation of an ordered, amphipathic supramolecular assembly. To our knowledge, this is the first report wherein proline is proposed as a protein folding aid.

The guanidinium hydrochloride (GdnHCl)-induced unfolding of an all ␤-sheet protein, the human acidic fibroblast growth factor (hFGF-1), is studied using a variety of biophysical techniques including multidimensional NMR spectroscopy.

Effect(s) of organic solvents on an all beta-sheet protein are investigated to understand the influence of backbone conformation on protein aggregation. Results obtained in the present study reveal that protein aggregation is accompanied by the formation of non-native beta-sheet conformation. In contrast, induction of non-native helical segments in the protein is found to inhibit aggregation. The differential effects of the secondary structures on protein aggregation are proposed to stem from the disparity in the nature of the hydrogen bonds and packing of the side chains of hydrophobic residues in the beta-sheet and alpha-helix conformation. In our opinion, the results of the present study provide useful hints to develop methods to alleviate the problems of both in vitro and in vivo protein aggregation.

The structure and dynamics of equilibrium intermediate in the unfolding pathway of the human acidic fibroblast growth factor (hFGF-1) are investigated using a variety of biophysical techniques including multidimensional NMR spectroscopy. 15 N spin relaxation experiments show that many residues located in ␤-strands IX, X, and XI exhibit conformational motions in the micro-to millisecond time scale. Analysis of 15 N relaxation data in conjunction with the amide proton exchange kinetics suggests that residues in the ␤-strands II, VIII, and XII possibly constitute the stability core of the protein in the near-native intermediate state.Understanding the mechanism by which a disordered polypeptide chain folds to a unique three-dimensional structure is one of the major challenges in molecular structural biology (1-4). It is elucidated that folding of moderate size proteins (Ͼ10-kDa) proceeds along well defined intermediates similar to a chemical reaction (5-9). Formation of partially folded intermediate states during the protein-folding process is believed to aid in avoiding search of large conformation space on the energy landscape (10, 11). In addition to their role in protein folding, partially structured intermediates are proposed to be involved in a number of biological processes such as interaction with molecular chaperones, translocation across biological membranes, formation of amyloid, and dissociation of supramolecular complexes (12)(13)(14). Thus, investigation of the structural features of equilibrium folding/unfolding intermediates would not only enhance our understanding of the mechanism of protein folding but is also expected to provide strong clues on the interplay of molecular forces in many natural and disease-related processes.Dynamic information about a protein on different time and length scales is important to gain useful knowledge on the mechanism of the protein folding process (15-17). In addition, investigation of the conformational dynamics in the partially structured state(s) can provide valuable information about the interaction potential energy landscape, which is crucial for understanding protein stability and rationalizing protein design (18). In this context, NMR spectroscopy is an apt technique to probe protein folding landscape, because it provides a unique opportunity to study the conformational dynamics of unfolded, partially folded, and native state(s) at the level of individual amino acids (3, 19 -21). Amide proton exchange kinetics and 15 N-relaxation measurements (using NMR) have been successfully used to probe the structural and dynamic features of partially structured intermediate state(s) of proteins (17,(22)(23)(24)(25).Acidic human fibroblast growth factor (hFGF-1) 1 is a 16-kDa, all ␤-sheet protein, devoid of disulfide bonds. The secondary structural elements in the protein include 12 ␤-strands arranged antiparallel into a ␤-barrel architecture (Fig. 1 (26 -31). hFGF-1 is a potent mitogen and plays crucial roles in important cellular processes such as morphogenesis, develo...

Acidic fibroblast growth factors from human (hFGF-1) and newt (nFGF-1) (Notopthalamus viridescens) are 16-kDa, all beta-sheet proteins with nearly identical three-dimensional structures. Guanidine hydrochloride (GdnHCl)-induced unfolding of hFGF-1 and nFGF-1 monitored by fluorescence and far-UV circular dichroism (CD) shows that the FGF-1 isoforms differ significantly in their thermodynamic stabilities. GdnHCl-induced unfolding of nFGF-1 follows a two-state (Native state to Denatured state(s)) mechanism without detectable intermediate(s). By contrast, unfolding of hFGF-1 monitored by fluorescence, far-UV circular dichroism, size-exclusion chromatography, and NMR spectroscopy shows that the unfolding process is noncooperative and proceeds with the accumulation of stable intermediate(s) at 0.96 M GdnHCl. The intermediate (in hFGF-1) populated maximally at 0.96 M GdnHCl has molten globule-like properties and shows strong binding affinity to the hydrophobic dye, 1-Anilino-8-naphthalene sulfonate (ANS). Refolding kinetics of hFGF-1 and nFGF-1 monitored by stopped-flow fluorescence reveal that hFGF-1 and nFGF-1 adopts different folding mechanisms. The observed differences in the folding/unfolding mechanisms of nFGF-1 and hFGF-1 are proposed to be either due to differential stabilizing effects of the charged denaturant (Gdn(+) Cl(-)) on the intermediate state(s) and/or due to differences in the structural interactions stabilizing the native conformation(s) of the FGF-1 isoforms.

The caspase-recruitment domain (CARD) is known to play an important role in apoptosis and inflammation as an essential protein-protein interaction domain. The CARD of the cytosolic pathogen receptor Nod1 was overexpressed in Escherichia coli and purified by affinity chromatography and gel filtration. The purified CARD was crystallized at 277 K using the microseeding method. X-ray diffraction data were collected to 1.9 Å resolution. The crystals belong to space group P3 1 or P3 2 , with unit-cell parameters a = b = 79.1, c = 80.9 Å . Preliminary analysis indicates that there is one dimeric CARD molecule in the asymmetric unit.