Mapping local structural perturbations in the native state of stefin B (cystatin B) under amyloid forming conditions

Unlike a number of amyloid-forming proteins, stefins, and in particular stefin B (cystatin B) form amyloids under conditions where the native state predominates. In order to trigger oligomerization processes, the stability of the protein needs to be compromised, favoring structural re-arrangement however, accelerating fibril formation is not a simple function of protein stability. We report here on how optimal conditions for amyloid formation lead to the destabilization of dimeric and tetrameric states of the protein in favor of the monomer. Small, highly localized structural changes can be mapped out that allow us to visualize directly areas of the protein which eventually become responsible for triggering amyloid formation. These regions of the protein overlap with the Cu (II)-binding sites which we identify here for the first time. We hypothesize that in vivo modulators of amyloid formation may act similarly to painstakingly optimized solvent conditions developed in vitro. We discuss these data in the light of current structural models of stefin B amyloid fibrils based on H-exchange data, where the detachment of the helical part and the extension of loops were observed

Unfolding of apomyoglobin from Aplysia limacina: The effect of salt and pH on the cooperativity of folding

The equilibrium unfolding pathway of Aplysia apomyoglobin has been studied under various solvent conditions. The protein exhibits a single unfolding transition in acid in contrast to the two transitions observed for the mammalian apomyoglobins with which it shares a common fold but a low level of sequence identity (24%). This acid-unfolded species has considerable residual structure as evidenced by both tryptophan fluorescence and far-UV CD spectroscopy. It remains 40% α-helical under low salt conditions (2 mM citrate, 4°C); the folded form is 65% helical. A similar species is observed for the mammalian globins in mild acid conditions. Titration with GdnHCl at pH 7 reveals two unfolding transitions, the first having common features with that observed in acid and the second resulting in a completely unfolded state. Under the same conditions, urea unfolds the protein completely in an apparently single cooperative transition. Assuming a simple three-state model (F⇆I⇆U), data from GdnHCl and urea titrations over a range of pH conditions were used to derive values for the apparent stability (ΔG(w(app))) and solvent accessibility (n((app))) of the folded (F) and intermediate (I) forms of the protein. Urea titrations were then repeated over a range of KCl concentrations in order to understand the contribution of Cl- to the different unfolding activity of GdnHCl. A three-state scheme is justified when changes in ΔG(w(app)) occur without changes in n((app)). The change in free energy of folding of I⇆F (ΔG(w(F/I))) decreases to 0 at pH 4 as expected from the acid unfolding curve. ΔG(w(I/U)) reaches its maximum at pH 4.5, the isoelectric point of the protein. Variations of this value with pH and chloride are as much as 3 kcal mol-1 and correlate closely with changes in n((app)) although there is no change in the α-helical content of I across the pH range. This observation is interpreted here as a deviation of the unfolding of the I state of Aplysia apomyoglobin from a cooperative behaviour

A new folding intermediate of apomyoglobin from Aplysia limacina: Stepwise formation of a molten globule

Apomyoglobin from Aplysia limacina (al-apoMb), despite having only 20% sequence identity with the more commonly studied mammalian globins (m-apoMbs), properties which result in an increased number of hydrophobic contacts and a loss of most internal salt bridges, shares a number of features of their folding profiles. We show here that it contains an unusually stable core which resists unfolding even at 70 degrees C. The equilibrium intermediate (I-T) at this high temperature is distinct from the acid unfolded state I-A which has many properties in common with the acid intermediate observed for the mammalian apoproteins (I-AGH). It contains a smaller amount of secondary structure (27% alpha-helical instead of 35%) and is more highly solvated as evidenced from its fluorescence spectrum (lambda(max)=344 nm instead of 338 nm). Its stability is greatly increased (Delta Delta G(w) = - 6.75 kcal mol(-1)) in the presence of high salt (2 M KCl), lending support to the view that hydrophobic interactions are responsible for its stability. Kinetic data show classical two-state kinetics between I-A and the folded state both in the presence and absence of salt. Both I-A and I-T can be populated within the dead time of the stopped-flow apparatus, since initiating the refolding reaction from I-T or I-A rather than the completely unfolded state does not affect the observed refolding time-course. Our conclusion is that al-apoMb, as other "apo" proteins (including for example alpha-lactalbumin in the absence of Ca2+), may be described as "uncoupled" with an unusually high and exploitable tendency to populate partially folded states. (C) 2000 Academic Press

A method for the reversible trapping of proteins in non-native conformations

High-dilution equilibrium macrocyclization is developed as a general approach to trapping proteins in a non-native state with a synthetic cross-linking agent. The approach is illustrated using the N-terminal domain of phosphoglycerate kinase and a synthetic reagent containing two maleimide groups, for selective attachment to cysteines introduced onto the protein surface through mutagenesis, and an aromatic disulfide that can be chemically or photochemically cleaved. Following functionalization of the cysteine residues, thiol-disulfide exchange chemistry under strongly unfolding conditions was used to achieve intramolecular cyclization and a high yield of the cross-linked protein. H-1 NMR, CD, and fluorescence spectroscopies indicate that the conformation of the cross-linked protein is non-native. Chemical cleavage of the aromatic disulfide cross-link by a reducing agent results in the acquisition of a nativelike conformation for the reduced protein. Thus, the cross-link acts as a reversible switch of protein folding

The major transition state in folding need not involve the immobilization of side chains

During protein folding in which few, if any, definable kinetic intermediates are observable, the nature of the transition state is central to understanding the course of the reaction. Current experimental data does not distinguish the relative contributions of side chain immobilization and dehydration phenomena to the major rate-limiting transition state whereas this distinction is central to theoretical models that attempt to simulate the behavior of proteins during folding. Renaturation of the small proteinase inhibitor cystatin under oxidizing versus reducing conditions is the first experimental case in which these processes can be studied independently. Using this example, we show that sidechain immobilization occurs downstream of the major folding transition state. A consequence of this is the existence of states with disordered side chains, which are distinct from kinetic protein folding intermediates and which lie within the folded state free energy well

Three-dimensional domain swapping in the folded and molten-globule states of cystatins, an amyloid-forming structural superfamily

Cystatins, an amyloid-forming structural superfamily, form highly stable, domain-swapped dimers at physiological protein concentrations. In chicken cystatin, the active monomer is a kinetic trap en route to dimerization, and any changes in solution conditions or mutations that destabilize the folded state shorten the lifetime of the monomeric form. In such circumstances, amyloidogenesis will start from conditions where a domain-swapped dimer is the most prevalent species. Domain swapping occurs by a rearrangement of loop I, generating the new intermonomer interface between strands 2 and 3. The transition state for dimerization has a high level of hydrophobic group exposure, indicating that gross conformational perturbation is required for domain swapping to occur. Dimerization also occurs when chicken cystatin is in its reduced, molten-globule state, implying that the organization of secondary structure in this state mirrors that in the folded state and that domain swapping is not limited to the folded states of proteins. Although the interface between cystatin-fold units is poorly defined for cystatin A, the dimers are the appropriate size to account for the electron-dense regions in amyloid protofilaments