Shin-ichi Segawa

Efficient in vitro folding of the three-disulfide derivatives of hen lysozyme in the presence of glycerol

Four derivatives of hen lysozyme, each lacking one native disulfide bond of the four in authentic lysozyme, were produced in Escherichia coli by expressing synthetic mutant genes. In the reoxidation reaction of the reduced derivatives purified from inclusion bodies, the addition of glycerol significantly enhanced the efficiency of folding and ‘correct’ disulfide bond formation. This enabled simple chromatographical purification of refolded materials. Purified 3SS‐derivatives all showed lytic activities and secondary structures comparable to authentic lysozyme, which directly showed that none of the four native disulfide bonds is a prerequisite for ‘correct’ in vitro folding.

Modulation of structure and dynamics by disulfide bond formation in unfolded states.

During oxidative folding, the formation of disulfide bonds has profound effects on guiding the protein folding pathway. Until now, comparatively little is known about the changes in the conformational dynamics in folding intermediates of proteins that contain only a subset of their native disulfide bonds. In this comprehensive study, we probe the conformational landscape of non-native states of lysozyme containing a single native disulfide bond utilizing nuclear magnetic resonance (NMR) spectroscopy, small-angle X-ray scattering (SAXS), circular dichroism (CD) data, and modeling approaches. The impact on conformational dynamics varies widely depending on the loop size of the single disulfide variants and deviates significantly from random coil predictions for both NMR and SAXS data. From these experiments, we conclude that the introduction of single disulfides spanning a large portion of the polypeptide chain shifts the structure and dynamics of hydrophobic core residues of the protein so that these regions exhibit levels of order comparable to the native state on the nanosecond time scale.

Characterisation of disulfide-bond dynamics in non-native states of lysozyme and its disulfide deletion mutants by NMR.

This report describes NMR‐spectroscopic investigations of the conformational dynamics of disulfide bonds in hen‐egg‐white lysozyme substitution mutants. The following four systems have been investigated: 2SSα, a lysozyme variant that contains C64A, C76A, C80A and C94A substitutions, was studied in water at pH 2 and 3.8 and in urea (8 M, pH 2); 2SSβ lysozyme, which has C6S, C30A, C115A and C127A substitutions, was studied in water (pH 2) and urea (8 M, pH 2). The NMR analysis of heteronuclear 15N‐relaxation rates shows that the barrier to disulfide‐bond isomerisation can vary substantially in different lysozyme mutants and depends on the residual structure present in these states. The investigations reveal cooperativity in the modulation of micro‐ to millisecond dynamics that is due to the presence of multiple disulfide bridges in lysozyme. Mutation of cysteines in one of the two structural domains substantially diminishes the barrier to rotational isomerisation in the other domain. However, the interactions between hydrophobic clusters within and across the domains remains intact.

Seven cysteine-deficient mutants depict the interplay between thermal and chemical stabilities of individual cysteine residues in mitogen-activated protein kinase c-Jun N-terminal kinase 1

Intracellular proteins can have free cysteines that may contribute to their structure, function, and stability; however, free cysteines can lead to chemical instabilities in solution because of oxidation-driven aggregation. The MAP kinase, c-Jun N-terminal kinase 1 (JNK1), possesses seven free cysteines and is an important drug target for autoimmune diseases, cancers, and apoptosis-related diseases. To characterize the role of cysteine residues in the structure, function, and stability of JNK1, we prepared and evaluated wild-type JNK1 and seven cysteine-deficient JNK1 proteins. The nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments showed that the chemical stability of JNK1 increased as the number of cysteines decreased. The contribution of each cysteine residue to biological function and thermal stability was highly susceptible to the environment surrounding the particular cysteine mutation. The mutations of solvent-exposed cysteine to serine did not influence biological function and increased the thermal stability. The mutation of the accessible cysteine involved in the hydrophobic pocket did not affect biological function, although a moderate thermal destabilization was observed. Cysteines in the loosely assembled hydrophobic environment moderately contributed to thermal stability, and the mutations of these cysteines had a negligible effect on enzyme activity. The other cysteines are involved in the tightly filled hydrophobic core, and mutation of these residues was found to correlate with thermal stability and enzyme activity. These findings about the role of cysteine residues should allow us to obtain a stable JNK1 and thus promote the discovery of potent JNK1 inhibitors.

A two-dimensional NMR study of exchange behavior of amide hydrogens in a lysozyme derivative with an extra cross-link between Glu35 and Trp 108—quenching of cooperative fluctuations and effects on the protein stability

Two-dimensional nmr spectra [correlated spectroscopy (COSY), homonuclear Hartmann-Hahn (HOHAHA), nuclear Overhauser effect spectroscopy (NOESY)] have been observed for cross-linked lysozyme, a chemically modified lysozyme derivative with an extra ester cross-link between residues E35 and W108. Eight shifted cross-peaks were found in the fingerprint region of COSY spectra. By searching COSY, HOHAHA and NOESY spectra, they have been assigned to A32, E35, S36, 158, A107, W108, V109, and A110. The NOE connectivities (dNN and d alpha N) found for the cross-linked lysozyme are quite similar to those for the intact lysozyme. Exchange behavior of amide hydrogens has been studied for both intact and cross-linked lysozymes by observing the fingerprint region of COSY spectra. Hydrogen exchange reactions were carried out at pH 7.0 and at several temperatures. There exist 41 amide hydrogens whose exchange reactions are detectable under this experimental condition. Not only exchange rates but also their activation enthalpies were determined for individual amide hydrogens. They are classified into two groups, which are called categories III and IV. Category III hydrogens are distributed in relatively flexible peripheral parts of protein, and category IV hydrogens are deeply buried in the core region of protein. Category III hydrogens are exchanged through localized unfolding around their sites with a low activation enthalpy ranging from 10 to 25 kcal/mol. The formation of an extra cross-link affects neither the exchange rate nor the activation enthalpy of category III hydrogens. However, amide hydrogens of residues 34-39 in the vicinity of the hinge are exceptions. They are easily exchanged in the intact lysozyme but their exchange rates are drastically retarded by cross-linking. In the intact lysozyme, structural fluctuations mediating the exchange of category IV hydrogens are highly cooperative with a large activation enthalpy. These large-scale structural fluctuations are the global unfolding of the overall structure and also concerted motions within a domain. Especially near 38 degrees C, it was found that the dominant fluctuation occurring in the alpha-domain is different from that in the beta-domain. However, these concerted motions are strongly quenched by the formation of the cross-link because of the cooperativity of such a large-scale fluctuation. The stabilization of a localized area of protein by cross-linking results in the great suppression of large-scale and concerted motions. The exchange rates of category IV hydrogens are extremely retarded in the cross-linked lysozyme, so that they are exchanged through the so-called penetration mechanism characterized by a low activation enthalpy. These experimental results are discussed with regard to the contribution of cross-linking to the stabilization of the folded structure of protein.

Glycerol-induced folding of unstructured disulfide-deficient lysozyme into a native-like conformation

2SS[6‐127,64‐80] variant of lysozyme which has two disulfide bridges, Cys6‐Cys127 and Cys64‐Cys80, and lacks the other two disulfide bridges, Cys30‐Cys115 and Cys76‐Cys94, was quite unstructured in water, but a part of the polypeptide chain was gradually frozen into a native‐like conformation with increasing glycerol concentration. It was monitored from the protection factors of amide hydrogens against H/D exchange. In solution containing various concentrations of glycerol, H/D exchange reactions were carried out at pH* 3.0 and 4°C. Then, 1H‐15N‐HSQC spectra of partially deuterated protein were measured in a quenching buffer for H/D exchange (95% DMSO/5% D2O mixture at pH* 5.5 adjusted with dichloroacetate). In a solution of 10% glycerol, the protection factors were nearly equal to 10 at most of residues. With increasing glycerol concentration, some selected regions were further protected, and their protection factors reached about a 1000 in 30% glycerol solution. The highly protected residues were included in A‐, B‐, and C‐helices and β3‐strand, and especially centered on Ile 55 and Leu 56. In 2SS[6‐127,64‐80], long‐range interactions were recovered due to the preferential hydration by glycerol in the hydrophobic box of the α‐domain. Glycerol‐induced recovering of the native‐like structure is discussed from the viewpoint of molten globules growing with the protein folding.

Glycerol‐enhanced detection of a preferential structure latent in unstructured 1SS‐variants of lysozyme

Four species of 1SS‐varinats of lysozyme were almost unstructured in water, judged from their near‐UV CD and 1H‐15N‐HSQC spectra. Some preferential structure might exist in such a disordered state, but the population of molecules in such a conformation must have been too small to be detected by spectroscopic methods. Indeed, our previous study showed that the addition of 30% glycerol induced the unstructured 2SS‐variant of lysozyme to form a native‐like structure. To extend this method to more disordered proteins, we attempted to detect some preferential structure latent in unstructured 1SS‐variants by the glycerol‐enhanced detection. Only in one molecular species of the four 1SS‐variants, 1SS[6‐127] containing a single disulfide bridge of Cys6‐Cys127, a preferential structure was found in the presence of 50% glycerol. It was detected by near‐UV CD measurements and the H/D exchange method combined with the NMR spectroscopy. The glycerol‐induced structure in 1SS[6‐127] was not localized only in the vicinity of Cys6‐Cys127, and largely protected regions distributed themselves among A‐, B‐, and C‐helices and Ile55 and Leu56. It was similar to the glycerol‐induced structure in 2SS[6‐127, 64‐80] containing two disulfide bridges of Cys6‐Cys127 and Cys64‐Cys80, although the former was less rigid than the latter. The role of A‐helix (residues 4–15) is proposed as an origin of excellent potential of Cys6‐Cys127 for inducing a tertiary structure in the α‐domain.

NMR Analysis of a Kinetically Trapped Intermediate of a Disulfide-Deficient Mutant of the Starch-Binding Domain of Glucoamylase

A thermally unfolded disulfide-deficient mutant of the starch-binding domain of glucoamylase refolds into a kinetically trapped metastable intermediate when subjected to a rapid lowering of temperature. We attempted to characterise this intermediate using multidimensional NMR spectroscopy. The (1)H-(15)N heteronuclear single quantum coherence spectrum after a rapid temperature decrease (the spectrum of the intermediate) showed good chemical shift dispersion but was significantly different from that of the native state, suggesting that the intermediate adopts a nonnative but well-structured conformation. Large chemical shift changes for the backbone amide protons between the native and the intermediate states were observed for residues in the β-sheet consisting of strands 2, 3, 5, 6, and 7 as well as in the C-terminal region. These residues were found to be in close proximity to aromatic residues, suggesting that the chemical shift changes are mainly due to ring current shifts caused by the aromatic residues. The two-dimensional nuclear Overhauser enhancement (NOE) spectroscopy experiments showed that the intermediate contained substantial, native-like NOE connectivities, although there were fewer cross peaks in the spectrum of the intermediate compared with that of the native state. It was also shown that there were native-like interresidue NOEs for residues buried in the protein, whereas many of the NOE cross peaks were lost for the residues involved in a surface-exposed aromatic cluster. These results suggest that, in the intermediate, the aromatic cluster at the surface is structurally less organised, whereas the interior of the protein has relatively rigid, native-like side-chain packing.

The confirmation of the denatured structure of pyrrolidone carboxyl peptidase under nondenaturing conditions: difference in helix propensity of two synthetic peptides with single amino acid substitution.

In the denatured state (D1 state) of cystein‐free pyrrolidone carboxyl peptidase (PCP‐0SH) from Pyrococcus furiosus, a hyperthermophile under nondenaturing conditions, a fairly stable α‐helix (α6‐helix) has been determined from H/D exchange‐NMR experiments. On the other hand, the α6‐helix region of the proline‐mutant at position 199 (A199P) was unstructured in the D1 state unlike that of the wild‐type PCP‐0SH, although the folded conformations of both proteins were almost identical to each other. This finding has been deduced from the information regarding the remaining amide hydrogens in the HSQC spectra after H/D exchanges in the D1 state. To confirm this inference, we examined the helical propensities of two synthetic peptides from their NMR structural analysis in the presence of trifluoroethanol (TFE). One is an 18‐residue peptide called the wild‐type H6‐peptide corresponding to the α6‐helix (from Ser188 to Glu205) of the wild‐type PCP‐0SH, and the other is the mutant H6‐peptide corresponding to the α6‐helix region of A199P. The NOE‐contact information obtained from the 2D‐1H‐NOESY spectra measured for both peptides in the presence of 30% TFE clearly demonstrated that the wild‐type H6‐peptide had a high helical propensity, but the mutant H6‐peptide was almost totally unstructured. The TFE‐induced helical propensities for these peptide fragments confirmed the conclusions deduced from the H/D exchange data measured in the D1 states of two proteins. Proteins 2008.

NMR and CD analysis of an intermediate state in the thermal unfolding process of mouse lipocalin-type prostaglandin D synthase.

We previously reported that the thermal unfolding of mouse lipocalin-type prostaglandin D synthase (L-PGDS) is a completely reversible process under acidic conditions and follows a three-state pathway, including an intermediate state (I) between native state (N) and unfolded state. In the present study, we investigated the intermediate state of mouse C65A L-PGDS and clarified the local conformational changes in the upper and bottom regions by using NMR and CD spectroscopy. The (1)H-(15)N HSQC measurements revealed that the backbone conformation was disrupted in the upper region of the β-barrel at 45°C, which is around the T(m) value for the N ↔ I transition, but that the signals of the residues located at the bottom region of L-PGDS remained at 54°C, where the maximum accumulation of the intermediate state was found. (1)H-NMR and CD measurements showed that the T(m) values obtained by monitoring Trp54 at the upper region and Trp43 at the bottom region of the β-barrel were 41.4 and 47.5°C, respectively, suggesting that the conformational change in the upper region occurred at a lower temperature than that in the bottom region. These findings demonstrate that the backbone conformation of the bottom region is still maintained in the intermediate state.