Native-like partially folded conformations and folding process revealed in the N-terminal large fragments of staphylococcal nuclease: a study by NMR spectroscopy

The N-terminal large fragments of staphylococcal nuclease (SNase), SNase110 (1-110 residues), SNase121 (1-121 residues), and SNase135 (1-135 residues), and the fragment mutants G88W110, G88W121, V66W110 and V66W121 were studied by heteronuclear multidimensional NMR spectroscopy. Ensembles of co-existent native-like partially folded and unfolded states were observed for fragments. The persistent native-like tertiary interaction drives fragments to be in partially folded states, which reveal native-like beta-barrel conformations. G88W and V66W mutations modulate the extent of inherent native-like tertiary interaction in fragment molecules, and in consequence, fragment mutants fold into native-like beta-subdomain conformations. In cooperation with the inherent tertiary interaction, 2 M TMAO (trimethylamine N-oxide) can promote the folding reaction of fragments through the changes of unfolding free energy, and a native-like beta-subdomain conformation is observed when the chain length contains 135 residues. Heterogeneous partially folded conformations of 1-121 and 1-135 fragments due to cis and trans X-prolyl bond of Lys116-Pro117 make a non-unique folding pathway of fragments. The folding reaction of fragments can be characterized as a hierarchical process.     

Loop propensity of the sequence YKGQP from staphylococcal nuclease: implications for the folding of nuclease.

Recently we performed molecular dynamics (MD) simulations on the folding of the hairpin peptide DTVKLMYKGQPMTFR from staphylococcal nuclease in explicit water. We found that the peptide folds into a hairpin conformation with native and nonnative hydrogen-bonding patterns. In all the folding events observed in the folding of the hairpin peptide, loop formation involving the region YKGQP was an important event. In order to trace the origins of the loop propensity of the sequence YKGQP, we performed MD simulations on the sequence starting from extended, polyproline II and native type I' turn conformations for a total simulation length of 300 ns, using the GROMOS96 force field under constant volume and temperature (NVT) conditions. The free-energy landscape of the peptide YKGQP shows minima corresponding to loop conformation with Tyr and Pro side-chain association, turn and extended conformational forms, with modest free-energy barriers separating the minima. To elucidate the role of Gly in facilitating loop formation, we also performed MD simulations of the mutated peptide YKAQP (Gly --> Ala mutation) under similar conditions starting from polyproline II conformation for 100 ns. Two minima corresponding to bend/turn and extended conformations were observed in the free-energy landscape for the peptide YKAQP. The free-energy barrier between the minima in the free-energy landscape of the peptide YKAQP was also modest. Loop conformation is largely sampled by the YKGQP peptide, while extended conformation is largely sampled by the YKAQP peptide. We also explain why the YKGQP sequence samples type II turn conformation in these simulations, whereas the sequence as part of the hairpin peptide DTVKLMYKGQPMTFR samples type I' turn conformation both in the X-ray crystal structure and in our earlier simulations on the folding of the hairpin peptide. We discuss the implications of our results to the folding of the staphylococcal nuclease.     

The kinetic basis for the stabilization of staphylococcal nuclease by xylose.

The effect of xylose on the rates of folding and unfolding of staphylococcal nuclease (nuclease) have been investigated using fluorescence-detected pressure-jump relaxation kinetics in order to establish the kinetic basis for the observed stabilization of nuclease by this sugar (Frye KJ, Perman CS, Royer CA, 1996, Biochemistry 35:10234-10239). The activation volumes for both folding and unfolding and the equilibrium volume change for folding were all positive. Their values were within experimental error of those reported previously (Vidugiris GJA, Markley JL, Royer CA, 1995, Biochemistry 34:4909-4912) and were independent of xylose concentration. The major effect of xylose concentration was to increase significantly the rate of folding. The large positive activation volume for folding was interpreted previously as indicating that the rate-limiting step in nuclease folding involves dehydration of a significant amount of surface area. A large effect of xylose on the rate constant for folding provides strong support for this interpretation, because xylose, an osmolyte, stabilizes the folded state of proteins through surface tension effects. These studies further characterize the transition state in nuclease folding as lying closer to the folded, rather than the unfolded state along the folding coordinate in terms of the degree of burial of surface area. The image of the transition state that emerges is consistent with a dry molten globule.     

NMR analysis of staphylococcal nuclease thermal quench refolding kinetics.

Thermally unfolded staphylococcal nuclease has been rapidly quenched to temperatures near 0 degree C and the refolding behavior examined using an NMR kinetic experiment. Unfolded protein, exhibiting random coil chemical shifts, persists following the quench and refolds in two distinct kinetic phases. A protein folding intermediate with a trans Lys 116-Pro 117 peptide bond is transiently overpopulated and relaxes to the predominantly cis native cis-trans equilibrium. The rate of trans-->cis isomerization in the native-like nuclease intermediate is approximately 100-fold faster than that observed in a Lys-Pro model peptide. The activation enthalpy of 20 kcal/mol observed for the nuclease Lys 116-Pro 117 peptide bond is comparable to that observed for other X-Pro isomerizations.     

Compact dimension of denatured states of staphylococcal nuclease

Fluorescence and circular dichroism stopped-flow have been widely used to determine the kinetics of protein folding including folding rates and possible folding pathways. Yet, these measurements are not able to provide spatial information of protein folding/unfolding. Especially, conformations of denatured states cannot be elaborated in detail. In this study, we apply the method of fluorescence energy transfer with a stopped-flow technique to study global structural changes of the staphylococcal nuclease (SNase) mutant K45C, where lysine 45 is replaced by cysteine, during folding and unfolding. By labeling the thiol group of cysteine with TNB (5,5'-dithiobis-2-nitrobenzoic acid) as an energy acceptor and the tryptophan at position 140 as a donor, distance changes between the acceptor and the donor during folding and unfolding are measured from the efficiency of energy transfer. Results indicate that the denatured states of SNase are highly compact regardless of how the denatured states (pH-induced or GdmCl-induced) are induced. The range of distance changes between two probes is between 25.6 and 25.4 A while it is 20.4 A for the native state. Furthermore, the folding process consists of three kinetic phases while the unfolding process is a single phase. These observations agree with our previous sequential model: N(0) left arrow over right arrow D(1) left arrow over right arrow D(2) left arrow over right arrow D(3) (Chen et al., J Mol Biol 1991;220:771-778). The efficiency of protein folding may be attributed to initiating the folding process from these compact denatured structures.     

Hierarchical protein folding pathways: a computational study of protein fragments

We have previously presented a building block folding model. The model postulates that protein folding is a hierarchical top-down process. The basic unit from which a fold is constructed, referred to as a hydrophobic folding unit, is the outcome of combinatorial assembly of a set of "building blocks." Results obtained by the computational cutting procedure yield fragments that are in agreement with those obtained experimentally by limited proteolysis. Here we show that as expected, proteins from the same family give very similar building blocks. However, different proteins can also give building blocks that are similar in structure. In such cases the building blocks differ in sequence, stability, contacts with other building blocks, and in their 3D locations in the protein structure. This result, which we have repeatedly observed in many cases, leads us to conclude that while a building block is influenced by its environment, nevertheless, it can be viewed as a stand-alone unit. For small-sized building blocks existing in multiple conformations, interactions with sister building blocks in the protein will increase the population time of the native conformer. With this conclusion in hand, it is possible to develop an algorithm that predicts the building block assignment of a protein sequence whose structure is unknown. Toward this goal, we have created sequentially nonredundant databases of building block sequences. A protein sequence can be aligned against these, in order to be matched to a set of potential building blocks.     

金黄色葡萄球菌核酸酶A与不同外源DNA片段融合表达的作用比较

金黄色葡萄球菌核酸酶A(Staphylococcal nuclease A,SNA)可用于菌蜕制备中宿主菌的进一步灭活及残存遗传物质的去除。关于SNA在去除了信号肽后是否仍能分泌至胞外以及是否需要融合其他氨基酸片段才能在宿主菌胞质中发挥作用尚存争议。为厘清这一争议,分别构建一系列含SNA、SNA与λ噬菌体cro基因或结核杆菌脲酶基因部分序列的融合片段c SNA或u SNA的温控质粒并对其在大肠杆菌内的作用进行评价。结果显示,SNA、c SNA和u SNA的表达产物对大肠杆菌的4 h灭活率分别为99.9%、99.8%和74.2%。升温诱导30 min后,SNA和c SNA即可在宿主菌胞内被检出,而u SNA需1 h;相较之下,SNA和c SNA在培养液上清中的被检出时间要晚1 h,u SNA则晚2 h。对三者的核酸酶活性检测均为:SNA﹥c SNA﹥u SNA,且胞外活性都显著低于胞内。此外,SNA和c SNA在诱导2 h后即将宿主基因组成功降解,而u SNA则在整个实验期间均未能使宿主基因组完全降解。这表明SNA、c SNA和u SNA的表达产物均具有核酸酶活性,可被动释放至胞外,外源DNA片段的融入反而会降低SNA的核酸酶活性。     

Genetic control of immune response to staphylococcal nuclease. XII: Analysis of nuclease antigenic determinants using anti-nuclease monoclonal antibodies

Summary SJL mice, which are high responders to Staphylococcal nuclease (nuclease), were immunized and used to produce hybridoma cell lines secreting anti-nuclease monoclonal antibodies (mAb). Ten stable clones were derived from a single fusion. Seven of these produced antibodies of the IgG_1, isotype and were more precisely characterized for antigenic specificity. Only one hybridoma cell line (54-10-4) produced anti-nuclease antibodies capable of inhibiting enzymatic activity of nuclease. Binding inhibition analyses strongly suggest that the other monoclonal antibodies, which failed to inhibit nuclease activity detect two different antigenic regions, or epitopes, of the molecule: epitope cluster 1 domain is defined by hybridomas 54-2-7, 54-5-2, 54-9-8, and 54-10-8; epitope cluster 2 by 54-5-1 and 54-1-9. Because of its capacity to inhibit nuclease enzymatic activity mAb 54-10-4 was considered specific for a third epitope of the nuclease molecule called epitope 3. Binding studies of these monoclonal antibodies were extended to peptide fragments of the nuclease molecule in order to examine possible cross-reactions with such fragments, as has previously been reported for antibodies purified from polyclonal antisera. Monoclonal antibodies specific for epitope cluster 1 on the native molecule also bound to the fragments 1–126 and 49–149 but failed to bind to fragment 99–149, suggesting that the corresponding epitope(s) is determined by amino acids localized between residues 49 and 99. The epitope clusters 2 and 3 appeared to be expressed only on the native molecule. Monoclonal antibodies of different clusters exhibited very different migration patterns on isoelectric focusing while monoclonal antibodies of the same cluster were indistinguishable, which suggests that they may have originated from the same B cell precursor. Taken together these data suggest that this panel of monoclonal antibodies detects at least three distinct epitopes of the nuclease molecule, one of which could be involved in the determination of the enzymatic site.     

Analysis of long-range interactions in a model denatured state of staphylococcal nuclease based on correlated changes in backbone dynamics.

An expanded, highly dynamic denatured state of staphylococcal nuclease exhibits a native-like topology in the apparent absence of tight packing and fixed hydrogen bonds (Gillespie JR, Shortle D, 1997, J Mol Biol 268:158-169, 170-184). To address the physical basis of the long-range spatial ordering of this molecule, we probe the effects of perturbations of the sequence and solution conditions on the local chain dynamics of a denatured 101-residue fragment that is missing the first three beta strands. Structural interactions between chain segments are inferred from correlated changes in the motional behavior of residues monitored by 15N NMR relaxation measurements. Restoration of the sequence corresponding to the first three beta strands significantly increases the average order of all chain segments that form the five strand beta barrel including loops but has no effect on the carboxy terminal 30 residues. Addition of the denaturing salt sodium perchlorate enhances ordering over the entire sequence of this fragment. Analysis of seven different substitution mutants points to a complex set of interactions between the hydrophobic segment corresponding to beta strand 5 and the remainder of the chain. General patterns in the data suggest there is a hierarchy of native-like interactions that occur transiently in the denatured state and are consistent with the overall topology of the denatured state ensemble being determined by many coupled local interactions rather than a few highly specific long-range interactions.     

Early formation of a beta hairpin during folding of staphylococcal nuclease H124L as detected by pulsed hydrogen exchange

Pulsed hydrogen exchange methods were used to follow the formation of structure during the refolding of acid-denatured staphylococcal nuclease containing a stabilizing Leu substitution at position 124 (H124L SNase). The protection of more than 60 backbone amide protons in uniformly (15)N-labeled H124L SNase was monitored as a function of refolding time by heteronuclear two-dimensional NMR spectroscopy. As found in previous studies of staphylococcal nuclease, partial protection was observed for a subset of amide protons even at the earliest folding time point (10 msec). Protection indicative of marginally stable hydrogen-bonded structure in an early folding intermediate was observed at over 30 amide positions located primarily in the beta-barrel and to a lesser degree in the alpha-helical domain of H124L SNase. To further characterize the folding intermediate, protection factors for individual amide sites were measured by varying the pH of the labeling pulse at a fixed refolding time of 16 msec. Protection factors >5.0 were observed only for amide positions in a beta-hairpin formed by strands 2 and 3 of the beta-barrel domain and a single site near the C-terminus. The results indicate that formation of stable hydrogen-bonded structure in a core region of the beta-sheet is among the earliest structural events in the folding of SNase and may serve as a nucleation site for further structure formation.     

Characterization of the stable, acid-induced, molten globule-like state of staphylococcal nuclease.

Titration of a salt-free solution of native staphylococcal nuclease by HCl leads to an unfolding transition in the vicinity of pH 4, as determined by near- and far-UV circular dichroism. At pH 2-3, the protein is substantially unfolded. The addition of further HCl results in a second transition, this one to a more structured species (the A state) with the properties of an expanded molten globule, namely substantial secondary structure, little or no tertiary structure, relatively compact size as determined by hydrodynamic radius, and the ability to bind the hydrophobic dye 1-anilino-8-naphthalene sulfonic acid. The addition of anions, in the form of neutral salts, to the acid-unfolded state at pH 2 also causes a transition leading to the A state. Fourier transform infrared analysis of the amide I band was used to compare the amount and type of secondary structure in the native and A states. A significant decrease in alpha-helix structure, with a corresponding increase in beta or extended structure, was observed in the A state, compared to the native state. A model to account for such compact denatured states is proposed.     

Biosynthetic incorporation of tryptophan analogues into staphylococcal nuclease: effect of 5-hydroxytryptophan and 7-azatryptophan on structure and stability.

5-Hydroxytryptophan (5HW) and 7-azatryptophan (7AW) are analogue of tryptophan that potentially can be incorporated biosynthetically into proteins and used as spectroscopic probes for studying protein-DNA and protein-protein complexes. The utility of these probes will depend on the extent to which they can be incorporated and the demonstration that they cause minimal perturbation of a protein's structure and stability. To investigate these factors in a model protein, we have incorporated 5HW and 7AW biosynthetically into staphylococcal nuclease A, using a trp auxotroph Escherichia coli expression system containing the temperature-sensitive lambda cI repressor, Both tryptophan analogues are incorporated into the protein with good efficiency. From analysis of absorption spectra, we estimate approximately 95% incorporation of 5HW into position 140 of nuclease, and we estimate approximately 98% incorporation of 7AW, CD spectra of the nuclease variants are similar to that of the tryptophan-containing protein, indicating that the degree of secondary structure is not changed by the tryptophan analogues. Steady-state fluorescence data show emission maxima of 338 nm for 5HW-containing nuclease and 355 nm for 7AW-containing nuclease. Time-resolved fluorescence intensity and anisotropy measurements indicate that the incorporated 5HW residue, like tryptophan at position 140, has a dominant rotational correlation time that is approximately the value expected for global rotation of the protein. Guanidine-hydrochloride-induced unfolding studies show the unfolding transition to be two-state for 5HW-containing protein, with a free energy change for unfolding that is equal to that of the tryptophan-containing protein. In contrast, the guanidine-hydrochloride-induced unfolding of 7AW-containing nuclease appears to show a non-two-state transition, with the apparent stability of the protein being less than that of the tryptophan form.     

Thermal unfolding of staphylococcal nuclease and several mutant forms thereof studied by differential scanning calorimetry.

The effects of eight mutations on the thermodynamics of the reversible thermal unfolding of staphylococcal nuclease have been determined over a range of pH and protein concentration by means of differential scanning calorimetry. Variation of the protein concentration was included in our study because we found a significant dependence of the thermodynamics of protein unfolding on concentration. Values for the change in the standard free energy of unfolding, delta delta G0d, produced by the mutations in the pH range 5.0-7.0 varied from 1.9 kcal mol-1 (apparent stabilization) for H124L to -2.8 kcal mol-1 (apparent destabilization) for L25A. As has been observed in numerous other cases, there is no correlation in magnitude or sign between delta delta G0d and the corresponding values for delta delta Hd and T delta delta S0d, the latter quantities being in most cases much larger in magnitude than delta delta G0d. This fact emphasizes the difficulty in attempting to correlate the thermodynamic changes with structural changes observed by X-ray crystallography.     

Mechanism of induced folding: Both folding before binding and binding before folding can be realized in staphylococcal nuclease mutants

A considerable number of functional proteins are unstructured under physiological condition. These "intrinsically disordered" proteins exhibit induced folding when they bind their targets. The induced folding comprises two elementary processes: folding and binding. Two mechanisms are possible for the induced folding: either folding before binding or binding before folding. We found that these two mechanisms can be distinguished by the target-concentration dependence of folding kinetics. We also created two types of mutants of staphylococcal nuclease showing the different inhibitor-concentration dependence of induced folding kinetics. One mutant obeys the scheme of binding before folding, while the other the folding before binding. This is the first experimental evidence demonstrating that both mechanisms are realized for a single protein. Binding before folding is possible, when the protein lacks essential nonlocal interaction to stabilize the native conformation. The results cast light on the protein folding mechanism involved in the intrinsically disordered proteins.     

Development of cell lines stably expressing staphylococcal nuclease fused to dengue 2 virus capsid protein for CTVI

Dengue virus (DV) is one of the most significanthuman viral pathogens transmitted by arthropod vectorsand now present in over 100 countries. Half of the world’spopulation live in areas at risk of dengue virus infection[1]. The infection of DV causes a spectrum of diseasesranging from a debilitating, self-limited illness (denguefever), and life-threaten syndromes (dengue haemorrhagicfever/dengue shock syndrome). Annually, the fourserotypes of DV collectively cause 50–100 million casesof inf…     

Thermodynamic characterization of an equilibrium folding intermediate of staphylococcal nuclease.

High-sensitivity differential scanning calorimetry and CD spectroscopy have been used to probe the structural stability and measure the folding/unfolding thermodynamics of a Pro117-->Gly variant of staphylococcal nuclease. It is shown that at neutral pH the thermal denaturation of this protein is well accounted for by a 2-state mechanism and that the thermally denatured state is a fully hydrated unfolded polypeptide. At pH 3.5, thermal denaturation results in a compact denatured state in which most, if not all, of the helical structure is missing and the beta subdomain apparently remains largely intact. At pH 3.0, no thermal transition is observed and the molecule exists in the compact denatured state within the 0-100 degrees C temperature interval. At high salt concentration and pH 3.5, the thermal unfolding transition exhibits 2 cooperative peaks in the heat capacity function, the first one corresponding to the transition from the native to the intermediate state and the second one to the transition from the intermediate to the unfolded state. As is the case with other proteins, the enthalpy of the intermediate is higher than that of the unfolded state at low temperatures, indicating that, under those conditions, its stabilization must be of an entropic origin. The folding intermediate has been modeled by structural thermodynamic calculations. Structure-based thermodynamic calculations also predict that the most probable intermediate is one in which the beta subdomain is essentially intact and the rest of the molecule unfolded, in agreement with the experimental data. The structural features of the equilibrium intermediate are similar to those of a kinetic intermediate previously characterized by hydrogen exchange and NMR spectroscopy.     

Probing the folding capacity and residual structures in 1-79 residues fragment of staphylococcal nuclease by biophysical and NMR methods

1-79 residues SNase fragment (SNase79) has chain length containing a sequence for helix alpha(1), omega-loop, beta(I)-sheet, and partial beta(II)-sheet of native SNase. The incomplete "beta-barrel" structural region of SNase79 makes this fragment to be interested in investigation of its conformation. For this study, we use CD, fluorescence, and NMR spectroscopy to probe the folding capacity and the residual structures in SNase79. The optical spectra obtained for SNase79 and its mutants reveal the presence of retained capacity for folding of the fragment. The NMR derived (13)C(alpha) secondary chemical shifts, (3)J(NH-Halpha) coupling constants, amide-proton temperature coefficients, interresidue NOEs, and (15)N relaxation data determine the intrinsic propensities for helix- and turn- or beta-sheet-like conformations of SNase79, which is not the result of stabilizing inter-molecular interactions by oligomerization effects. The residual turn- and helix-like structures may serve as potential local nucleation sites, whereas the residual beta(I)-sheet-like structure can be regarded as a potential non-local nucleation site in the folding of SNase79. The intrinsic local and non-local interactions in these potential initiation sites are insufficient to stabilize the folding of SNase79 due to the shortage of relevant long-range interactions from other part of the fragment. The conformational ensemble of SNase79 is a highly heterogeneous collection of interconverting conformations having transiently populated helix- and beta-sheet- or turn-like structures.     

Kinetic folding and unfolding of staphylococcal nuclease and its six mutants studied by stopped-flow circular dichroism

Kinetics of refolding and unfolding of staphylococcal nuclease and its six mutants, each carrying single or double amino acid substitutions, are studied by stopped-flow circular dichroism measurements. A transient kinetic intermediate formed within 10 ms after refolding starts possesses a substantial part of the N-domain core β-structure, whereas helices are formed at the later stages. The structure of the kinetic intermediate is less organized than the structure that is known to be formed by a nuclease 1-136 fragment. Only the refolding kinetics are affected by the mutations in all the mutants except two in which the mutations have changed the native structure. From this result and also from the locations of the mutation sites, the major N-terminal domain of the nuclease in the transition state of folding has a structure nearly identical to the native one. On the other hand, the minor C-terminal domain has previously been shown to be still disorganized in the transition state. The effects of the amino acid substitutions on the stability of the native and the transition states are in good agreement with the changes in the hydration free energy, expected for the corresponding amino acid replacements in the unfolded polypeptide. Since side chains of all the mutated residues are not accessible to solvent in the native structure, the result suggests that it is the unfolded state that is mainly affected by the mutations. © 1995 Wiley-Liss, Inc.     

Highly efficient production of the staphylococcal nuclease reporter in Lactobacillus bulgaricus governed by the promoter of the hlbA gene

Lactobacillus delbrueckii ssp. bulgaricus ( L. bulgaricus ) genome sequence analysis revealed the presence of two genes that encode histone-like HU proteins ( hlbA and hlbB ) showing extensive similarity to other bacterial homologues. These genes were found to be extremely conserved among several L. bulgaricus strains. The hlbA gene was shown to be constitutively transcribed from a unique promoter ( phlbA ) during normal growth, whereas hlbB did not seem to be expressed under usual laboratory conditions. Using a reporter cassette in which the staphylococcal nuclease was fused at its N-terminus to the lactococcal signal peptide Usp45 (SP Usp45), we have demonstrated that phlbA promotes high expression of the reporter in L. bulgaricus , which correlated with an abundant secretion of the mature nuclease in the supernatant fraction. Quantification of the exported enzyme reveals a secretion level approximately threefold higher when the expression of the reporter was under the control of phlbA compared with the lactococcal usp45 promoter. Together, these results indicate that phlbA is suitable for gene expression in L. bulgaricus , that SP Usp45 is functionally recognized and processed by the L. bulgaricus secretion machinery and that the nuclease reporter gene can be used for the identification of exported products in this bacterium.