Inhibitor binding increases the mechanical stability of staphylococcal nuclease

Staphylococcal nuclease (SNase) catalyzes the hydrolysis of DNA and RNA in a calcium-dependent fashion. We used AFM-based single-molecule force spectroscopy to investigate the mechanical stability of SNase alone and in its complex with an SNase inhibitor, deoxythymidine 3′,5′-bisphosphate. We found that the enzyme unfolds in an all-or-none fashion at ∼26 pN. Upon binding to the inhibitor, the mechanical unfolding forces of the enzyme-inhibitor complex increase to ∼50 pN. This inhibitor-induced increase in the mechanical stability of the enzyme is consistent with the increased thermodynamical stability of the complex over that of SNase. Because of its strong mechanical response to inhibitor binding, SNase, a model protein folding system, offers a unique opportunity for studying the relationship between enzyme mechanics and catalysis.     

Searching for folding initiation sites of staphylococcal nuclease: a study of N-terminal short fragments

The N-terminal short fragments of staphylococcal nuclease (SNase), SNase20, SNase28, and SNase36, corresponding to the sequence regions, Ala1-Gly20, Ala1-Lys28, and Ala1-Leu36, respectively, as well as an 8-residue peptide (Ala17-Ile18-Asp19-Gly20-Asp21-Thr22-Val23-Lys24) have been synthesized. The conformational states of these fragments were investigated using CD and NMR spectroscopy in aqueous solution and in trifluoroethanol (TFE)-H(2)O mixture. SNase20 containing a sequence corresponding to a bent peptide in native SNase shows a transient population of bend-like conformation around Ala12-Thr13-Leu14 in TFE-H(2)O mixture. The sequence region of Ala17-Thr22 of SNase28 displays a localized propensity for turn-like conformation in both aqueous solution and TFE-H(2)O mixture. The conformational ensemble of SNase36 in aqueous solution includes populated turn-like conformations localized in sequence regions Ala17-Thr22 and Tyr27-Gln30. The analysis suggests that these sequence regions, which form the regular secondary structures in native protein, may serve as the folding nucleation sites of SNase fragments of different chain lengths starting from the N-terminal end. Thus, the formation of bend- and turn-like conformations of these sequence regions may be involved in the early folding events of the SNase polypeptide chain in vitro.     

Inhibition of DNA Replication of Human Papillomavirus by Using Zinc Finger–Single-Chain FokI Dimer Hybrid

Previously, we reported that an artificial zinc-finger protein (AZP)–staphylococcal nuclease (SNase) hybrid (designated AZP–SNase) inhibited DNA replication of human papillomavirus type 18 (HPV-18) in mammalian cells by binding to and cleaving a specific HPV-18 ori plasmid. Although the AZP–SNase did not show any side effects under the experimental conditions, the SNase is potentially able to cleave RNA as well as DNA. In the present study, to make AZP hybrid nucleases that cleave only viral DNA, we switched the SNase moiety in the AZP–SNase to the single-chain FokI dimer (scFokI) that we had developed previously. We demonstrated that transfection with a plasmid expressing the resulting hybrid nuclease (designated AZP–scFokI) inhibited HPV-18 DNA replication in transient replication assays using mammalian cells more efficiently than AZP–SNase. Then, by linker-mediated PCR analysis, we confirmed that AZP–scFokI cleaved an HPV-18 ori plasmid around its binding site in mammalian cells. Finally, a modified MTT assay revealed that AZP–scFokI did not show any significant cytotoxicity. Thus, the newly developed AZP–scFokI hybrid is expected to serve as a novel antiviral reagent for the neutralization of human DNA viruses with less fewer potential side effects.     

SNase R与SNase酶学性质的比较研究

金黄色葡萄球菌核酸酶(SNase)的一种类似物SNase R在E.coli DH5x细胞中高效表达, 经磷酸纤维素离子交换柱层析纯化后.在SDS-PAGE上为一条分子量17.5KDa的蛋白带.本文对SNase R和SNase的某些酶学性质.酶动力学性质进行了比较研究.为蛋白质结构与功能及肽链折叠研究提供了又一种材料.     

Effect of a specific hydrogen bond (N138ND2-Q106O) on conformational integrity, stability, and activity of staphylococcal nuclease

There are two hydrogen bonding interactions (N138ND2-Q106O and Y54OH-S141OG) between the C-terminal region and the main body of staphylococcal nuclease (SNase). To examine the role of these hydrogen bonds, SNase(141) and its three mutants, SNase(141)N138D, SNase(141)S141A, and SNase(141)N138D/S141A, were created. The N138D mutation has the N138ND2-Q106O interaction deleted and the S141A mutation has the Y54OH-S141OG and S141OG-N138O interactions deleted. The conformational features, stability, and activity of the proteins have been compared by using circular dichroism, intrinsic and ANS-binding fluorescence, GdnHCl-induced denaturation, and activity assay. The results clearly show that the N138D mutation significantly alters the secondary and tertiary structures of the protein, producing a partially unfolding state; in contrast, the S141A mutation has no such effect on structure. These results strongly suggest that the specific hydrogen bond, N138ND2-Q106O, plays an important role in maintaining the conformational integrity and stability of the nuclease.     

Restricted backbone conformational and motional flexibilities of loops containing peptidyl-proline bonds dominate the enzyme activity of staphylococcal nuclease

The role of cis-trans isomerizations of peptidyl-proline bonds in the enzyme activity of staphylococcal nuclease (SNase) was examined by mutation of proline residues. The proline-free SNase ([Pro-]SNase), namely, P11A/P31A/P42A/P47T/P56A/P117G-mutant SNase, was adopted for elucidating the correlation between the nuclease activity and the backbone conformational and dynamic states of SNase. The 3D solution structure of [Pro-]SNase has been determined by heteronuclear NMR experiments. Comparing the structure of [Pro-]SNase with the structure of SNase revealed the conformational differences between the two proteins. In the structure of [Pro-]SNase, conformational rearrangements were observed for the loop of residues Ala112-His121 containing a trans Lys116-Gly117 peptide bond and for the C-terminal alpha-helical loop of residues Leu137-Glu142. Mutation of proline at position 117 also caused the conformational rearrangement of the p-loop (Asp77-Leu89), which is remote from the Ala112-His121 loop. The Ala112-His121 loop and p-loop are placed closer to each other in [Pro-]SNase than in SNase. The backbone dynamic features of the omega-loop (Pro42-Pro56) of SNase are different from those of [Pro-]SNase. The backbone of the omega-loop exhibits restricted flexibility with slow conformational exchange motions in SNase, but is highly flexible in [Pro-]SNase. The analysis indicates that the restrained backbone conformation of the Ala112-His121 loop and restricted flexibility of the omega-loop are two dominant factors determining the enzyme activity of SNase. Of the two factors, the former is correlated with the strained cis Lys116-Pro117 peptide bond and the latter is correlated with the cis-trans isomerizations of the His46-Pro47 peptide bond.     

Protein stabilization by osmolytes from hyperthermophiles: effect of mannosylglycerate on the thermal unfolding of recombinant nuclease a from Staphylococcus aureus studied by picosecond time-resolved fluorescence and calorimetry

2-O-alpha-Mannosylglycerate, a negatively charged osmolyte widely distributed among (hyper)thermophilic microorganisms, is known to provide notable protection to proteins against thermal denaturation. To study the mechanism responsible for protein stabilization, pico-second time-resolved fluorescence spectroscopy was used to characterize the thermal unfolding of a model protein, Staphylococcus aureus recombinant nuclease A (SNase), in the presence or absence of mannosylglycerate. The fluorescence decay times are signatures of the protein state, and the pre-exponential coefficients are used to evaluate the molar fractions of the folded and unfolded states. Hence, direct determination of equilibrium constants of unfolding from molar fractions was carried out. Van't Hoff plots of the equilibrium constants provided reliable thermodynamic data for SNase unfolding. Differential scanning calorimetry was used to validate this thermodynamic analysis. The presence of 0.5 m potassium mannosylglycerate caused an increase of 7 degrees C in the SNase melting temperature and a 2-fold increase in the unfolding heat capacity. Despite the considerable degree of stabilization rendered by this solute, the nature and population of protein states along unfolding were not altered in the presence of mannosylglycerate, denoting that the unfolding pathway of SNase was unaffected. The stabilization of SNase by mannosylglycerate arises from decreased unfolding entropy up to 65 degrees C and from an enthalpy increase above this temperature. In molecular terms, stabilization is interpreted as resulting from destabilization of the denatured state caused by preferential exclusion of the solute from the protein hydration shell upon unfolding, and stabilization of the native state by specific interactions. The physiological significance of charged solutes in hyperthermophiles is discussed.     

Monoclonal antibody, a novel probe for protein folding

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Two peptide fragments G55-I72 and K97-A109 from staphylococcal nuclease exhibit different behaviors in conformational preferences for helix formation

Two synthetic peptides, SNasealpha1 and SNasealpha2, corresponding to residues G55-I72 and K97-A109, respectively, of staphylococcal nuclease (SNase), are adopted for detecting the role of helix alpha1 (E57-A69) and helix alpha2 (M98-Q106) in the initiation of folding of SNase. The helix-forming tendencies of the two SNase peptide fragments are investigated using circular dichroism (CD) and two-dimensional (2D) nuclear magnetic resonance (NMR) methods in water and 40% trifluoroethanol (TFE) solutions. The coil-helix conformational transitions of the two peptides in the TFE-H2O mixture are different from each other. SNasealpha1 adopts a low population of localized helical conformation in water, and shows a gradual transition to helical conformation with increasing concentrations of TFE. SNasealpha2 is essentially unstructured in water, but undergoes a cooperative transition to a predominantly helical conformation at high TFE concentrations. Using the NMR data obtained in the presence of 40% TFE, an ensemble of alpha-helical structures has been calculated for both peptides in the absence of tertiary interactions. Analysis of all the experimental data available indicates that formation of ordered alpha-helical structures in the segments E57-A69 and M98-Q106 of SNase may require nonlocal interactions through transient contact with hydrophobic residues in other parts of the protein to stabilize the helical conformations in the folding. The folding of helix alpha1 is supposed to be effective in initiating protein folding. The formation of helix alpha2 depends strongly on the hydrophobic environment created in the protein folding, and is more important in the stabilization of the tertiary conformation of SNase.     

金黄色葡萄球菌核酸酶C末端去9肽对酶蛋白溶液构象的影响

通过多维异核核磁共振方法,结合运用荧光和圆二色等光谱方法,比较研究了V8菌株金黄色葡萄球菌核酸酶（含149个氨基酸残基）,酶蛋白1－140片段（SNase140)以及在TMP（thymidine 5′-monophosphate)和Ca^2 存在下的SNase140的溶液构象状态。探讨了酶蛋白C末端去9肽后对酶蛋白构象和活力的影响。研究指出,远离酶蛋白活性部位残基间相互作用的变化,将通过酶蛋白两个亚结构域之间所形成的氢键,影响酶蛋白活性部位的空间构象,从而影响酶蛋白的活力。     

Folding of the C-terminal fragment V111-D143 of staphylococcal nuclease in aqueous solution

Studies of conformational features of fragments SNase(111-143) and SNase(118-143) and segment E122-K136 in 1-139 fragment (SNase139) suggest that the high intrinsic helical propensity can drive segment E122-K136 fold into a stable helix only when the segments V111-H121 and L137-D143 flanked on segment E122-K136 in staphylococcal nuclease (SNase) have stable folding.     

den V gene of bacteriophage T4 codes for both pyrimidine dimer-DNA glycosylase and apyrimidinic endonuclease activities.

Recent studies have shown purified preparations of phage T4 UV DNA-incising activity (T4 UV endonuclease or endonuclease V of phage T4) contain a pyrimidine dimer-DNA glycosylase activity that catalyzes hydrolysis of the 5' glycosyl bond of dimerized pyrimidines in UV-irradiated DNA. Such enzyme preparations have also been shown to catalyze the hydrolysis of phosphodiester bonds in UV-irradiated DNA at a neutral pH, presumably reflecting the action of an apurinic/apyrimidinic endonuclease at the apyrimidinic sites created by the pyrimidine dimer-DNA glycosylase. In this study we found that preparations of T4 UV DNA-incising activity contained apurinic/apyrimidinic endonuclease activity that nicked depurinated form I simian virus 40 DNA. Apurinic/apyrimidinic endonuclease activity was also found in extracts of Escherichia coli infected with T4 denV+ phage. Extracts of cells infected with T4 denV mutants contained significantly lower levels of apurinic/apyrimidinic endonuclease activity; these levels were no greater than the levels present in extracts of uninfected cells. Furthermore, the addition of DNA containing apurinic or apyrimidinic sites to reactions containing UV-irradiated DNA and T4 enzyme resulted in competition for pyrimidine dimer-DNA glycosylase activity against the UV-irradiated DNA. On the basis of these results, we concluded that apurinic/apyrimidinic endonuclease activity is encoded by the denV gene of phage T4, the same gene that codes for pyrimidine dimer-DNA glycosylase activity.     

A molecular dynamics simulation of SNase and its hydration shell at high temperature and high pressure

Temperature- and pressure-induced unfolding of staphylococcal nuclease (SNase) was studied by Royer, Winter et al. using a variety of experimental techniques (SAXS, FT-IR and fluorescence spectroscopy, DSC, PPC, densimetry). For a more detailed understanding of the underlying mechanistic processes of the different unfolding scenarios, we have carried out a series of molecular dynamics (MD) computer simulations on SNase. We investigated the initial changes of the structure of the protein upon application of pressure (up to 5 kbar) and discuss volumetric and structural differences between the native and pressure pre-denatured state. Additionally, we have obtained the compressibility of the protein and hydration water and compare these data with experimental results. As water plays a crucial role in determining the structure, dynamics and function of proteins, we undertook a detailed analysis of the structure of the interfacial water and the protein-solvent H-bond network as well. Moreover, we report here also MD results on the temperature-induced unfolding of SNase. The time evolution of the protein volume and solvent accessible surface area during thermal unfolding have been investigated, and we present a detailed discussion of the temperature-induced unfolding pathway of SNase in terms of secondary and tertiary structural changes.     

Deletion of the omega-loop in the active site of staphylococcal nuclease. 1. Effect on catalysis and stability

The high-resolution X-ray structure of wild-type staphylococcal nuclease (E43 SNase) suggests that Glu 43 acts a general basic catalyst to assist the attack of water on a phosphodiester substrate [Loll, P., & Lattman, E. E. (1989) Proteins: Struct., Funct., Genet. 5, 183]. Glu 43 is located at the base of the solvent-exposed and conformationally mobile omega-loop in the active site of E43 SNase having the sequence Glu43-Thr44-Lys45-His46-Pro47-Lys48- Lys49-Gly50-Val51-Glu52, where the gamma-carboxylate of Glu 52 is hydrogen bonded to the amide hydrogen of Glu 43. With a metabolic selection for SNase activity produced in an Escherichia coli host, we detected an unexpected deletion of residues 44-49 of the omega-loop of E43 SNase in cassette mutagenesis experiments designed to randomize codons 44 and 45 in the omega-loop and increase the activity of the previously described E43D mutation (D43 SNase). A high-resolution X-ray structure of D43 SNase has revealed that the E43D substitution significantly changes the structure of the omega-loop, reduces the interaction of the essential Ca2+ ion with its active-site ligands, and diminishes the network of hydrogen-bonded water molecules in the active site [Loll, P., & Lattman, E. E. (1990) Biochemistry 29, 6866]. This deletion of six amino acids from the omega-loop generates a protein (E43 delta SNase) having a partially solvent-exposed, surface beta-turn with the sequence Glu43-Gly50-Val51-Glu52; the structure of this beta-turn is addressed in the following article [Baldisseri et al. (1991) Biochemistry (following paper in this issue)].(ABSTRACT TRUNCATED AT 250 WORDS)     

Deletion of the omega-loop in the active site of staphylococcal nuclease. 2. Effects on protein structure and dynamics

It has been shown (Poole et al., 1991) that deletion of residues 44-49 from the sequence of staphylococcal nuclease (E43 SNase) results in an enzyme (E43 delta SNase) that is significantly more active than D43 SNase, an enzyme that differs from the wild-type enzyme by deletion of a single methylene group. In addition, both E43 delta SNase and D43 delta SNase are significantly more stable than their respective parent enzymes. Herein we use high-resolution 2D and 3D NMR spectroscopy to characterize the solution conformations of the four enzymes in order to better understand their differences in stability and activity. The backbone assignments of E43 SNase were extended to the three mutant proteins (uniformly 15N-enriched) by using 2D HSQC, 3D HOHAHA-HMQC, and 3D NOESY-HMQC spectra. The NOE patterns observed for E43 and D43 SNase in solution are consistent with the crystal structures of these proteins. The NOESY data further show that the intact and deleted proteins have essentially the same structures except that (a) the disordered omega-loops in the intact proteins are replaced by tight type II' turns, formed by residues 43-50-51-52, in the deleted proteins and (b) the orientation of the D43 side chain in crystalline D43 SNase differs from that found for D43 delta SNase in solution. Except for regions neighboring the omega-loops, the intact and deleted proteins show nearly identical amide 15N and 1H chemical shifts. In contrast, there are widespread, small and similar, chemical shift differences (a) between E43 SNase and D43 SNase and (b) between E43 delta SNase and D43 delta SNase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Local stability identification and the role of a key aromatic amino acid residue in staphylococcal nuclease refolding

Staphylococcal nuclease (SNase) is a model protein that contains one domain and no disulfide bonds. Its stability in the native state may be maintained mainly by key amino acids. In this study, two point-mutated proteins each with a single base substitution [alanine for tryptophan (W140A) and alanine for lysine (K133A)] and two truncated fragment proteins (positions 1-139 [SNase(1-139) or W140O] and positions 1-141 [SNase(1-141) or E142O]) were generated. Differential scanning microcalorimetry in thermal denaturation experiments showed that K133A and E142O have nearly unchanged DeltaH(cal) relative to the wild-type, whereas W140A and W140O display zero enthalpy change (DeltaH(cal) approximately 0). Far-UV CD measurements indicate secondary structure in W140A but not W140O, and near-UV CD measurements indicate no tertiary structure in either W140 mutant. These observations indicate an unusually large contribution of W140 to the stability and structural integrity of SNase.     

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.     

Mannosylglycerate stabilizes staphylococcal nuclease with restriction of slow β-sheet motions

Mannosylglycerate is a compatible solute typical of thermophilic marine microorganisms that has a remarkable ability to protect proteins from thermal denaturation. This ionic solute appears to be a universal stabilizing agent, but the extent of protection depends on the specific protein examined. To understand how mannosylglycerate confers protection, we have been studying its influence on the internal motions of a hyperstable staphylococcal nuclease (SNase). Previously, we found a correlation between the magnitude of protein stabilization and the restriction of fast backbone motions. We now report the effect of mannosylglycerate on the fast motions of side-chains and on the slower unfolding motions of the protein. Side-chain motions were assessed by (13)CH(3) relaxation measurements and model-free analysis while slower unfolding motions were probed by H/D exchange measurements at increasing concentrations of urea. Side-chain motions were little affected by the presence of different concentrations of mannosylglycerate or even by the presence of urea (0.25M), and show no correlation with changes in the thermodynamic stability of SNase. Native hydrogen exchange experiments showed that, contrary to reports on other stabilizing solutes, mannosylglycerate restricts local motions in addition to the global motions of the protein. The protein unfolding/folding pathway remained undisturbed in the presence of mannosylglycerate but the solute showed a specific effect on the local motions of β-sheet residues. This work reinforces the link between solute-induced stabilization and restriction of protein motions at different timescales, and shows that the solute preferentially affects specific structural elements of SNase.     

Gene- and Protein-Delivered Zinc Finger–Staphylococcal Nuclease Hybrid for Inhibition of DNA Replication of Human Papillomavirus

Previously, we reported that artificial zinc-finger proteins (AZPs) inhibited virus DNA replication in planta and in mammalian cells by blocking binding of a viral replication protein to its replication origin. However, the replication mechanisms of viruses of interest need to be disentangled for the application. To develop more widely applicable methods for antiviral therapy, we explored the feasibility of inhibition of HPV-18 replication as a model system by cleaving its viral genome. To this end, we fused the staphylococcal nuclease cleaving DNA as a monomer to an AZP that binds to the viral genome. The resulting hybrid nuclease (designated AZP–SNase) cleaved its target DNA plasmid efficiently and sequence-specifically in vitro. Then, we confirmed that transfection with a plasmid expressing AZP–SNase inhibited HPV-18 DNA replication in transient replication assays using mammalian cells. Linker-mediated PCR analysis revealed that the AZP–SNase cleaved an HPV-18 ori plasmid around its binding site. Finally, we demonstrated that the protein-delivered AZP–SNase inhibited HPV-18 DNA replication as well and did not show any significant cytotoxicity. Thus, both gene- and protein-delivered hybrid nucleases efficiently inhibited HPV-18 DNA replication, leading to development of a more universal antiviral therapy for human DNA viruses.     

Conformational adjustments of SNase R and its N-terminal fragments probed by monoclonal antibodies

Two monoclonal antibodies specific for staphylococcal nuclease R (SNase R) (McAb2C9 and McAb1B8) were prepared and used to probe protein folding during peptide elongation, by measuring antibody binding to seven N-terminal fragments (SNR141, SNR135, SNR121, SNR110, SNR102, SNR79 and SNR52) of SNase R. Comparative studies of the conformations of the N-terminal fragments have shown that all seven fragments of SNase R have a certain amount of residual structure, indicating that folding may occur during elongation of the nascent peptide chain. We show that the binding abilities of the intact enzyme and its seven fragments to the monoclonal antibodies are not simply proportional to the length of the peptide chain, suggesting that there may be continuous conformational adjustment in the nascent peptide chain as new C-terminal amino acids are added. A folding intermediate close in structure to the native state but with structural features in common with SNR121 is highly populated in 0.6 M GuHCl, and is also formed transiently during folding.