Internal solvation of protein was studied by site-directed mutagenesis, with which an intrinsically fluorescent probe,tryptophan, is inserted into the desired position inside a protein molecule for ultrafast spectrosc...Internal solvation of protein was studied by site-directed mutagenesis, with which an intrinsically fluorescent probe,tryptophan, is inserted into the desired position inside a protein molecule for ultrafast spectroscopic study. Here we review this unique method for protein dynamics research. We first introduce the frontiers of protein solvation, site-directed mutagenesis, protein stability and characteristics, and the spectroscopic methods. Then we present time-resolved spectroscopic dynamics of solvation dynamics inside cavities of active sites. The studies are carried out on a globular protein, staphylococcal nuclease. The solvation at sites inside the protein molecule＇s cavities clearly reveals characteristics of the local environment. These solvation behaviors are directly correlated to enzyme activity.展开更多
Tryptophan （Trp） is an intrinsic fluorescent probe for detecting the site-specified dynamics inside/outside protein. It is found that the Trp can easily be inserted in desired sites of protein, which affects the int...Tryptophan （Trp） is an intrinsic fluorescent probe for detecting the site-specified dynamics inside/outside protein. It is found that the Trp can easily be inserted in desired sites of protein, which affects the integrity of the overall structure. To evaluate this effect, we design thirteen double point mutants of staphylococcal nuclease, each of which has a single Trp residue planted at an internal site. The studies on Trp fluorescence, ANS-binding fluorescence, far- and near-UV CD spectra, and enzymatic activity are carried out. It is found that the mutation at the hydrophobic core of protein generates molten globular state conformation, which is a loose structure compared to their original compactness in wild type （WT）. Its enzyme activity and surface hydrophobicity are also affected. The studies show that by proper site designing and external binding, Trp mutagenesis is a suitable method for carrying out the study on site specified dynamics of proteins.展开更多
The staphylococcal nuclease, encoded by the nucl gene, is an important virulence factor of Staphylococcus aureus. However, the physiological role of the nuclease has not been fully characterized. The current study obs...The staphylococcal nuclease, encoded by the nucl gene, is an important virulence factor of Staphylococcus aureus. However, the physiological role of the nuclease has not been fully characterized. The current study observed that biofilm development could be prevented in staphylococcal nuclease-producing strains of S. aureus; however, when the nucl gene was knocked out, the ability to form a biofilm significantly increased. Scanning electron and confocal scanning laser microscopy were used to evaluate the role of the nucl gene in biofilm formation. Moreover, the nucl gene product, staphylococcal nuclease, and re- combinant NUC1 protein were found to have a visible effect on other biofilm-forming bacteria, such as Pseudomonas aeru- ginosa, Actinobacillus pleuropneurnoniae, and Haernophilus parasuis. The current study showed a direct relationship between staphylococcal nuclease production and the prevention of biofilm development. The findings from this study underscore the important role of staphylococcal nuclease activity to prevent biofilm formation in S. aureus. They also provided evidence for the biological role of staphylococcal nucleases in other organisms.展开更多
STAPHYLOCOCCAL nuclease(SNase A,EC 3.1.4.7),which hydrolyzes the phosphodiesterbond of DNA and RNA,and releases 3’-phosphate mononucleotides and dinucleotides,con-sists of 149 amino acid residues(MW=16 807)without su...STAPHYLOCOCCAL nuclease(SNase A,EC 3.1.4.7),which hydrolyzes the phosphodiesterbond of DNA and RNA,and releases 3’-phosphate mononucleotides and dinucleotides,con-sists of 149 amino acid residues(MW=16 807)without sulfhydryl and disulfide groups.SNase A was originally derived from Staphylococcus aureus.Later the gene of the enzyme wascloned and inserted into several expression systems.Its crystal structure was detected展开更多
The acid-unfolded state(U_A)of staphylococcal nuclease(SNase)can fold into a state(Astate)in acidic solution of high NaCl concentration,that is,U_A→A.The transition curve of U_A→A inducedby the increasing of NaCl co...The acid-unfolded state(U_A)of staphylococcal nuclease(SNase)can fold into a state(Astate)in acidic solution of high NaCl concentration,that is,U_A→A.The transition curve of U_A→A inducedby the increasing of NaCl concentration was superimposed with that induced by the increasing of HClconcentration,indicating that the U_A → A transition is induced by the chloride anion being inclined to bind onsome parts of SNase molecules in A state.Circular dichroism(CD)spectra showed that A state hassubstantial secondary structure.Size exclusion chromatography measurement indicated that A state is compact involume.The A state can bind with 1-anilino-naphthalene-8-sulfonate(ANS),a fluorescent probe forhydrophobicity.These results indicated that A state of SNaseR is a molten globule-like state.Besides,ourresults showed that A state can be unfolded by guanidine hydrochloride(Gu · HCl),implying that thehydrophobic interaction between side chains in A state is responsible to its stabilization.展开更多
基金Project supported by the National Basic Research Program of China(Grant Nos.2013CB921904,2009CB930504,and 2013CB328700)the National Natural Science Foundation of China(Grant Nos.11074016,11121091,10934001,61177020,11134001,and 10828407)
文摘Internal solvation of protein was studied by site-directed mutagenesis, with which an intrinsically fluorescent probe,tryptophan, is inserted into the desired position inside a protein molecule for ultrafast spectroscopic study. Here we review this unique method for protein dynamics research. We first introduce the frontiers of protein solvation, site-directed mutagenesis, protein stability and characteristics, and the spectroscopic methods. Then we present time-resolved spectroscopic dynamics of solvation dynamics inside cavities of active sites. The studies are carried out on a globular protein, staphylococcal nuclease. The solvation at sites inside the protein molecule＇s cavities clearly reveals characteristics of the local environment. These solvation behaviors are directly correlated to enzyme activity.
基金Supported by the National Key Basic Research Program of China under Grant Nos 2013CB921904,2013CB328701-2013CB328706the National Natural Science Foundation of China under Grant Nos 11074016,11121091,61177020,11134001and 10828407
文摘Tryptophan （Trp） is an intrinsic fluorescent probe for detecting the site-specified dynamics inside/outside protein. It is found that the Trp can easily be inserted in desired sites of protein, which affects the integrity of the overall structure. To evaluate this effect, we design thirteen double point mutants of staphylococcal nuclease, each of which has a single Trp residue planted at an internal site. The studies on Trp fluorescence, ANS-binding fluorescence, far- and near-UV CD spectra, and enzymatic activity are carried out. It is found that the mutation at the hydrophobic core of protein generates molten globular state conformation, which is a loose structure compared to their original compactness in wild type （WT）. Its enzyme activity and surface hydrophobicity are also affected. The studies show that by proper site designing and external binding, Trp mutagenesis is a suitable method for carrying out the study on site specified dynamics of proteins.
基金supported by the National Natural Science Foundation of China (Grant Nos. 31071515 and 31070113)the Special Foundation for Young Scientists of Sichuan Province,China (Grant No. 2011JQ0043)the Fundamental Research Funds for the Central Universities,Southwest University for Nationalities (Grant No. 09NZYZJ04)
文摘The staphylococcal nuclease, encoded by the nucl gene, is an important virulence factor of Staphylococcus aureus. However, the physiological role of the nuclease has not been fully characterized. The current study observed that biofilm development could be prevented in staphylococcal nuclease-producing strains of S. aureus; however, when the nucl gene was knocked out, the ability to form a biofilm significantly increased. Scanning electron and confocal scanning laser microscopy were used to evaluate the role of the nucl gene in biofilm formation. Moreover, the nucl gene product, staphylococcal nuclease, and re- combinant NUC1 protein were found to have a visible effect on other biofilm-forming bacteria, such as Pseudomonas aeru- ginosa, Actinobacillus pleuropneurnoniae, and Haernophilus parasuis. The current study showed a direct relationship between staphylococcal nuclease production and the prevention of biofilm development. The findings from this study underscore the important role of staphylococcal nuclease activity to prevent biofilm formation in S. aureus. They also provided evidence for the biological role of staphylococcal nucleases in other organisms.
文摘STAPHYLOCOCCAL nuclease(SNase A,EC 3.1.4.7),which hydrolyzes the phosphodiesterbond of DNA and RNA,and releases 3’-phosphate mononucleotides and dinucleotides,con-sists of 149 amino acid residues(MW=16 807)without sulfhydryl and disulfide groups.SNase A was originally derived from Staphylococcus aureus.Later the gene of the enzyme wascloned and inserted into several expression systems.Its crystal structure was detected
文摘The acid-unfolded state(U_A)of staphylococcal nuclease(SNase)can fold into a state(Astate)in acidic solution of high NaCl concentration,that is,U_A→A.The transition curve of U_A→A inducedby the increasing of NaCl concentration was superimposed with that induced by the increasing of HClconcentration,indicating that the U_A → A transition is induced by the chloride anion being inclined to bind onsome parts of SNase molecules in A state.Circular dichroism(CD)spectra showed that A state hassubstantial secondary structure.Size exclusion chromatography measurement indicated that A state is compact involume.The A state can bind with 1-anilino-naphthalene-8-sulfonate(ANS),a fluorescent probe forhydrophobicity.These results indicated that A state of SNaseR is a molten globule-like state.Besides,ourresults showed that A state can be unfolded by guanidine hydrochloride(Gu · HCl),implying that thehydrophobic interaction between side chains in A state is responsible to its stabilization.
