Direct measurement of oligonucleotide binding stoichiometry of gene V protein by mass spectrometry.

The binding stoichiometry of gene V protein from bacteriophage f1 to several oligonucleotides was studied using electrospray ionization-mass spectrometry (ESI-MS). Using mild mass spectrometer interface conditions that preserve noncovalent associations in solution, gene V protein was observed as dimer ions from a 10 mM NH4OAc solution. Addition of oligonucleotides resulted in formation of protein-oligonucleotide complexes with stoichiometry of approximately four nucleotides (nt) per protein monomer. A 16-mer oligonucleotide gave predominantly a 4:1 (protein monomer: oligonucleotide) complex while oligonucleotides shorter than 15 nt showed stoichiometries of 2:1. Stoichiometries and relative binding constants for a mixture of oligonucleotides were readily measured using mass spectrometry. The binding stoichiometry of the protein with the 16-mer oligonucleotide was measured independently using size-exclusion chromatography and the results were consistent with the mass spectrometric data. These results demonstrate, for the first time, the observation and stoichiometric measurement of protein-oligonucleotide complexes using ESI-MS. The sensitivity and high resolution of ESI-MS should make it a useful too] in the study of protein-DNA interactions.     

Genetic fusion of subunits of a dimeric protein substantially enhances its stability and rate of folding.

The gene V protein of bacteriophage f1 is a single-stranded DNA and RNA-binding protein composed of two identical subunits. We have constructed single-chain variants of the protein using short peptide linkers of five or six amino acids to connect the carboxyl terminus of one monomer to the amino terminus of the second monomer. The resulting subunit-fusion gene V proteins were found to bind single-stranded DNA nearly as tightly as the wild-type protein. Denaturation measurements show that the subunit-fusion gene V proteins are 5 kcal/mol (1 kcal = 4.18 kJ) more stable than the wild-type protein at a protein concentration of 10 microM. The rate of unfolding of the protein is essentially unaffected by the fusion of monomeric subunits, whereas the rate of folding is greatly enhanced. Our results suggest a simple way of obtaining a substantial thermodynamic stabilization for some oligomeric proteins.     

Combined-information protein structure refinement: Potential energy-constrained real-space method for refinement with limited diffraction data

A potential energy-constrained real-space refinement method designed for use with x-ray diffraction data of low to moderate resolution has been developed. The number of adjustable parameters is severely restricted to ensure a reasonable ratio of data to parameters. Only dihedral angles are allowed to vary; bond lengths and bond angles are fixed at physically reasonable values. The structure of bovine pancreatic trypsin inhibitor was refined by using this method with data to only 2.5-Å resolution. Both the R-factor and the electron-density map improved throughout the refinement, and the final structure was a satisfactory approximation to the 1.5-Å structure.     

Val-->Ala mutations selectively alter helix-helix packing in the transmembrane segment of phage M13 coat protein.

Val-->Ala mutations within the effective transmembrane segment of a model single-spanning membrane protein, the 50-residue major coat (gene VIII) protein of bacteriophage M13, are shown to have sequence-dependent impacts on stabilization of membrane-embedded helical dimeric structures. Randomized mutagenesis performed on the coat protein hydrophobic segment 21-39 (YIGYAWAMV-VVIVGATIGI) produced a library of viable mutants which included those in which each of the four valine residues was replaced by an alanine residue. Significant variations found among these Val-->Ala mutants in the relative populations and thermal stabilities of monomeric and dimeric helical species observed on SDS/PAGE, and in the range of their alpha-helix-->beta-sheet transition temperatures confirmed that intramembranous valine residues are not simply universal contributors to membrane anchoring. Additional analyses of (i) nonmutatable sites in the mutant protein library, (ii) the properties of the double mutant V29A-V31A obtained by recycling mutant V31A DNA through mutagenesis procedures, and (iii) energy-minimized helical dimer structures of wild-type and mutant V31A transmembrane regions indicated that the transmembrane hydrophobic core helix of the M13 coat protein can be partitioned into alternating pairs of potential protein-interactive residues (V30, V31; G34, A35; G38, I39) and membrane-interactive residues (M28, V29; I32, V33; T36, I37). The overall results consitute an experimental approach to categorizing the distinctive contributions to structure of the residues comprising a protein-protein packing interface vs. those facing lipid and confirm the sequence-dependent capacity of specific residues within the transmembrane domain to modulate protein-protein interactions which underlie regulatory events in membrane proteins.     

Structure and function of disease-causing missense mutations in the PHEX gene

The PHEX gene that is mutated in patients with X-linked hypophosphatemia (XLH) encodes a protein homologous to the M13 family of zinc metallopeptidases. The present study was undertaken to assess the impact of nine PHEX missense mutations on cellular trafficking, endopeptidase activity, and protein conformation. Secreted forms of wild-type and mutant PHEX proteins were generated by PCR mutagenesis; these included C85R, D237G, Y317F, G579R, G579V, S711R, A720T, and F731Y identified in XLH patients, and E581V, which in neutral endopeptidase 24.11 abolishes catalytic activity but not plasma membrane localization. The wild-type and D237G, Y317F, E581V, and F731Y proteins were terminally glycosylated and secreted into the medium, whereas the C85R, G579R, G579V, S711R, and A720T proteins were trapped inside the transfected cells. Growing the cells at 26 C permitted the secretion of G579V, S711R, and A720T proteins, although the yield of rescued G579V was insufficient for further analysis. Endopeptidase activity of secreted and rescued PHEX proteins, assessed using a novel internally quenched fluorogenic peptide substrate, revealed that E581V and S711R are completely inactive; D237G and Y317F exhibit 50-60% of wild-type activity; and A720T and F731Y retain full catalytic activity. Conformational analysis by limited proteolysis demonstrated that F731Y is more sensitive to trypsin and D237G is more resistant to endoproteinase Glu-c than the wild-type protein. Thus, defects in protein trafficking, endopeptidase activity, and protein conformation account for loss of PHEX function in XLH patients harboring these missense mutations.     

Native-like in vivo folding of a circularly permuted jellyroll protein shown by crystal structure analysis.

A jellyroll beta-sandwich protein, the Bacillus beta-glucanase H(A16-M), is used to probe the role of N-terminal peptide regions in protein folding in vivo. A gene encoding H(A16-M) is rearranged to place residues 1-58 of the protein behind a signal peptide and residues 59-214. The rearranged gene is expressed in Escherichia coli. The resultant circularly permuted protein, cpA16M-59, is secreted into the periplasm, correctly processed, and folded into a stable and active enzyme. Crystal structure analysis at 2.0-A resolution, R = 15.3%, shows cpA16M-59 to have a three-dimensional structure nearly identical with that of the parent beta-glucanase. An analogous experiment based on the wild-type Bacillus macerans beta-glucanase, giving rise to the circularly permuted variant cpMAC-57, yields the same results. Folding of these proteins, therefore, is not a vectorial process depending on the conformation adopted by their native N-terminal oligopeptides after ribosomal synthesis and translocation through the cytoplasmic membrane.     

Accurate protein crystallography at ultra-high resolution: valence electron distribution in crambin

The charge density distribution of a protein has been refined experimentally. Diffraction data for a crambin crystal were measured to ultra-high resolution (0.54 A) at low temperature by using short-wavelength synchrotron radiation. The crystal structure was refined with a model for charged, nonspherical, multipolar atoms to accurately describe the molecular electron density distribution. The refined parameters agree within 25% with our transferable electron density library derived from accurate single crystal diffraction analyses of several amino acids and small peptides. The resulting electron density maps of redistributed valence electrons (deformation maps) compare quantitatively well with a high-level quantum mechanical calculation performed on a monopeptide. This study provides validation for experimentally derived parameters and a window into charge density analysis of biological macromolecules.     

副溶血性弧菌Vop T蛋白的表达及其抗体的毒力菌株特异性研究

目的 研究副溶血性弧菌VopT蛋白的菌群特异性,为建立致病性副溶血性弧菌的快速检测方法以及探讨VopT的作用机制奠定基础。方法 根据副溶血性弧菌VopT蛋白基因(VPA1327)序列设计合成引物,通过PCR从副溶血性弧菌致病株WX06152(血清型O3∶K6)中扩增出目的基因,构建原核表达载体pET-30a(+)-VPA1327并转化大肠杆菌BL21(DE3)。经IPTG诱导表达,重组蛋白应用MALDI-TOF-MS/MS鉴定和His镍柱纯化,并通过免疫BALB/c小鼠制备抗血清,建立副溶血性弧菌VopT蛋白的间接ELISA检测方法,并分析其敏感性和毒力菌株特异性。结果 成功表达了大小为33 kDa融合蛋白,肽指纹图谱与副溶血性弧菌VopT蛋白一致。重组VopT抗血清能够特异性检测副溶血性弧菌毒力株的全菌细胞及其裂解蛋白,与环境株及其它种属菌株无明显交叉反应,灵敏度达到10⁵CFU·mL^-1。结论 重组VopT的抗血清具有良好特异性,为探讨VopT致病机理和分泌机制研究提供了一定的参考依据。     

鸟分枝杆菌MAV2928基因编码蛋白的生物信息学分析

目的运用生物信息学方法分析鸟分枝杆菌PPE25-MAV蛋白的结构与功能。方法从NCBI数据库获取MAV2928基因编码蛋白氨基酸序列;使用protParam和PortScale工具分析该基因编码PPE25-MAV蛋白的理化性质以及亲疏水性;分别运用SignaIP 4.1 Server、TMHMM Server v.2.0和PSORT Prediction工具预测PPE25-MAV蛋白的信号肽、跨膜区、亚细胞定位;采用NetPhos 3.1 Servera预测磷酸化位点,采用NCBI-Conserved domains分析蛋白的保守域结构;采用SPOMA软件在线分析PPE25MAV蛋白二级结构,使用SWISS-MODEL建立三级结构模型。运用ABCpred软件和IEDB预测蛋白的B细胞抗原表位。结果MAV2928基因全长为1266 bp,编码蛋白PPE25-MAV含有421个氨基酸,为不稳定疏水蛋白,脂肪系数为72.66,无信号肽及跨膜区,定位于细胞质中。该蛋白含有52个磷酸位点,有1个保守域结构属于PPE超家族蛋白;二级结构以无规则卷曲为主,结构较松散。预测该蛋白含有7个B细胞优势抗原表位和24个Th细胞表位。结论PPE25-MAV蛋白含有多个磷酸化位点,参与细胞信号的转导,含有7个优势B细胞抗原表位,为该蛋白作为候选疫苗抗原提供了理论基础。     

The 3 A resolution structure of a D-galactose-binding protein for transport and chemotaxis in Escherichia coli.

X-ray diffraction studies of a D-galactose-binding protein essential for transport and chemotaxis in Escherichia coli have yielded a model of the polypeptide chain backbone. An initial polyalanine backbone trace was obtained at 3.2 A resolution by the molecular replacement technique, using a polyalanine search model derived from the refined structure of the L-arabinose-binding protein. Concurrently, a 3 A resolution electron-density map of the D-galactose receptor was determined from multiple isomorphous replacement (MIR) phases. The properly transformed initial polyalanine model superimposed on the MIR electron-density map proved to be an excellent guide in obtaining a final trace. The few changes made in the polyalanine model to improve the fit to the density were confined primarily to the COOH-terminal peptide and some loops connecting the elements of the secondary structure. Despite the lack of significant sequence homology, the overall course of the polypeptide backbone of the D-galactose-binding protein is remarkably similar to that of the L-arabinose-binding protein, the first structure in a series to be solved from this family of binding proteins. Both structures are elongated (axial ratios of 2:1) and composed of two globular domains. For both proteins, the arrangements of the elements of the secondary structure in both domains are identical; both lobes contain a core of beta-pleated sheet with a pair of helices on either side of the plane of the sheet. The four major hydrophobic clusters that stabilize the structure of the L-arabinose-binding protein are also present in the D-galactose-binding protein.     

Structure and activity of the axon guidance protein MICAL

During development, neurons are guided to their targets by short- and long-range attractive and repulsive cues. MICAL, a large multidomain protein, is required for the combined action of semaphorins and plexins in axon guidance. Here, we present the structure of the N-terminal region of MICAL (MICAL(fd)) determined by x-ray diffraction to 2.0 A resolution. The structure shows that MICAL(fd) is an FAD-containing module structurally similar to aromatic hydroxylases and amine oxidases. In addition, we present biochemical data that show that MICAL(fd) is a flavoenzyme that in the presence of NADPH reduces molecular oxygen to H(2)O(2) (K(m,NAPDH) = 222 microM; k(cat) = 77 sec(-1)), a molecule with known signaling properties. We propose that the H(2)O(2) produced by this reaction may be one of the signaling molecules involved in axon guidance by MICAL.     

A joint x-ray and neutron study on amicyanin reveals the role of protein dynamics in electron transfer

The joint x-ray/neutron diffraction model of the Type I copper protein, amicyanin from Paracoccus denitrificans was determined at 1.8 Å resolution. The protein was crystallized using reagents prepared in D₂O. About 86% of the amide hydrogen atoms are either partially or fully exchanged, which correlates well with the atomic depth of the amide nitrogen atom and the secondary structure type, but with notable exceptions. Each of the four residues that provide copper ligands is partially deuterated. The model reveals the dynamic nature of the protein, especially around the copper-binding site. A detailed analysis of the presence of deuterated water molecules near the exchange sites indicates that amide hydrogen exchange is primarily due to the flexibility of the protein. Analysis of the electron transfer path through the protein shows that residues in that region are highly dynamic, as judged by hydrogen/deuterium exchange. This could increase the rate of electron transfer by transiently shortening through-space jumps in pathways or by increasing the atomic packing density. Analysis of C-H⋯X bonding reveals previously undefined roles of these relatively weak H bonds, which, when present in sufficient number can collectively influence the structure, redox, and electron transfer properties of amicyanin.     

Applications of synchrotron radiation to protein crystallography: preliminary results.

X-ray diffraction photographs of protein single crystals have been obtained using synchrotron radiation produced by an electron-positron storage ring. The diffracted intensities observed with this unconventional source are a factor of at least 60 greater than those obtained with a sealed x-ray tube using the same crystal and instrumental parameters. Diffraction data have been collected by the precession method to higher resolution and using smaller protein crystals than would have been possible with a conventional source. The crystal decay rate in the synchrotron beam for several proteins appears to be substantially less than that observed with Ni-filtered Cu radiation. The tunable nature of the source (which allows selective optimization of anomalous contributions to the scattering factors) and the low angular divergence of the beam make the source very useful for single crystal protein diffraction studies.     

Purification of the cdc8 protein of Saccharomyces cerevisiae by complementation in an aphidicolin-sensitive in vitro DNA replication system.

DNA synthesis in vitro in Brij-treated Saccharomyces cerevisiae requires the product of the CDC8 gene (Hereford, L. M. & Hartwell, L. H. (1971) Nature (London) New Biol. 234, 171-172). Extracts of wild-type A364a yeast restore DNA synthesis in Brij-treated cdc8, a mutant containing a thermolabile cdc8 gene product. This constitutes a complementation assay by which the cdc8 gene product can be monitored during purification. A heat-stable protein responsible for this complementation has been partially purified from both wild-type A364a cells and from a cdc8 temperature-sensitive mutant. The complementation activity from the mutant is thermolabile when compared to the wild-type activity, indicating that CDC8 is the structural gene for the protein.     

YgbQ, a cell division protein in Escherichia coli and Vibrio cholerae, localizes in codependent fashion with FtsL to the division site

YgbQ is a cell division protein in Escherichia coli and Vibrio cholerae. In E. coli the ygbQ gene was discovered as a result of a computer search of the E. coli genome designed to find potential interacting partners for cell division protein FtsL. In V. cholerae, ygbQ was identified as an essential gene by using a transposon that fuses genes to an arabinose promoter. The role of YgbQ in cell division is supported by the following. Cells depleted of YgbQ in both organisms form long filaments, but DNA segregation is not affected. YgbQ localizes to the constriction site in wild-type E. coli cells. Localization of E. coli YgbQ to the constriction site depends on cell division proteins FtsQ and FtsL but not FtsW and FtsI, placing YgbQ in the sequential dependency order of proteins localizing to the division site. Localization of green fluorescent protein-FtsL also depends on YgbQ, indicating that FtsL and YgbQ colocalize to the division site in E. coli. Our results show colocalization of proteins to the bacterial midcell in E. coli and raise the possibility that these proteins interact in a coiled-coil structure.     

Asp361Val Mutant of alkaline phosphatase found in patients with dominantly inherited hypophosphatasia inhibits the activity of the wild-type enzyme

Hypophosphatasia is characterized by the hypomineralization of bone associated with the mutation of the tissue-nonspecific alkaline phosphatase (TNSALP) gene. Although the disease is usually autosomal recessive, an autosomal dominant form is also recognized. Approximately 50 mutations have been found in the TNSALP gene in patients with hypophosphatasia. However, the mutations identified to date do not seem to account for the dominantly inherited form of the disease. We have examined a German family in which the father and all 4 children were affected with hypophosphatasia, whereas the mother was healthy. The affected members of this family showed premature loss of deciduous teeth at or shortly before 2 yr of age and low levels of serum ALP with elevated levels of urinary phosphoethanolamine. DNA analysis by direct sequencing revealed a heterozygous missense mutation that caused the conversion of amino acid Asp to Val at position 361 (D361V) in the patients. Another substitution was detected in exon 12 (Val to Ala conversion at codon 505: V505A) in 1 allele of the mother and 3 children, indicating no association of the substitution with the disease. Reconstruction experiments demonstrated that the D361V mutant protein lost its enzymatic activity and that it inhibited the function of wild-type enzyme when coexpressed in COS-7 cells. On the other hand, the V505A mutant exhibited enzymatic activities equal to those of the wild-type ALP. It is likely that the mutant D361V protein forms dimers with the wild-type protein, and the protein-protein interaction contributes to the dominant effect of the mutant D361V. The mutation that causes D361V is the first one proven to be associated with the dominant form of hypophosphatasia.     

Codon insertion mutants of the adenovirus terminal protein.

A series of codon insertion mutants was isolated following restriction site-directed linker insertion mutagenesis of the open reading frame for the type 5 adenovirus terminal protein precursor. The conditionally lethal mutant H5sub100 bears an insertion mutation upstream of the first AUG in the reading frame, fails to replicate its DNA under nonpermissive conditions, and was assigned to the terminal protein complementation group. These data establish that terminal protein is an essential polypeptide required for DNA replication in vivo and indicate that the NH2-terminal region of the precursor is encoded in an upstream mRNA leader. The extended eclipse period of the viral replication cycle in H5in179-infected cells is probably a consequence of delayed onset of DNA replication. Analysis of DNA replication in coinfections with wild-type virus shows that the in179 mutation has cis and trans effects. The trans-dominant, negative-complementing in179 terminal protein precursor inhibits wild-type DNA replication in a dose-dependent manner. Replication of parental in179 templates is not stimulated by an excess of coinfecting wild-type virus, indicating that the mutant terminal protein covalently bound to the in179 template in some way interferes with the replication of that template. The implications of these results for the structure and function of the terminal protein are discussed.     

Energetics of repacking a protein interior.

To test whether interactions in the hydrophobic core of a protein can be adequately modeled based on the properties of a liquid hydrocarbon, we measured the unfolding free energies of the wild-type bacteriophage f1 gene V protein and 29 mutants with apolar substitutions at positions 35 and 47. Stability changes arising from identical mutations at these two buried sites are quite different, suggesting that one site is more rigid than the other. Reversals of residues at positions 35 and 47 confirm that their environments are distinct. Mutants containing weakly polar residues at these two sites suggest that the protein interior is more polar than a liquid hydrocarbon. Interactions between residues at the two sites appear to be minimal. These observations are compatible with a view of protein interiors that incorporates properties of liquid hydrocarbons but also includes polar interactions and a site-dependent "packing energy" associated with changes in internal structure.