Capturing protein dynamics with time-resolved luminescence spectroscopy

The presented doctoral research utilizes time-resolved spectroscopy to characterize protein dynamics and folding mechanisms. We resolve millisecond-timescale folding by coupling time-resolved fluorescence energy transfer (trFRET) to a continuous flow microfluidic mixer to obtain intramolecular distance distributions throughout the folding process. We have elucidated the folding mechanisms of two cytochromes---one that exhibits two-state folding (cytochrome cb₅₆₂) and one that has both a kinetic refolding intermediate ensemble and a distinct equilibrium unfolding intermediate (cytochrome c₅₅₂). Our data reveal that the distinct structural features of cytochrome c₅₅₂ contribute to its thermostability.

We have also investigated intrachain contact dynamics in unfolded cytochrome cb₅₆₂ by monitoring electron transfer, which occurs as the heme collides with a ruthenium photosensitizer, covalently bound to residues along the polypeptide. Intrachain diffusion for chemically denatured proteins proceeds on the microsecond timescale with an upper limit of 0.1 microseconds. The power-law dependence (slope = -1.5) of the rate constants on the number of peptide bonds between the heme and Ru complex indicate that cytochrome cb₅₆₂ is minimally frustrated.

In addition, we have explored the pathway dependence of electron tunneling rates between metal sites in proteins. Our research group has converted cytochrome b₅₆₂ to a c-type cytochrome with the porphyrin covalently bound to cysteine sidechains. We have investigated the effects of the changes to the protein structure (i.e., increased rigidity and potential new equatorial tunneling pathways) on the electron transfer rates, measured by transient absorption, in a series of ruthenium photosensitizer-modified proteins.

Fish species identification in Canned Tuna by DNA Analysis (PCR-SSCP)

DNA in canned tuna is degraded into short fragments of a rew hundred base pairs. The polymerase chain reaction (PCR) was used to amplify short sequences of mitochondrial DNA, which were denatured and analysed by polyacrylamide gel electrophoresis (native PAGE) for detection of single strand conformation polymorphisms. Species specific patterns of DNA bands were obtained for a number of tuna and bonito species. DE: In Thunfischkonserven liegt die DNA in Form kurzkettiger Fragmente von wenigen Hundert Basenpaaren Länge vor. Mit Hilfe der Polymerase-Kettenreaktion (PCR) wurden kurze Sequenzen der mitochondrialen DNA vervielfältigt. Anschließend wurde die gebildete DNA in Einzelsträngen überführt, die durch eine native Polyacrylamidgel-Elektrophorese (PAGE) aufgetrennt wurde. Für eine Reihe von Thunfischen und Boniten ergaben die Einzelstränge artspezifische Bandenmuster, die auf unterschiedliche Konformationen der DNA-Stränge der einzelnen Fischarten zurückzuführen sind.

The structure specificity of alpha-chymotrypsin. I. Polypeptides as substrates. II. N-acylated peptide esters as substrates. III. Some reactive esters of N-acylated amino acids as substrates

The structural specificity of α-chymotrypsin for polypeptides and denatured proteins has been examined. The primary specificity of the enzyme for these natural substrates is shown to closely correspond to that observed for model substrates. A pattern of secondary specificity is proposed.

A series of N-acetylated peptide esters of varying length have been evaluated as substrates of α-chymotrypsin. The results are interpreted in terms of proposed specificity theories.

The α-chymotrypsin-catalyzed hydrolyses of a number of N-acetylated dipeptide methyl esters were studied. The results are interpreted in terms of the available specificity theories and are compared with results obtained in the study of polypeptide substrates. The importance of non-productive binding in determining the kinetic parameters of these substrates is discussed. A partial model of the locus of the active site which interacts with the R’₁CONH- group of a substrate of the form R’₁CONHCHR₂COR’₃ is proposed.

Finally, some reactive esters of N-acetylated amino acids have been evaluated as substrates of α-chymotrypsin. Their reactivity and stereo-chemical behavior are discussed in terms of the specificity theories available. The importance of a binding interaction between the carboxyl function of the substrate and the enzyme is suggested by the results obtained.

The interactions of basic proteins and DNA

I. ELECTROPHORESIS OF THE NUCLEIC ACIDS

A zone electrophoresis apparatus using ultraviolet optics has been constructed to study nucleic acids at concentrations less than 0.004%. Native DNA has a mobility about 15% higher than denatured DNA over a range of conditions. Otherwise, the electrophoretic mobility is independent of molecular weight, base composition or source. DNA mobilities change in the expected way with pH but the fractional change in mobility is less than the calculated change in charge. A small decrease in mobility accompanies an increase in ionic strength. RNA’s from various sources have mobilities slightly lower than denatured DNA except for s-RNA which travels slightly faster. The important considerations governing the mobility of nucleic acids appear to be the nature of the hydrodynamic segment, and the binding of counterions. The differences between electrophoresis and sedimentation stem from the fact that all random coil polyelectrolytes are fundamentally free draining in electrophoresis.

II. THE CYTOCHROME C/DNA COMPLEX

The basic protein, cytochrome c, has been complexed to DNA. Up to a cytochrome:DNA mass ratio of 2, a single type of complex is formed. Dissociation of this complex occurs between 0.05F and 0.1F NaCl. The complexing of cytochrome to DNA causes a slight increase in the melting temperature of the DNA, and a reduction of the electrophoretic mobility proportional to the decrease in net charge. Above a cytochrome:DNA mass ratio of 2.5, a different type of complex is formed. The results suggest that complexes such as are formed in the Kleinschmidt technique of electron microscopy would not exist in bulk solution and are exclusively film phenomena.

III. STUDIES OF THE ELECTROPHORESIS AND MELTING BEHAVIOUR OF NUCLEOHISTONES

Electrophoresis studies on reconstituted nucleohistones indicate that the electrophoretic mobility for these complexes is a function of the net charge of the complex. The mobility is therefore dependent on the charge density of the histone complexing the DNA, as well as on the histone/DNA ratio. It is found that the different histones affect the transition from native to denatured DNA in different ways. It appears that histone I is exchanging quite rapidly between DNA molecules in 0.01 F salt, while histone II is irreversibly bound. Histone III-IV enhances the capacity of non-strand separated denatured DNA to reanneal. Studies on native nucleoproteins indicate that there are no gene-sized uncomplexed DNA regions in any preparations studied.

IV. THE DISSOCIATION OF HISTONE FROM CALF THYMUS CROMATIN

Calf thymus nucleoprotein was treated with varying concentrations of NaCl. The identity of the histones associated and dissociated from the DNA at each salt concentration was determined by gel electrophoresis. It was found that there is no appreciable histone dissociation below 0.4 F NaCl. The lysine rich histones dissociate between 0.4 and 0.5 F NaCl. Their dissociation is accompanies by a marked increase in the solubility of the chromatin. The moderately lysine rich histones dissociate mainly between 0.8 and 1.1 F NaCl. There are two arginine rich histone components: the first dissociates between 0.8 F and 1.1 F NaCl, but the second class is the very last to be dissociated from the DNA (dissociation beginning at 1.0 F NaCl). By 2.0 F NaCl, essentially all the histones are dissociated.

The properties of the extracted nucleoprotein were studied. The electrophoretic mobility increases and the melting temperature decreases as more histones are dissociated from the DNA. A comparison with the dissociation of histones from DNA in NaClO₄ shows that to dissociate the same class of histones, the concentration of NaCl required is twice that of NaClO₄.

Isolation and characteristics of a bacteriophage for bacillus stearothermophilus

A bacteriophage (TØ3) which infects the thermophilic bacterium Bacillus stearothermophilus ATCC 8005 was isolated and characterized. Infection of the bacterium by the bacteriophage was carried out at 60°C, the optimum growth temperature of the host. At 60°C the phage has a latent period of 18 minutes and a burst size of about 200. The phage is comparatively thermostable in broth. The half life of the phage is 400 minutes at 60°C, 120 minutes at 65°C, 40 minutes at 70°C and 12 minutes at 75°C. The activation energy for the heat inactivation of TØ3 is 56,000 cal. The buoyant density of TØ3 in a cesium chloride density gradient is 1.526.

Electron micrographs of TØ3 indicate that the phage has a regular hexagonal shaped head 57 mμ long. The morphology of the head is compatible with icosahedral symmetry. Each edge of the head is 29 mμ long, and there are 6 or 7 subunits along each edge. The tail of TØ3 is 125 mμ long and 10 mμ wide. There are about 30 cross striations that are spaced at 3.9 mμ intervals along the tail.

The DNA of phage TØ3 has a melting temperature of 88.5°C. Heat denatured TØ3 DNA can be extensively annealed in a high ionic strength environment. The buoyant density of TØ3 DNA in a cesium chloride density gradient is 1.695. TØ3 DNA contains: 42.7% guanine plus cytosine, as determined from the melting temperature; 43% guanine plus cytosine, as determined from the buoyant density; and 40.2% guanine plus cytosine, as determined by chromatographic separation and spectrophotometric estimation of the bases. The molecular weight of TØ3 DNA is 16.7 X 10⁶ as determined from the band width of the TØ3 DNA concentration distribution in a cesium chloride density gradient. Electron microscopy of TØ3 DNA revealed a single linear molecule that is 11.7 μ long. This corresponds to a molecular weight of 22.5 X 10⁶.

Heat denatured TØ3 DNA forms two bands in a cesium chloride density gradient, one at a density of 1.707 and the other at a density of 1.715. After the separated bands are mixed and annealed in the centrifuge cell, the renatured TØ3 DNA forms a single band at a density of 1.699. These results indicate that the two complementary strands of TØ3 DNA have different buoyant densities in cesium chloride, presumably because they have different base compositions.

The characteristics of TØ3 are compared with those of other phages. A hypothesis is presented for a relationship between the base composition of one strand of TØ3 DNA and the amino acid composition of the proteins of TØ3.

Observations on changes in the major protein nitrogen fraction of prawns and sardines during ice storage

Changes in the major protein nitrogen fractions (sarcoplasmic, myofibrillar, stroma) have been studied in two species of prawns and in oil sardine held in ice storage. Myofibrillar proteins were observed to get denatured at a rapid rate as determined by salt extractability method. The sarcoplasmic proteins were not denatured to any considerable extent. With sardine however, the extraction of myofibrillar proteins was inhibited rather in the uniced condition itself presumably owing to the presence of free fatty acids.

PCR-DGGE Fingerprinting Analysis of Plankton Communities and Its Relationship to Lake Trophic Status

Plankton communities in eight lakes of different trophic status near Yangtze, China were characterized by using denatured gradient gel electrophoresis (DGGE). Various water quality parameters were also measured at each collection site. Following extraction of DNA from plankton communities, 16S rRNA and 18S rRNA genes were amplified with specific primers for prokaryotes and eukaryotes, respectively; DNA profiles were developed by DGGE. The plankton community of each lake had its own distinct DNA profile. The total number of bands identified at 34 sampling stations ranged from 37 to 111. Both prokaryotes and eukaryotes displayed complex fingerprints composed of a large number of bands: 16 to 59 bands were obtained with the prokaryotic primer set; 21 to 52 bands for the eukaryotic primer set. The DGGE-patterns were analyzed in relation to water quality parameters by canonical correspondence analysis (CCA). Temperature, pH, alkalinity, and the concentration of COD, TP and TN were strongly correlated with the DGGE patterns. The parameters that demonstrated a strong correlation to the DGGE fingerprints of the plankton community differed among lakes, suggesting that differences in the DGGE fingerprints were due mainly to lake trophic status. Results of the present study suggest that PCR-DGGE fingerprinting is an effective and precise method of identifying changes to plankton community composition, and therefore could be a useful ecological tool for monitoring the response of aquatic ecosystems to environmental perturbations.

Isolation and identification of a novel WSSV nucleocapsid protein by cDNA phage display using an scFv antibody

In a previous study, a scFv phage display library against white spot syndrome virus (WSSV) was constructed and yielded a clone designated A I with conformational specificity against native but not denatured viral antigen. Although the clone A1 has been used successfully as a diagnostic antibody, its precise target antigen has not been elucidated. A different strategy was adopted involving the construction of a second T7 phage display library utilizing mRNA isolated from shrimp infected with WSSV. Following RT-PCR and T7 phage library construction, phages displaying the candidate epitope were selected with A I scFv. Since successive enrichment steps were not associated with an increased titer of the phages, enrichment after successive tests was confirmed by PCR resulting in the prefer-red selection of a specific DNA sequence encoding a novel nucleocapsid protein WSSV388. Immune electron microscopy revealed that WSSV388 is located on the nucleocapsid. This result demonstrated that unknown antigen could be identified by phage display using the epitope conformation dependent scFv. (c) 2006 Elsevier B.V. All rights reserved.

Real-Time Study of Genomic DNA Structural Changes upon Interaction with Small Molecules Using Dual-Polarization Interferometry

The structural changes of genomic DNA upon interaction with small molecules have been studied in real time using dual-polarization interferometry (DPI). Native or thermally denatured DNA was immobilized on the silicon oxynitride surface via a preadsorbed poly(ethylenimine) (PEI) layer. The mass loading was similar for both types of DNA, however, native DNA formed a looser and thicker layer due to its rigidity, unlike the more flexible denatured DNA, which mixed with PEI to form a denser and thinner layer. Ethidium bromide (EtBr), a classical intercalator, induced the large thickness decrease and density increase of native DNA (double-stranded), but a slight increase in both the thickness and density of denatured DNA (partial single-stranded).

Label free electrochemiluminescence protocol for sensitive DNA detection with a tris(2,2'-bipyridyl)ruthenium(II) modified electrode based on nucleic acid oxidation

Label free electrochemiluminescence (ECL) DNA detection based on catalytic guanine and adenine bases oxidation using tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)(3)(2+)] modified glassy carbon (GC) electrode was demonstrated in this work. The modified GC electrode was prepared by casting carbon nanotubes (CNT)/Nafion/Ru(bpy)(3)(2+) composite film on the electrode surface. ECL signals of doublestranded DNA and their thermally denatured counterparts can be distinctly discriminated using cyclic voltammetry (CV) with a low concentration (3.04 x 10(-8) mol/L for Salmon Testes-DNA). Most importantly, sensitive single-base mismatch detection of p53 gene sequence segment was realized with 3.93 x 10(-10) mol/L employing CV stimulation (ECL signal of C/A mismatched DNA oligonucleotides was 1.5-fold higher than that of fully base-paired DNA oligonucleotides). Label free, high sensitivity and simplicity for single-base mismatch discrimination were the main advantages of the present ECL technique for DNA detection over the traditional DNA sensors.

Electrochemiluminescence-based DNA detection using guanine oxidation at electrostatic self-assembly of Ru(bpy)(3)(2+)-doped silica nanoparticles on indium tin oxide electrode

A Ru(bpy)(3)(2+)-doped silica nanoparticle-[Ru@Silica] modified indium tin oxide electrode was prepared by simple electrostatic self-assembly technique, and one-electron catalytic oxidation of guanine bases in double-strand and denatured DNA was realized using the electrochemiluminescence detection means.

Co-electrospun poly(lactide-co-glycolide), gelatin, and elastin blends for tissue engineering scaffolds

In this study, we describe composite scaffolds composed of synthetic and natural materials with physicochemical properties suitable for tissue engineering applications. Fibrous scaffolds were co-electrospun from a blend of a synthetic biodegradable polymer (poly(lactic-co-glycolic acid), PLGA, 10% solution) and two natural proteins, gelatin (denatured collagen, 8% solution) and (x-elastin (20% solution) at ratios of 3:1:2 and 2:2:2 (v/v/v). The resulting PLGA-gelatin-elastin (PGE) fibers were homogeneous in appearance with an average diameter of 380 80 mn, which was considerably smaller than fibers made under identical conditions from the starting materials (PLGA, 780 +/- 200 nm; gelatin, 447 +/- 1.23 nm; elastin, 1060 170 nm). Upon hydration, PGE fibers swelled to an average fiber diameter of 963 +/- 132 nm, but did not disintegrate. Importantly, PGE scaffolds were stable in an aqueous environment without crosslinking, and were more elastic than those made of pure elastin fibers. To investigate the cytocompatibility of PGE, we cultured H9c2 rat cardiac myoblasts and rat bone marrow stromal cells (BMSCs) on fibrous PGE scaffolds. We found that myoblasts grew equally as well or slightly better on the scaffolds than on tissue-culture plastic. Microscopic evaluation confirmed that myoblasts reached confluence on the scaffold surfaces while simultaneously growing into the scaffolds.

Direct electron transfer between hemoglobin and a glassy carbon electrode facilitated by lipid-protected gold nanoparticles

We synthesized a kind of gold nanoparticle protected by a synthetic lipid (didodecyidimethylammonium bromide, DDAB). With the help of these gold nanoparticles, hemoglobin can exhibit a direct electron transfer (DET) reaction. The formal potential locates at -169 mV vs. Ag/AgCl. Spectral data indicated the hemoglobin on the electrode was not denatured. The lipid-protected gold nanoparticles were very stable (for at least 8 months). Their average diameter is 6.42 nm. It is the first time to use monolayer-protected nanoparticles to realize the direct electrochemistry of protein.

Study on adsorption and oxidation of calf thymus DNA at glassy carbon electrode

The oxidation and adsorption of the temperature-denatured DNA at GC electrode are studied by differential pulse voltammetry and in situ FTIR spectroelectrochemistry. The temperature-denatured DNA is adsorbed and formed a DNA multilayer at electrode surface. The temperature-denatured DNA showing partly reversible process was first observed based on the reduction peaks appearing at negative scans and the reversible spectral change. The oxidation product of the temperature-denatured DNA can not diffuse away from the electrode surface easily due to the impediment of the DNA multilayer, so it can be partly reduced.

Scanning tunnelling microscopy observation of cytochrome-c denaturation induced by bromopyrogal red on highly oriented pyrolytic graphite

The denaturation of cytochrome-e (cyt-c) induced by bromopyrogal red (BPR) was studied by scanning tunnelling microscopy (STM) on the electrochemically pretreated highly oriented pyrolytic graphite (HOPG) surface. STM images reveal that denatured cyt-c molecules exist in variable states including aggregates, globular compact, partially unfolded and combined with BPR molecule. The apparently low image contrast of denatured cyt-c observed in this experiment comparing to that of native cyt-c molecules, and the relative low image contrast of the unfolded part comparing with the compact globular part, are ascribed to the unfavourable tunnelling paths for the conformational variations of denatured cyt-c molecules. (C) 1997 Elsevier Science B.V.