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• W2057960025 abstract "Terminase enzymes are common to double-stranded DNA viruses. These enzymes “package” the viral genome into a pre-formed capsid. Terminase from bacteriophage λ is composed of gpA (72.4 kDa) and gpNu1 (20.4 kDa) subunits. We have described the expression and biochemical characterization of gpNu1ΔK100, a construct comprising the N-terminal 100 amino acids of gpNu1 (Yang, Q., de Beer, T., Woods, L., Meyer, J., Manning, M., Overduin, M., and Catalano, C. E. (1999) Biochemistry 38, 465–477). Here we present a biophysical characterization of this construct. Thermally induced loss of secondary and tertiary structures is fully reversible. Surprisingly, although loss of tertiary structure is cooperative, loss of secondary structure is non-cooperative. NMR and limited proteolysis data suggest that ≈30 amino acids of gpNu1ΔK100 are solvent-exposed and highly flexible. We therefore constructed gpNu1ΔE68, a protein consisting of the N-terminal 68 residues of gpNu1. gpNu1ΔE68 is a dimer with no evidence of dissociation or further aggregation. Thermally induced unfolding of gpNu1ΔE68 is reversible, with concomitant loss of both secondary and tertiary structure. The melting temperature increases with increasing protein concentration, suggesting that dimerization and folding are, at least in part, coupled. The data suggest that gpNu1ΔE68 represents the minimal DNA binding domain of gpNu1. We further suggest that the C-terminal ≈30 residues in gpNu1ΔK100 adopt a pseudo-stable α-helix that extends from the folded core of the protein. A model describing the role of this helix in the assembly of the packaging apparatus is discussed. Terminase enzymes are common to double-stranded DNA viruses. These enzymes “package” the viral genome into a pre-formed capsid. Terminase from bacteriophage λ is composed of gpA (72.4 kDa) and gpNu1 (20.4 kDa) subunits. We have described the expression and biochemical characterization of gpNu1ΔK100, a construct comprising the N-terminal 100 amino acids of gpNu1 (Yang, Q., de Beer, T., Woods, L., Meyer, J., Manning, M., Overduin, M., and Catalano, C. E. (1999) Biochemistry 38, 465–477). Here we present a biophysical characterization of this construct. Thermally induced loss of secondary and tertiary structures is fully reversible. Surprisingly, although loss of tertiary structure is cooperative, loss of secondary structure is non-cooperative. NMR and limited proteolysis data suggest that ≈30 amino acids of gpNu1ΔK100 are solvent-exposed and highly flexible. We therefore constructed gpNu1ΔE68, a protein consisting of the N-terminal 68 residues of gpNu1. gpNu1ΔE68 is a dimer with no evidence of dissociation or further aggregation. Thermally induced unfolding of gpNu1ΔE68 is reversible, with concomitant loss of both secondary and tertiary structure. The melting temperature increases with increasing protein concentration, suggesting that dimerization and folding are, at least in part, coupled. The data suggest that gpNu1ΔE68 represents the minimal DNA binding domain of gpNu1. We further suggest that the C-terminal ≈30 residues in gpNu1ΔK100 adopt a pseudo-stable α-helix that extends from the folded core of the protein. A model describing the role of this helix in the assembly of the packaging apparatus is discussed. kilobase(s) cohesive end site, the junction between individual genomes in immature concatemeric λ DNA the large subunit of phage λ terminase the small subunit of phage λ terminase a gpNu1 construct truncated at Glu68 a gpNu1 construct truncated at Lys100 a gpNu1 construct truncated at Pro141 polyacrylamide gel electrophoresis polymerase chain reaction N-terminal domain C-terminal domain circular dichroism Terminase enzymes are common to many of the double-stranded DNA bacteriophage and eukaryotic DNA viruses such as adenovirus and the herpesvirus groups (2Black L.W. Annu. Rev. Microbiol. 1989; 43: 267-292Crossref PubMed Scopus (365) Google Scholar, 3Roizman, B., Sears, A. E., Fields Viriology, 2nd Ed., Fields, B. N., Knipe, D. M., Howley, P. M., 2, 1996, 2231, 2297, Lippencott-Raven, New York.Google Scholar, 4Casjens S.R. Casjens S.R. Virus Structure and Assembly. Jones and Bartlett Publishers, Inc., Boston, MA1985: 1-28Google Scholar). These enzymes function to insert a viral genome into the confines of a preformed, empty capsid. The terminase enzyme from bacteriophage λ is composed of two virally encoded proteins, gpNu1 (181 amino acids) and gpA (640 amino acids), in a gpA1·gpNu12 holoenzyme complex (5Gold M. Becker A. J. Biol. Chem. 1983; 258: 14619-14625Abstract Full Text PDF PubMed Google Scholar, 6Tomka M.A. Catalano C.E. J. Biol. Chem. 1993; 268: 3056-3065Abstract Full Text PDF PubMed Google Scholar). Terminase holoenzyme possesses site-specific nuclease (6Tomka M.A. Catalano C.E. J. Biol. Chem. 1993; 268: 3056-3065Abstract Full Text PDF PubMed Google Scholar, 7Woods L. Terpening C. Catalano C.E. Biochemistry. 1997; 36: 5777-5785Crossref PubMed Scopus (27) Google Scholar, 8Rubinchik S. Parris W. Gold M. J. Biol. Chem. 1994; 269: 13575-13585Abstract Full Text PDF PubMed Google Scholar, 9Parris W. Rubinchik S. Yang Y.-C. Gold M. J. Biol. Chem. 1994; 269: 13564-13574Abstract Full Text PDF PubMed Google Scholar), ATPase (10Tomka M.A. Catalano C.E. Biochemistry. 1993; 32: 11992-11997Crossref PubMed Scopus (56) Google Scholar, 11Hwang Y. Catalano C.E. Feiss M. Biochemistry. 1995; 35: 2796-2803Crossref Scopus (61) Google Scholar, 12Woods L. Catalano C.E. Biochemistry. 1999; 38: 14624-14630Crossref PubMed Scopus (19) Google Scholar, 13Rubinchik S. Parris W. Gold M. J. Biol. Chem. 1994; 269: 13586-13593Abstract Full Text PDF PubMed Google Scholar), DNA strand separation (9Parris W. Rubinchik S. Yang Y.-C. Gold M. J. Biol. Chem. 1994; 269: 13564-13574Abstract Full Text PDF PubMed Google Scholar, 14Yang Q. Catalano C.E. Biochemistry. 1997; 36: 10638-10645Crossref PubMed Scopus (28) Google Scholar), and DNA translocase (15Hwang Y. Feiss M. Virology. 1995; 211: 367-376Crossref PubMed Scopus (39) Google Scholar, 16Rubinchik S. Parris W. Gold M. J. Biol. Chem. 1995; 270: 20059-20066Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) catalytic activities that work in concert to package viral DNA. All of the terminase enzymes characterized to date possess a similar holoenzyme composition (small and large subunits) and catalytic activities (5Gold M. Becker A. J. Biol. Chem. 1983; 258: 14619-14625Abstract Full Text PDF PubMed Google Scholar, 17Fujisawa H. Morita M. Genes Cells. 1997; 2: 537-545Crossref PubMed Scopus (150) Google Scholar, 18Guo P. Grimes S. Anderson D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 3505-3509Crossref PubMed Scopus (138) Google Scholar, 19Rao V.B. Black L.W. J. Mol. Biol. 1988; 200: 475-488Crossref PubMed Scopus (104) Google Scholar, 20Dröge A. Tavares P. J. Mol. Biol. 2000; 296: 103-105Crossref PubMed Scopus (39) Google Scholar, 21Catalano C.E. Cell. Mol. Life Sci. 2000; 57: 128-148Crossref PubMed Scopus (147) Google Scholar). Replication of λ DNA proceeds through a rolling circle mechanism that gives rise to linear concatemers of the viral genome linked in a head to tail fashion (22Furth M.E. Wickner S.H. Hendrix R.W. Roberts J.W. Stahl F.W. Weisberg R.A. Lambda II. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1983: 145-155Google Scholar, 23Skalka A.M. Arber W. Henle W. Hofschneider P.H. Humphrey J.H. Klein J. Koldovsky P. Koprowski H. Maaløe O. Melchers F. Rott R. Schweiger H.G. Syrucek L. Vogt P.K. Current Topics in Mircobiology and Immunology. 78. Springer-Verlag, New York, NY1977: 201-238Google Scholar). Packaging of viral DNA requires the excision of an individual genome from the concatemer, and packaging of the 48.5-kb1 duplex within the capsid. Genome packaging by λ terminase has been described in detail (21Catalano C.E. Cell. Mol. Life Sci. 2000; 57: 128-148Crossref PubMed Scopus (147) Google Scholar, 24Catalano C.E. Cue D. Feiss M. Mol. Microbiol. 1995; 16: 1075-1086Crossref PubMed Scopus (171) Google Scholar, 25Murialdo H. Annu. Rev. Biochem. 1991; 60: 125-153Crossref PubMed Scopus (88) Google Scholar, 26Becker A. Murialdo H. J. Bacteriol. 1990; 172: 2819-2824Crossref PubMed Google Scholar, 27Feiss M. Trends Genet. 1986; 2: 100-104Abstract Full Text PDF Scopus (106) Google Scholar) and is summarized here. Packaging initiates with the assembly of the holoenzyme at a cos site in the concatemer. This site represents the junction between the left and right ends of individual genomes within the concatemer (Fig. 1 A). Site-specific assembly at cos is mediated by cooperative gpNu1 binding to three repeated R-elements in the cosBsubsite of cos. Assembly of gpNu1 at cosBpromotes the assembly of a gpA dimer symmetrically disposed atcosN, yielding a stable pre-nicking complex. Site-specific nicking of the duplex at cosN, followed by an ATP-dependent separation of the nicked strands, yields complex I, the next stable intermediate. This nucleoprotein complex next binds an empty capsid, which triggers the transition to a mobile, ATP-driven translocation complex that inserts DNA into the capsid. Upon arrival at the next downstream cos site, terminase again nicks the duplex, and strand separation results in release of the DNA-filled capsid and re-generation of complex I. The gpA subunit of λ terminase appears to possess all of the catalytic activities required for genome packaging, but the efficiency of each reaction is strongly stimulated by the smaller gpNu1 subunit (7Woods L. Terpening C. Catalano C.E. Biochemistry. 1997; 36: 5777-5785Crossref PubMed Scopus (27) Google Scholar, 8Rubinchik S. Parris W. Gold M. J. Biol. Chem. 1994; 269: 13575-13585Abstract Full Text PDF PubMed Google Scholar, 12Woods L. Catalano C.E. Biochemistry. 1999; 38: 14624-14630Crossref PubMed Scopus (19) Google Scholar, 13Rubinchik S. Parris W. Gold M. J. Biol. Chem. 1994; 269: 13586-13593Abstract Full Text PDF PubMed Google Scholar, 14Yang Q. Catalano C.E. Biochemistry. 1997; 36: 10638-10645Crossref PubMed Scopus (28) Google Scholar, 16Rubinchik S. Parris W. Gold M. J. Biol. Chem. 1995; 270: 20059-20066Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 28Hwang Q. Woods L. Feiss M. Catalano C.E. J. Biol. Chem. 1999; 274: 15305-15314Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Moreover, gpNu1 is required for specific and high affinity gpA DNA binding interactions (29Yang Q. Hanagan A. Catalano C.E. Biochemistry. 1997; 36: 2744-2752Crossref PubMed Scopus (39) Google Scholar) and likely contributes to the exceptional stability of the pre-nicking complex and complex I (30Cue D. Feiss M. J. Mol. Biol. 1992; 228: 72-87Crossref PubMed Scopus (26) Google Scholar, 31Cai A.-H. Hwang Y. Cue D. Catalano C. Feiss M. J. Bacteriol. 1997; 179: 2479-2485Crossref PubMed Google Scholar). Our laboratory is interested in the biochemical and biophysical mechanisms of DNA packaging by phage λ terminase. Central to the packaging process is the cooperative assembly of gpNu1 and gpA atcos (Fig. 1 A). To define this assembly process at a molecular level requires an understanding of the structural features governing physical interaction between the enzyme subunits and with DNA. Toward this end, we have sought to define the properties governing intrinsic and cooperative DNA binding by gpNu1. Unfortunately, the isolated gpNu1 subunit shows a strong tendency to aggregate upon concentration (9Parris W. Rubinchik S. Yang Y.-C. Gold M. J. Biol. Chem. 1994; 269: 13564-13574Abstract Full Text PDF PubMed Google Scholar, 28Hwang Q. Woods L. Feiss M. Catalano C.E. J. Biol. Chem. 1999; 274: 15305-15314Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 32Meyer J.D. Hanagan A. Manning M.C. Catalano C.E. Int. J. Biol. Macromol. 1998; 23: 27-36Crossref PubMed Scopus (19) Google Scholar, 33Hanagan A. Meyer J.D. Johnson L. Manning M.C. Catalano C.E. Int. J. Biol. Macromol. 1998; 23: 37-48Crossref PubMed Scopus (14) Google Scholar, 34Parris W. Davidson A. Keeler C.L. Gold M. J. Biol. Chem. 1988; 263: 8413-8419Abstract Full Text PDF PubMed Google Scholar), a feature that has hampered structural and biophysical characterization of the protein. We recently described the construction and characterization of two deletion mutants of gpNu1, gpNu1ΔP141 (35Yang Q. Berton N. Manning M.C. Catalano C.E. Biochemistry. 1999; 38: 14238-14247Crossref PubMed Scopus (22) Google Scholar) and gpNu1ΔK100 (1Yang Q. de Beer T. Woods L. Meyer J. Manning M. Overduin M. Catalano C.E. Biochemistry. 1999; 38: 465-477Crossref PubMed Scopus (24) Google Scholar), which are proteins truncated at Pro141 and Lys100 of full-length gpNu1, respectively. Studies of these constructs led to a model where the C-terminal 40 residues of the protein are required for interactions with the gpA subunit to form a catalytically competent holoenzyme complex (Fig. 1 B). Residues 100–140 promote self-association interactions that mediate cooperative DNA binding. The N-terminal 100 residues of the protein represented by gpNu1ΔK100 contain the putative helix-turn-helix DNA binding motif postulated to play a direct role in DNA binding. Indeed, the construct is folded in solution and binds cos-containing DNA with reasonable specificity (1Yang Q. de Beer T. Woods L. Meyer J. Manning M. Overduin M. Catalano C.E. Biochemistry. 1999; 38: 465-477Crossref PubMed Scopus (24) Google Scholar). Preliminary NMR experiments suggested that gpNu1ΔK100 would be amenable to structural studies (1Yang Q. de Beer T. Woods L. Meyer J. Manning M. Overduin M. Catalano C.E. Biochemistry. 1999; 38: 465-477Crossref PubMed Scopus (24) Google Scholar). Here we present a detailed biophysical characterization of gpNu1ΔK100. These studies suggest that the construct consists of a functional N-terminal domain that possess a pseudo-stable C-terminal helix extending from the folded core of the protein. We further describe the construction and characterization of a shorter construct that clearly demonstrates the unusual biophysical characteristics of gpNu1ΔK100 result from this extended C-terminal helix. The biological significance of these results is discussed. Tryptone, yeast extract, and agar were purchased from DIFCO. Restriction enzymes were purchased fromPromega. DEAE-Sepharose FF and SP-Sepharose FF chromatography resins were purchased from Amersham Pharmacia Biotech. Restriction enzymes were purchased from Promega. Guanidinium hydrochloride was purchased from Mallinckrodt. All other materials were of the highest quality commercially available. Bacterial cultures were grown in shaker flasks utilizing a New Brunswick Scientific series 25 incubator-shaker. All protein purifications utilized a Amersham Pharmacia Biotech fast-protein liquid chromatography system that consisted of two P500 pumps, a GP250-plus controller, a V7 injector, and a Uvicord SII variable-wavelength detector. UV-visible absorbance spectra were recorded on a Hewlett-Packard HP8452A spectrophotometer. Fluorescence spectra were recorded at room temperature on a PTI QuantaMaster spectrofluorometer. A protein concentration of 10 μg/ml in 10 mm potassium phosphate buffer, pH 7.4, was used, and a buffer blank was subtracted from the fluorescence spectrum. Circular dichroism (CD) spectra were recorded on an Aviv model 62DS circular dichroism spectropolarimeter equipped with a Brinkmann Lauda RM6 circulating water bath and a thermostated cell holder. Near-UV CD spectra utilized a protein concentration of 1 mg/ml in a 0.1-cm strain-free cuvette. Data were typically collected between 250 and 350 nm at 0.5-nm intervals using a bandwidth of 1.5 nm and a dwell time of 30 s. Far-UV CD spectra utilized a protein concentration of 100 μg/ml in a 0.1-cm strain-free cuvette. Data were typically collected from 180 to 260 nm at 0.5-nm intervals using a bandwidth of 1.5 nm and a dwell time of 30 s. The raw spectra were converted to molar ellipticity using,θ=θobs*MRW10*b*cEquation 1 where θ is the molar ellipticity (degrees-cm2/dmol), θobs is the ellipticity recorded by the instrument (millidegrees), MRW is the mean residue weight (formula weight divided by the total number of residues in the protein), b is the cell path length, and c is the protein concentration in mg/ml (36Woody, R. W. (1985) (Hruby, V. J., ed) Vol. 7, 15–114, Academic Press, Orlanco, FL.Google Scholar). Protein secondary matrix-assisted laser desorption time-of-flight mass spectra were obtained from the University of Colorado Health Sciences Center Macromolecular Resource Center. Automated DNA sequence analysis was performed by the University of Colorado Cancer Center Macromolecular Resources Core facility. Both strands of the duplex were examined to ensure the expected DNA sequence. Prediction of protein secondary structures based upon primary sequence data was performed by the method of Chou and Fasman (37Sreerama N. Woody R.W. Anal. Biochem. 1993; 209: 32-44Crossref PubMed Scopus (946) Google Scholar), using the DNASIS program (Macintosh version 2.0). Calculation of protein secondary structures based upon the far-UV CD data was performed using the SELCON program. Escherichia coli BL21(DE3) cells were a generous gift of D. Kroll (University of Colorado Health Sciences Center, Denver, CO). All synthetic oligonucleotides used in this study were purchased from Life Technologies, Inc. and were used without further purification. Plasmids pSF1 and pAFP1, kindly provided by M. Feiss (University of Iowa, Iowa City, IA), were purified from theE. coli cell lines C600[pSF1] and JM107[pAFP1], respectively, using Qiagen DNA prep columns. All of our purified proteins were homogenous as determined by SDS-PAGE and densitometric analysis using a Molecular Dynamics laser densitometer and the ImageQuaNT data analysis package. Unless otherwise indicated, protein concentrations were determined spectrally using millimolar extinction coefficients (1Yang Q. de Beer T. Woods L. Meyer J. Manning M. Overduin M. Catalano C.E. Biochemistry. 1999; 38: 465-477Crossref PubMed Scopus (24) Google Scholar, 32Meyer J.D. Hanagan A. Manning M.C. Catalano C.E. Int. J. Biol. Macromol. 1998; 23: 27-36Crossref PubMed Scopus (19) Google Scholar). A truncated Nu1 gene was amplified by PCR using pSF1 as a DNA template. This plasmid contains the wild-type Nu1 gene cloned into a pBR322 background (38Feiss M. Siegele D.A. Rudolph C.F. Frackman M. Gene. 1982; 17: 123-130Crossref PubMed Scopus (33) Google Scholar). Primers were designed such that EcoRI andBamHI restriction sequences were present at the 5′ and 3′ ends, respectively, of the PCR product. The primer sequences used to amplify pNu1ΔE68 were as follows. Forward primer: 5′-CCT CTC CCT TTC TCC GAA TTC ATG GAA GTC AAC AAA AAG C-3′; reverse primer: 5-CTT CCT GGA TTC TTA TTC TTC AAC CTC CCG GCG-3′. The EcoRI andBamHI restriction sequences in the above primers are indicated in italics, whereas the f-MET (forward primer) and stop (reverse primer) codons are shown in boldface. Sequences complementary to the Nu1 gene areunderlined. The stop codon present in the reverse PCR primer yields, upon amplification, a truncated Nu1 gene that expresses only the first 68 amino acids of the protein. PCR amplification, isolation of the PCR product, and construction of the overexpression plasmid (pNu1ΔE68) was performed as described previously (1Yang Q. de Beer T. Woods L. Meyer J. Manning M. Overduin M. Catalano C.E. Biochemistry. 1999; 38: 465-477Crossref PubMed Scopus (24) Google Scholar, 28Hwang Q. Woods L. Feiss M. Catalano C.E. J. Biol. Chem. 1999; 274: 15305-15314Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 35Yang Q. Berton N. Manning M.C. Catalano C.E. Biochemistry. 1999; 38: 14238-14247Crossref PubMed Scopus (22) Google Scholar). Four liters of 2X-YT media containing 50 μg/ml ampicillin, 25 mmpotassium phosphate, pH 7.5, and 5 mm glucose were inoculated with a 40 ml of overnight culture of BL21(DE3)[pNu1ΔE68] derived from an isolated colony. The cultures were maintained at 37 °C until an optical density 1.0 (A600 nm) was obtained, at which point isopropyl-1-thio-β-d-galactopyranoside (1.2 mm) was added. The cells were maintained at 37 °C for an additional 3 h, and then harvested by centrifugation. Unless otherwise indicated, all subsequent steps were performed at 0–4 °C. The cell pellet was resuspended in ice-cold buffer A (20 mmTris, pH 8.0, 2 mm EDTA, 7 mm2-mercaptoethanol, and 10% glycerol) containing 100 mmNaCl, and the cells were disrupted by sonification. Insoluble cellular debris was removed by centrifugation (12,000 × g, 30 min), and solid ammonium sulfate was added to the clarified supernatant to 50% saturation. Insoluble protein was removed by centrifugation (12,000 × g, 30 min), and proteins were then precipitated with the addition of ammonium sulfate to 90% saturation followed by centrifugation. gpNu1ΔE68 was found in the 50–90% ammonium sulfate-precipitated fractions. The ammonium sulfate pellet was taken into buffer A and, after dialysis against the same buffer, loaded onto a DEAE-Sepharose column (200 ml) also equilibrated with buffer A. The column was developed with a salt gradient with gpNu1ΔE68 eluting at ≈300 mm NaCl. Column fractions were examined by SDS-PAGE, and the appropriate fractions were pooled, dialyzed against buffer A, and loaded onto an SP-Sepharose column equilibrated with the same buffer. The column was developed with a salt gradient with gpNu1ΔE68 eluting at ≈280 mm NaCl. As before, column fractions were examined by SDS-PAGE, the appropriate fractions were pooled, dialyzed against buffer A containing 20% glycerol, and stored at −80 °C. As required, the proteins were concentrated and/or buffer exchanged using an Ultrafree-15 centrifugal filter device according to the manufacturer's instructions (Millipore). Experiments were carried out with a Beckman XL-A analytical ultracentrifuge equipped with a Ti-60 four-hole rotor with six-channel, 12-mm path-length centerpieces. Absorbance optics were used throughout. Three different protein concentrations were used with ratios of 10:3:1, with the highest protein concentrations of 150 μm (≈1.2 mg/ml). Samples were dialyzed against the appropriate buffer and then diluted to the concentrations indicated in each experiment. Sample volumes were 100 μl with the inert oil FC-43 used to displace samples from the base of the cells. Samples were allowed to equilibrate at 20,000, 30,000, and 40,000 rpm. Samples were judged to be at equilibrium by successive subtraction of scans. The density of each buffer solution was calculated based on the salt composition and equilibrium temperature. The partial specific volume of gpNu1ΔE68 was calculated by summing the partial specific volumes of the individual amino acids (39Cohn E.J. Edsall J.T. Proteins, Amino Acids and Peptides. Reinhold, New York, NY1943Google Scholar). Data chosen for analysis had an absorbance between 0.1 and 1.5 optical density units. Each data point was an average of four scans taken every 0.001 cm. Data were selected for analysis using the program REEDIT (generously provided by Dr. David Yphantis). Individual and simultaneous analyses of nine channels (three concentrations at three speeds) were carried out to resolve assembly stoichiometry. Data were analyzed using the appropriate functions by non-linear least-squares parameter estimation (40Johnson M.L. Frasier S.G. Methods Enzymol. 1985; 117: 301-342Crossref Scopus (511) Google Scholar) to determine the best-fit model-dependent parameters that minimize the variance. The program NONLIN was used (Ref. 41Johnson M.L. Correia J.A. Yphantis D.A. Halvorson H.R. Biophys. J. 1981; 36: 575-588Abstract Full Text PDF PubMed Scopus (778) Google Scholar; kindly donated by Dr. David Yphantis). Confidence intervals (67%) correspond to approximately one standard deviation. Non-ideality was not considered, because there was no evidence for non-ideal effects. Models incorporating different assembly stoichiometries were based upon the general equation,Y(r)=δ+αexp[(ς(r2−ro2)]+∑αNKNexp[Nς(r2−ro2)]Equation 2 where Y(r) is the absorbance at radiusr, δ the baseline offset, and α the monomer absorbance at reference radius ro. ς is the reduced molecular weight (ς = M(1 − νρ)ω2/RT], N is the stoichiometry of the reaction, and KN is the association constant of the reaction NM ⇌MN. Thermally induced protein denaturation experiments were performed as described previously (32Meyer J.D. Hanagan A. Manning M.C. Catalano C.E. Int. J. Biol. Macromol. 1998; 23: 27-36Crossref PubMed Scopus (19) Google Scholar,33Hanagan A. Meyer J.D. Johnson L. Manning M.C. Catalano C.E. Int. J. Biol. Macromol. 1998; 23: 37-48Crossref PubMed Scopus (14) Google Scholar, 35Yang Q. Berton N. Manning M.C. Catalano C.E. Biochemistry. 1999; 38: 14238-14247Crossref PubMed Scopus (22) Google Scholar). Each data set represents the average of at least two independent experiments. The fraction of protein in the denatured state (FD) was determined using, FD=θN−θTθN−θDEquation 3 where θT is the ellipticity at temperatureT, and θN and θD represent the ellipticity for the native and denatured protein, respectively. Baseline corrections were not performed to demonstrate temperature-induced alterations in the pre-transition baseline slopes. The unfolding curves were analyzed using a complex sigmoidal curve function,FD=(mD*T−bD)−(mN*T−bN)1+TTmmT+(mN*T−bN)Equation 4 where (mN*T − bN) and (mD*T −bD) describe the linear portion of the pre-transition and post-transition baselines, respectively, at temperature T, mT is the slope of curve within the transition region, and Tm is the melting temperature for the transition. All data sets were fit to the above equations by non-linear regression methods using the IGOR graphics/analysis package (WaveMetrics, Lake Oswego, OR). We have previously described the construction, expression, and biochemical characterization of gpNu1ΔK100, a deletion construct of the small terminase subunit (1Yang Q. de Beer T. Woods L. Meyer J. Manning M. Overduin M. Catalano C.E. Biochemistry. 1999; 38: 465-477Crossref PubMed Scopus (24) Google Scholar). The construct is a dimer in the concentration range of 5 μm to 2 mm, with no evidence for dissociation or further aggregation. Preliminary NMR experiments suggested that the construct might be amenable to structural studies, and we therefore sought to further characterize the physical properties of this construct. Thermally induced unfolding of gpNu1ΔK100 secondary and tertiary structural elements is reversible, as indicated in Fig. 2, A and B, respectively. Moreover, the loss of tertiary structure (near-UV CD signal) is cooperative, consistent with a folded and stable construct (Fig. 2 C). 2gpNu1ΔK100 possesses two tryptophan (Trp22 and Trp49) and three tyrosine (Tyr41, Tyr50, and Tyr84) residues. The thermal unfolding data provide no indication for multiple unfolding transitions, which would indicate local unfolding in the vicinity of these residues. We thus interpret the loss of the near-UV CD signal as reflecting global unfolding of the protein. It is feasible, however, that the melting curves reflect regional versus global unfolding of the protein. Salt and protons stabilize protein tertiary structure, as evidenced by the significant increase in the Tm for the transition (Table I). Despite the observed cooperative loss of tertiary structure, thermally induced loss of secondary structural elements (far-UV CD signal) appears essentially non-cooperative (Fig. 2 C). The steep pre-transition baseline observed in these data make it difficult to accurately calculate theTm for this transition. Nevertheless, it is clear that salt and protons similarly affect theTm for the unfolding transition, whether monitored in the far-UV or near-UV region of the CD spectrum (Table I). Interestingly, salt and pH strongly affect the pre-transition baselines obtained in the far-UV CD melting curves, but not the near-UV CD melting curves (Table II).Table ISalt and protons affect the thermal stability of gpNu1ΔK100pH[NaCl]TmTertiary structure, near-UV CDSecondary structure, far-UV CD°C7.2052.2 ± 0.447.3 ± 0.77.2150 mmND 1-aND, not done.52.7 ± 0.87.2500 mm70.2 ± 1.666.2 ± 2.88.0045.7 ± 1.644.2 ± 0.47.2052.2 ± 0.447.3 ± 0.76.0062.0 ± 1.555.9 ± 0.71-a ND, not done. Open table in a new tab Table IISalt and protons affect the pre-transition baseline for thermally induced gpNu1ΔK100 secondary structure losspH[NaCl]Pre-transition baselineTertiary structure, near-UV CDSecondary structure, far-UV CDdeg−1 × 1037.206.0 ± 0.316.6 ± 0.37.2150 mmND 2-aND, not done.10.0 ± 0.47.2500 mm6.0 ± 0.58.6 ± 0.48.007.0 ± 0.610.4 ± 0.47.206.0 ± 0.316.6 ± 0.36.006.0 ± 0.77.8 ± 0.32-a ND, not done. Open table in a new tab Evaluation of the line widths and chemical shifts in a 1H-15N correlation spectrum of gpNu1ΔK100 suggested that ≈30 residues of the construct were solvent exposed and highly flexible. 3T. de Beer and C. E. Catalano, unpublished. Primary sequence analysis predicts strong α-helical character in the region spanning residues ≈50 and ≈115 of gpNu1 (Fig.1 B). We postulated that this putative helix might be partially disrupted in the gpNu1ΔK100 construct, leading to the unusual unfolding properties of the protein. If this were the case, limited proteolysis of gpNu1ΔK100 would be expected to degrade the exposed portion of the helix, yielding a fully folded domain suitable for structural characterization. This was indeed correct. Limited proteolysis of the gpNu1ΔK100 with a number of proteases consistently yielded two predominant products (data not shown). Analysis of these products by SDS-PAGE and matrix-assisted laser desorption time-of-flight mass spectrometry yielded molecular masses of ≈7.5 kDa and ≈10 kDa, respectively. Proteolysis studies, NMR spectral" @default.
• W2057960025 created "2016-06-24" @default.
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• W2057960025 creator A5022126884 @default.
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• W2057960025 date "2001-01-01" @default.
• W2057960025 modified "2023-09-27" @default.
• W2057960025 title "Biophysical Characterization of the DNA Binding Domain of gpNu1, a Viral DNA Packaging Protein" @default.
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