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ABSTRACT: Mutations that confer the loss of a single biochemical property (separation-of-function mutations) can often uncover a previously unknown role for a protein in a particular biological process. However, most mutations are identified based on loss-of-function (LOF) phenotypes, which cannot differentiate between separation-of-function alleles vs. mutations that encode unstable/unfolded proteins. An alternative approach is to use over-expression dominant negative (ODN) phenotypes to identify mutant proteins that disrupt function in an otherwise wild type strain when over-expressed. This is based on the assumption that such mutant proteins retain an overall structure which is comparable to that of the wild type protein and are able to compete with the endogenous protein (Herskowitz 1987). To test this, the in vivo phenotypes of mutations in the Est3 telomerase subunit from Saccharomyces cerevisiae were compared with the in vitro secondary structure of these mutant proteins as analyzed by circular dichroism spectroscopy, which demonstrates that ODN is a more sensitive assessment of protein stability than the commonly used method of monitoring protein levels from extracts. Reverse mutagenesis of EST3 which targeted different categories of amino acids also showed that mutating highly conserved charged residues to the oppositely charged amino acid had an increased likelihood of generating a severely defective est3(-) mutation which nevertheless encoded a structurally stable protein. These results suggest that charge swap mutagenesis directed at a limited subset of highly conserved charged residues, combined with ODN screening to eliminate partially unfolded proteins, may provide a widely applicable and efficient strategy for generating separation-of-function mutations.
Genetics 01/2013; · 4.01 Impact Factor
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ABSTRACT: Pot1 is the protein responsible for binding to and protecting the 3' single-stranded DNA (ssDNA) overhang at most eukaryotic telomeres. Here, we present the crystal structure of one of the two oligonucleotide/oligosaccharide-binding folds (Pot1pC) that make up the ssDNA-binding domain in S. pombe Pot1. Comparison with the homologous human domain reveals unexpected structural divergence in the mode of ligand binding that explains the differing ligand requirements between species. Despite the presence of apparently base-specific hydrogen bonds, Pot1pC is able to bind a wide range of ssDNA sequences with thermodynamic equivalence. To address how Pot1pC binds ssDNA with little to no specificity, multiple structures of Pot1pC bound to noncognate ssDNA ligands were solved. These structures reveal that this promiscuity is implemented through new binding modes that thermodynamically compensate for base-substitutions through alternate stacking interactions and new H-bonding networks.
Structure 11/2012; · 6.35 Impact Factor
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ABSTRACT: The purpose of this study was to probe the fate of a model antigen, a cysteine-free mutant of bacteriophage T4 lysozyme, to the level of fine structural detail, as a consequence of its interaction with an aluminum (Al)-containing adjuvant. Fluorescence spectroscopy and differential scanning calorimetry were used to compare the thermal stability of the protein in solution versus adsorbed onto an Al-containing adjuvant. Differences in accessible hydrophobic surface areas were investigated using an extrinsic fluorescence probe, 8-Anilino-1-naphthalenesulfonic acid (ANS). As has been observed with other model antigens, the apparent thermal stability of the protein decreased following adsorption onto the adjuvant. ANS spectra suggested that adsorption onto the adjuvant caused an increase in exposure of hydrophobic regions of the protein. Electrostatic interactions drove the adsorption, and disruption of these interactions with high ionic strength buffers facilitated the collection of two-dimensional (15) N heteronuclear single quantum coherence nuclear magnetic resonance data of protein released from the adjuvant. Although the altered stability of the adsorbed protein suggested changes to the protein's structure, the fine structure of the desorbed protein was nearly identical to the protein's structure in the adjuvant-free formulation. Thus, the adjuvant-induced changes to the protein that were responsible for the reduced thermal stability were not observed upon desorption.
Journal of Pharmaceutical Sciences 12/2011; 101(4):1391-9. · 3.06 Impact Factor
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ABSTRACT: The ends of eukaryotic chromosomes consist of long tracts of repetitive GT-rich DNA with variable sequence homogeneity between and within organisms. Telomeres terminate in a conserved 3'-ssDNA overhang that, regardless of sequence variability, is specifically and tightly bound by proteins of the telomere-end protection family. The high affinity ssDNA-binding activity of S. pombe Pot1 protein (SpPot1) is conferred by a DNA-binding domain consisting of two subdomains, Pot1pN and Pot1pC. Previous work has shown that Pot1pN binds a single repeat of the core telomere sequence (GGTTAC) with exquisite specificity, while Pot1pC binds an extended sequence of nine nucleotides (GGTTACGGT) with modest specificity requirements. We find that full-length SpPot1 binds the composite 15mer, (GGTTAC)(2)GGT, and a shorter two-repeat 12mer, (GGTTAC)(2), with equally high affinity (<3 pM), but with substantially different kinetic and thermodynamic properties. The binding mode of the SpPot1/15mer complex is more stable than that of the 12mer complex, with a 2-fold longer half-life and increased tolerance to nucleotide and amino acid substitutions. Our data suggest that SpPot1 protection of heterogeneous telomeres is mediated through 5'-sequence recognition and the use of alternate binding modes to maintain high affinity interaction with the G-strand, while simultaneously discriminating against the complementary strand.
Biochemistry 08/2011; 50(35):7503-13. · 3.42 Impact Factor
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ABSTRACT: Benzyl alcohol, a preservative commonly added to multidose therapeutic protein formulations, can accelerate aggregation of recombinant human interleukin-1 receptor antagonist (rhIL-1ra). To investigate the interactions between benzyl alcohol and rhIL-1ra, we used nuclear magnetic resonance to observe the effect of benzyl alcohol on the chemical shifts of amide resonances of rhIL-1ra and to measure hydrogen-deuterium exchange rates of individual rhIL-1ra residues. Addition of 0.9% benzyl alcohol caused significant chemical shifts of amide resonances for residues 90-97, suggesting that these solvent-exposed residues participate in the binding of benzyl alcohol. In contrast, little perturbation of exchange rates was observed in the presence of either sucrose or benzyl alcohol. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci.
Journal of Pharmaceutical Sciences 05/2011; · 3.06 Impact Factor
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ABSTRACT: The Est3 subunit of yeast telomerase, which adopts a predicted OB-fold, is essential for telomere replication. To assess the possible contributions that Est3 might make to enzyme catalysis, we compared telomerase activity from wild type and est3-Delta strains of Saccharomyces castellii, which revealed that loss of the Est3 subunit results in a 2- to 3-fold decline in nucleotide addition. This effect was not primer-specific, based on assessment of a panel of primers that spanned the template of the S. castellii telomerase RNA. Furthermore, using nuclear magnetic resonance chemical shift perturbation, no chemical shift change was observed at any site in the protein upon addition of single-stranded DNA, arguing against a role for Est3 in recognition of telomeric substrates by telomerase. Addition of exogenous Est3 protein, including mutant Est3 proteins that are severely impaired for telomere replication in vivo, fully restored activity in est3-Delta telomerase reactions. Thus, Est3 performs an in vivo regulatory function in telomere replication, which is distinct from any potential contribution that Est3 might make to telomerase activity.
Nucleic Acids Research 04/2010; 38(7):2279-90. · 8.03 Impact Factor
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ABSTRACT: Telomeres must be capped to preserve chromosomal stability. The conserved Stn1 and Ten1 proteins are required for proper capping of the telomere, although the mechanistic details of how they contribute to telomere maintenance are unclear. Here, we report the crystal structures of the C-terminal domain of the Saccharomyces cerevisiae Stn1 and the Schizosaccharomyces pombe Ten1 proteins. These structures reveal striking similarities to corresponding subunits in the replication protein A complex, further supporting an evolutionary link between telomere maintenance proteins and DNA repair complexes. Our structural and in vivo data of Stn1 identify a new domain that has evolved to support a telomere-specific role in chromosome maintenance. These findings endorse a model of an evolutionarily conserved mechanism of DNA maintenance that has developed as a result of increased chromosomal structural complexity.
Proceedings of the National Academy of Sciences 11/2009; 106(46):19298-303. · 9.68 Impact Factor
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ABSTRACT: Appropriate control of the chromosome end-replicating enzyme telomerase is crucial for maintaining telomere length and genomic stability. The essential telomeric DNA-binding protein Cdc13p both positively and negatively regulates telomere length in budding yeast. Here we test the effect of purified Cdc13p on telomerase action in vitro. We show that the full-length protein and its DNA-binding domain (DBD) inhibit primer extension by telomerase. This inhibition occurs by competitive blocking of telomerase access to DNA. To further understand the requirements for productive telomerase 3'-end access when Cdc13p or the DBD is bound to a telomerase substrate, we constrained protein binding at various distances from the 3'-end on two sets of increasingly longer oligonucleotides. We find that Cdc13p inhibits the action of telomerase through three distinct biochemical modes, including inhibiting telomerase even when a significant tail is available, representing a novel 'action at a distance' inhibitory activity. Thus, while yeast Cdc13p exhibits the same general activity as human POT1, providing an off switch for telomerase when bound near the 3'-end, there are significant mechanistic differences in the ways telomere end-binding proteins inhibit telomerase action.
Nucleic Acids Research 12/2008; 37(2):354-67. · 8.03 Impact Factor
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ABSTRACT: Linear chromosomes terminate in specialized nucleoprotein structures called telomeres, which are required for genomic stability and cellular proliferation. Telomeres end in an unusual 3' single-strand overhang that requires a special capping mechanism to prevent inappropriate recognition by the DNA damage machinery. In Schizosaccharomyces pombe, this protective function is mediated by the Pot1 protein, which binds specifically and with high affinity to telomeric ssDNA. We have characterized the thermodynamics and accommodation of both cognate and noncognate telomeric single-stranded DNA (ssDNA) sequences by Pot1pN, an autonomous ssDNA-binding domain (residues 1-187) found in full-length S. pombe Pot1. Direct calorimetric measurements of cognate telomeric ssDNA binding to Pot1pN show favorable enthalpy, unfavorable entropy, and a negative heat-capacity change. Thermodynamic analysis of the binding of noncognate telomeric ssDNA to Pot1pN resulted in unexpected changes in free energy, enthalpy, and entropy. Chemical-shift perturbation and structural analysis of these bound noncognate sequences show that these thermodynamic changes result from the structural rearrangement of both Pot1pN and the bound oligonucleotide. These data suggest that the ssDNA-binding interface is highly dynamic and, in addition to the conformation observed in the crystal structure of the Pot1pN/d(GGTTAC) complex, capable of adopting alternative thermodynamically equivalent conformations.
Biochemistry 05/2008; 47(15):4345-58. · 3.42 Impact Factor
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ABSTRACT: The Saccharomyces cerevisiae protein Cdc13 tightly and specifically binds the conserved G-rich single-stranded overhang at telomeres and plays an essential role in telomere end-protection and length regulation. The 200 residue DNA-binding domain of Cdc13 (Cdc13-DBD) binds an 11mer single-stranded representative of the yeast telomeric sequence [Tel11, d(GTGTGGGTGTG)] with a 3 pM affinity and specificity for three bases (underlined) at the 5' end. The structure of the Cdc13-DBD bound to Tel11 revealed a large, predominantly aromatic protein interface with several unusual features. The DNA adopts an irregular, extended structure, and the binding interface includes a long ( approximately 30 amino acids) structured loop between strands beta2-beta3 (L(2-3)) of an OB-fold. To investigate the mechanism of ssDNA binding, we studied the free and bound states of Cdc13-DBD using NMR spectroscopy. Chemical shift changes indicate that the basic topology of the domain, including L(2-3), is essentially intact in the free state. Changes in slow and intermediate time scale dynamics, however, occur in L(2-3), while conformational changes distant from the DNA interface suggest an induced fit mechanism for binding in the 'hot spot' for binding affinity and specificity. These data point to an overall binding mechanism well adapted to the heterogeneous nature of yeast telomeres.
Nucleic Acids Research 04/2008; 36(5):1624-33. · 8.03 Impact Factor
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ABSTRACT: The cores of globular proteins are densely packed, resulting in complicated networks of structural interactions. These interactions in turn give rise to dynamic structural correlations over a wide range of time scales. Accurate analysis of these complex correlations is crucial for understanding biomolecular mechanisms and for relating structure to function. Here we report a highly accurate technique for inferring the major modes of structural correlation in macromolecules using likelihood-based statistical analysis of sets of structures. This method is generally applicable to any ensemble of related molecules, including families of nuclear magnetic resonance (NMR) models, different crystal forms of a protein, and structural alignments of homologous proteins, as well as molecular dynamics trajectories. Dominant modes of structural correlation are determined using principal components analysis (PCA) of the maximum likelihood estimate of the correlation matrix. The correlations we identify are inherently independent of the statistical uncertainty and dynamic heterogeneity associated with the structural coordinates. We additionally present an easily interpretable method ("PCA plots") for displaying these positional correlations by color-coding them onto a macromolecular structure. Maximum likelihood PCA of structural superpositions, and the structural PCA plots that illustrate the results, will facilitate the accurate determination of dynamic structural correlations analyzed in diverse fields of structural biology.
PLoS Computational Biology 03/2008; 4(2):e43. · 5.22 Impact Factor
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ABSTRACT: At relatively high protein concentrations (i.e., up to 100 mg/mL), recombinant human interleukin-1 receptor antagonist (rhIL-1ra) was found to exist in a monomer-dimer equilibrium controlled by solution ionic strength. Sedimentation equilibrium at 25 degrees C was used to measure the increase in the dimer dissociation constant (K(d)) as a function of ionic strength. K(d) increased from 2.0 to 12.6 mM as the solution ionic strength was increased from 0.011 to 0.184 molal. These K(d) values were used with both static light scattering and membrane osmometry data collected over a protein concentration range of 1-100 mg/mL to determine second osmotic virial coefficients. Expanding the second osmotic virial coefficient model to account for separate monomer-monomer (B(22)), monomer-dimer (B(23)), and dimer-dimer (B(33)) interactions reveals net monomer-dimer interactions are attractive, whereas the others are repulsive. Lastly, isothermal titration calorimetry dilution experiments showed that rhIL-1ra dimerization is enthalpically driven (DeltaH(dimerization) < 0), which is consistent with intermolecular cation-pi interactions previously proposed as the monomer-monomer contact sites in dimers.
Journal of Pharmaceutical Sciences 10/2007; 97(8):3035-50. · 3.06 Impact Factor
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ABSTRACT: Two critical interactions within the poliovirus RNA replication complex are those of the RNA-dependent RNA polymerase 3D with the viral proteins 3AB and VPg. 3AB is a membrane-binding protein responsible for the localization of the polymerase to the membranous vesicles at which replication occurs. VPg (a peptide comprising the 3B region of 3AB) is the 22-residue soluble product of 3AB cleavage and serves as the protein primer for RNA replication. The detailed interactions of these proteins with the RNA-dependent RNA polymerase 3D were analyzed to elucidate the precise roles of 3AB and VPg in the viral RNA replication complex. Using a membrane-based pull-down assay, we have identified a binding "hot-spot" spanning residues 100 to 104 in the 3B (VPg) region of 3AB which plays a critical role in mediating the interaction of 3AB with the polymerase. Isothermal titration calorimetry shows that the interaction of VPg with 3D is enthalpically driven, with a dissociation constant of 11 microM. Mutational analyses of VPg indicate that a subset of the residues important for 3AB-3D binding are also important for VPg-3D binding. Two residues in particular, P14 and R17, were shown to be absolutely critical for the binding interaction. This work provides the direct characterization of two binding interactions critical for the replication of this important class of viruses and identifies a conserved polymerase binding sequence responsible for targeting the polymerase.
Journal of Virology 07/2007; 81(12):6369-78. · 5.40 Impact Factor
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ABSTRACT: Procrustes analysis involves finding the optimal superposition of two or more "forms" via rotations, translations, and scalings. Procrustes problems arise in a wide range of scientific disciplines, especially when the geometrical shapes of objects are compared, contrasted, and analyzed. Classically, the optimal transformations are found by minimizing the sum of the squared distances between corresponding points in the forms. Despite its widespread use, the ordinary unweighted least-squares (LS) criterion can give erroneous solutions when the errors have heterogeneous variances (heteroscedasticity) or the errors are correlated, both common occurrences with real data. In contrast, maximum likelihood (ML) estimation can provide accurate and consistent statistical estimates in the presence of both heteroscedasticity and correlation. Here we provide a complete solution to the nonisotropic ML Procrustes problem assuming a matrix Gaussian distribution with factored covariances. Our analysis generalizes, simplifies, and extends results from previous discussions of the ML Procrustes problem. An iterative algorithm is presented for the simultaneous, numerical determination of the ML solutions.
Proceedings of the National Academy of Sciences 01/2007; 103(49):18521-7. · 9.68 Impact Factor
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ABSTRACT: The ends of eukaryotic linear chromosomes are unique structures that require special management by the cell. If left unattended, the ends are inappropriately processed, leading to genomic instability and problems with proliferation. Telomeres are specialized nucleoprotein structures that restore chromosome stability by protecting and maintaining chromosome ends. Proper telomere function is facilitated, in part, by the telomere-end protection (TEP) family of proteins, which targets the 3' single-stranded (ss) overhang region of the telomere via a specialized ssDNA-binding domain (DBD). With the recent availability of the structures of these DBDs, the ssDNA-binding characteristics of TEP proteins can be compared and the common underlying mechanisms of ssDNA recognition identified, thus providing insights into telomere function.
Trends in Biochemical Sciences 10/2006; 31(9):516-25. · 10.85 Impact Factor
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ABSTRACT: THESEUS is a command line program for performing maximum likelihood (ML) superpositions and analysis of macromolecular structures. While conventional superpositioning methods use ordinary least-squares (LS) as the optimization criterion, ML superpositions provide substantially improved accuracy by down-weighting variable structural regions and by correcting for correlations among atoms. ML superpositioning is robust and insensitive to the specific atoms included in the analysis, and thus it does not require subjective pruning of selected variable atomic coordinates. Output includes both likelihood-based and frequentist statistics for accurate evaluation of the adequacy of a superposition and for reliable analysis of structural similarities and differences. THESEUS performs principal components analysis for analyzing the complex correlations found among atoms within a structural ensemble. AVAILABILITY: ANSI C source code and selected binaries for various computing platforms are available under the GNU open source license from http://monkshood.colorado.edu/theseus/ or http://www.theseus3d.org.
Bioinformatics 10/2006; 22(17):2171-2. · 5.47 Impact Factor
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ABSTRACT: The protection of telomeres 1 (Pot1) proteins specifically recognize the single-stranded 3' end of the telomere, an activity essential for sustained cellular viability and proliferation. The current model for the telomeric single-stranded DNA (ssDNA) binding activity of Schizosaccharomyces pombe Pot1 is based on a 20 kDa fragment, Pot1pN. Recent biochemical studies suggest that SpPot1 contains a larger ssDNA-binding domain and we have identified a novel ssDNA-binding domain similar in size to the human Pot1 domain. This domain, Pot1(1-389), binds extremely tightly to an oligonucleotide consisting of two conserved hexameric S. pombe telomere repeats, d(GGTTACGGTTAC), with an affinity approximately 4000-fold tighter than Pot1pN binds its cognate ssDNA. The Pot1(1-389)/ssDNA complex exhibits a half-life of 53 min, consistent with that estimated for full-length SpPot1 and significantly longer than that of Pot1pN. Single nucleotide substitutions reveal that, in contrast to Pot1pN, tandem trinucleotide repeats (GTT) within d(GGTTACGGTTAC) are specifically recognized by Pot1(1-389). Interestingly, certain single nucleotide substitutions that impacted Pot1pN binding exhibited no effect on binding affinity by Pot1(1-389). However, these substitutions reduced binding affinity when simultaneously substituted in each hexameric repeat. The non-additive nature of these substitutions suggests that certain nucleotides are coupled through the ability of the flexible ssDNA oligonucleotide to adopt alternate, thermodynamically equivalent conformations. The biochemical behavior of Pot1(1-389) is more similar to that of the full-length SpPot1 protein than to that of Pot1pN, making Pot1(1-389) a valuable domain for the future study of how full-length SpPot1 interacts with telomeric ssDNA.
Journal of Molecular Biology 09/2006; 361(1):80-93. · 4.00 Impact Factor
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ABSTRACT: The single-strand overhang present at telomeres plays a critical role in mediating both the capping and telomerase regulation functions of telomeres. The telomere end-binding proteins, Cdc13 in Saccharomyces cerevisiae, Pot1 in higher eukaryotes, and TEBP in the ciliated protozoan Oxytricha nova, exhibit sequence-specific binding to their respective single-strand overhangs. S. cerevisiae telomeres are composed of a heterogeneous mixture of GT-rich telomeric sequence, unlike in higher eukaryotes which have a simple repeat that is maintained with high fidelity. In yeast, the telomeric overhang is recognized by the essential protein Cdc13, which coordinates end-capping and telomerase activities at the telomere. The Cdc13 DNA-binding domain (Cdc13-DBD) binds these telomere sequences with high affinity (3 pM) and sequence specificity. To better understand the basis for this remarkable recognition, we have investigated the binding of the Cdc13-DBD to a series of altered DNA substrates. Although an 11-mer of GT-rich sequence is required for full binding affinity, only three of these 11 bases are recognized with high specificity. This specificity differs from that observed in the other known telomere end-binding proteins, but is well suited to the specific role of Cdc13 at yeast telomeres. These studies expand our understanding of telomere recognition by the Cdc13-DBD and of the unique molecular recognition properties of ssDNA binding.
Biochemistry 02/2006; 45(3):871-9. · 3.42 Impact Factor
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ABSTRACT: Many dissimilar protein sequences fold into similar structures. A central and persistent challenge facing protein structural analysis is the discrimination between homology and convergence for structurally similar domains that lack significant sequence similarity. Classic examples are the OB-fold and SH3 domains, both small, modular beta-barrel protein superfolds. The similarities among these domains have variously been attributed to common descent or to convergent evolution. Using a sequence profile-based phylogenetic technique, we analyzed all structurally characterized OB-fold, SH3, and PDZ domains with less than 40% mutual sequence identity. An all-against-all, profile-versus-profile analysis of these domains revealed many previously undetectable significant interrelationships. The matrices of scores were used to infer phylogenies based on our derivation of the relationships between sequence similarity E-values and evolutionary distances. The resulting clades of domains correlate remarkably well with biological function, as opposed to structural similarity, indicating that the functionally distinct sub-families within these superfolds are homologous. This method extends phylogenetics into the challenging "twilight zone" of sequence similarity, providing the first objective resolution of deep evolutionary relationships among distant protein families.
Journal of Molecular Biology 01/2006; 354(3):722-37. · 4.00 Impact Factor
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ABSTRACT: Telomerase is a ribonucleoprotein complex responsible for extending the ends of eukaryotic chromosomes. Structural and biophysical studies of this enzyme have been limited by the inability to produce large amounts of recombinant protein. Here we perform a high-throughput screen to map regions of the Tetrahymena thermophila TERT (Telomerase Reverse Transcriptase) protein that are overexpressed in a soluble form in Escherichia coli using a GFP-fusion system. Many of the soluble protein domains identified do not coincide with domains inferred from multiple sequence alignment, so screening for fluorescent colonies provided information not otherwise readily obtained. The method revealed an essential, independently folded N-terminal domain that was expressed and purified with high yield and found to be suitable for structural analysis. These results provide a tool for future structural and biophysical studies of TERT.
Protein Science 09/2005; 14(8):2051-8. · 2.80 Impact Factor
