bluuebunny/crossref_metadata_2025_split huggingface dataset:
Construction of an overproduction vector containing the novel srp (sterically repressed) promoter
Authors: Ezaz‐Nikpay Khosro , Uchino Ken , Lerner Rachel E. , Verdine Gregory L. 
Published: July 2010

AbstractWe report the design, synthesis, and evaluation of a novel Escherichia coli promoter intended for use in overproduction of proteins that are deleterious to the host. In this sterically repressed promoter (srp), the lac operator site is positioned between the ‐10 and ‐35 elements, where it can interfere sterically with RNA polymerase and thereby prevent assembly of a poised transcriptional complex. An srp‐containing phagemid, pKEN1, and a tac‐containing phagemid, pHN1, which has been widely used in protein overproduction but is often unstable, are compared with respect to levels of uninduced and induced protein expression. The level of uninduced protein synthesis by the srp promoter in vivo is ∼ 50% of that observed with tac, whereas the leveIs of induced protein synthesis with the 2 vectors are approximately equal. A remarkable increase in stability of overproduction and growth was observed when the toxic Ada protein was overproduced in pKEN1, demonstrating the potential utility of this vector in overproducing toxic proteins.
Do salt bridges stabilize proteins? A continuum electrostatic analysis
Authors: Hendsch Zachary S. , Tidor Bruce 
Published: July 2010

AbstractThe electrostatic contribution to the free energy of folding was calculated fo 21 salt bridges in 9 r X‐ray crystal structures using a continuum electrostatic approach with the DELPHI computer‐program package. The majority (17) were found to be electrostatically destabilizing; the average free energy change, which is analogous to mutation of salt bridging side chains to hydrophobic isosteres, was calculated to be 3.5 kcal/mol. This is fundamentally different from stability measurements using pKa shifts, which effectively measure the strength of a salt bridge relative to 1 or more charged hydrogen bonds. The calculated effect was due to a large, unfavorable desolvation contribution that was not fully compensated by favorable interactions within the salt bridge and between salt‐bridge partners and other polar and charged groups in the folded protein. Some of the salt bridges were studied in further detail to determine the effect of the choice of values for atomic radii, internal protein dielectric constant, and ionic strength used in the calculations. Increased ionic strength resulted in little or no change in calculated stability for 3 of 4 salt bridges over a range of 0.1‐0.9 M.The results suggest that mutation of salt bridges, particularly those that are buried, to “hydrophobic bridges” (that pack at least as well as wild type) can result in proteins with increased stability. Due to the large penalty for burying uncompensated ionizable groups, salt bridges could help to limit the number of low free energy conformations of a molecule or complex and thus play a role in determining specificity (i.e., the uniqueness of a protein fold or protein‐ligand binding geometry).
Cysteine scanning mutagenesis of the N‐terminal 32 amino acid residues in the lactose permease of Escherichia coli
Authors: Sahin‐Tóth Miklós , Persson Bengt , Schwieger Jeremy , Cohan Pejman , Kaback H.Ronald 
Published: July 2010

AbstractUsing a functional lactose permease mutant devoid of Cys residues (C‐less permease), each amino acid residue in the hydrophilic N‐terminus and the first putative transmembrane helix was systematically replaced with Cys (from Tyr‐2 to Trp‐33). Twenty‐three of 32 mutants exhibit high lactose accumulation (70‐100% or more of C‐less), and an additional 8 mutants accumulate to lower but highly significant levels, Surprisingly, Cys replacement for Gly‐24 or Tyr‐26 yields fully active permease molecules, and permease with Cys in place of Pro‐28 also exhibits significant transport activity, although previous mutagenesis studies on these residues suggested that they may be required for lactose transport. As expected, Cys replacement for Pro‐31 completely inactivates, in agreement with previous findings indicating that “helix‐breaking” propensity at this position is necessary for full activity (Consler TG, Tsolas 0, Kaback HR, 1991, Biochemistry 30:1291‐1297). Twenty‐nine mutants are present in the membrane in amounts comparable to C‐less permease, whereas membrane levels of mutants Tyr‐3 → Cys and Phe‐12 → Cys are slightly reduced, as judged by immunological techniques. Dramatically, mutant Phe‐9 →Cys is hardly detectable when expressed from the lac promoter/operator at a relatively low rate, but is present in the membrane in a stable form when expressed at a high rate from the T7 promoter. Finally, studies with N‐ethylmaleimide show that 6 Cys‐replacement mutants that cluster at the C‐terminal end of putative helix I are inactivated significantly. The results demonstrate that although no residue per se in this region is essential for activity, the structural integrity of the periplasmic half of the first transmembrane helix is important for active lactose transport.
Structures of larger proteins, protein‐ligand and protein‐DNA complexes by multidimensional heteronuclear NMR
Authors: Clore G. Marius , Gronenborn Angela M. 
Published: July 2010

AbstractThe recent development of a whole panoply of multidimensional heteronuclear‐edited and ‐filtered NMR experiments has revolutionized the field of protein structure determination by NMR, making it possible to extend the methodology from the IO‐kDa limit of conventional 2‐dimensional NMR to systems up to potentially 35‐40 kDa. The basic strategy for solving 3‐dimensional structures of larger proteins and protein‐ligand complexes in solution using 3‐ and 4‐dimensional NMR spectroscopy is summarized, and the power of these methods is illustrated using 3 examples: interleukin‐lβ, the complex of calmodulin with a target peptide, and the specific complex of the transcription factor GATA‐1 with its cognate DNA target site.
Accommodation of insertion mutations on the surface and in the interior of staphylococcal nuclease
Authors: Keefe Lisa J. , Quirk Stephen , Gittis Apostolos , Sondek John , Lattman Eaton E. 
Published: July 2010

AbstractAlignment of homologous amino acid sequences reveals that insertion mutations are fairly common in evolution. Hitherto, the structural consequences of insertion mutations on the surface and in the interior of proteins of known structure have received little attention. We report here the high‐resolution X‐ray crystal structures of 2 site‐directed insertion mutants of staphylococcal nuclease. The structure of the first insertion mutant, in which 2 glycine residues were inserted on the protein surface in the amino‐terminal β‐strand, has been solved to 1.70 Å resolution and refined to a crystallographic R value of 0.182. The inserted residues are accommodated in a special 3‐residue β‐bulge. A bridging water molecule in the newly created cavity satisfies the hydrogen bonding requirements of the β‐sheet by forming a bifurcated hydrogen bond to 1 β‐strand, and a single hydrogen bond to the other β‐strand. The second insertion mutant contains a single leucine residue inserted at the end of the third β‐strand. The structure was solved to 2.0 Å resolution and refined to a final R value of 0.196. The insertion is accommodated in a register shift that changes the conformation of the flexible loop portion of the molecule, relaxing and widening the omega turn. This structural alteration results in changes in position and coordination of a bound calcium ion important for catalysis. These structures illustrate important differences in how amino acid insertions are accommodated: as localized bulges, and as extensive register shifts.
Autonomous folding of the excised coenzyme‐binding domain of D‐glyceraldehyde 3‐phosphate dehydrogenase from Thermotoga maritima
Authors: Jecht Monika , Tomschy Andrea , Kirschner Kasper , Jaenicke Rainer 
Published: July 2010

AbstractAn important question in protein folding is whether compact substructures or domains are autonomous units of folding and assembly. The protomer of the tetrameric D‐glyceraldehyde‐3‐phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima has a complex coenzyme‐binding domain, in which residues 1‐146 form a compact substructure with the last 31 residues (313‐333). Here it is shown that the gene of a single‐chain protein can be expressed in Escherichia coli after deleting the 163 codons corresponding to the interspersed catalytic domain (150‐312). The purified gene product is a soluble, monomeric protein that binds both NAD+ and NADH strongly and possesses the same unfolding transition induced by guanidinium chloride as the native tetramer. The autonomous folding of the coenzyme‐binding domain has interesting implications for the folding, assembly, function, and evolution of the native enzyme.
Characteristics of a de novo designed protein
Authors: Tanaka Toshiki , Kimura Hiromi , Hayashi Mayumi , Fujiyoshi Yosinori , Fukuhara Ken‐Ichi , Nakamura Haruki 
Published: July 2010

AbstractA series of 204 amino acid proteins intended to form TIM (triose phosphate isomerase) barrel structures were designed de novo. Each protein was synthesized by expression of the synthetic gene as a fusion protein with a portion of human growth hormone in an Escherichia coli host. After BrCN treatment, the protein was purified to homogeneity. The refolded proteins are globular and exist as monomers. One of the designed proteins is stable toward guanidine hydrochloride (GuHCl) denaturation, with a midpoint of 2.6 M determined from CD and tryp‐tophan fluorescence measurements. The GuHCl denaturation is well described by a 2‐state model. The NMR spectra, the thermal denaturation curves, and the 1‐anilino‐8‐naphthalene sulfonic acid binding imply that the stability of the protein arises mainly from hydrophobic interactions, which are probably of a nonspecific nature. The protein has a similar shape to that of rabbit triosephosphate isomerase, as determined by electron microscopy.
Circular dichroism and crosslinking studies of the interaction between four neurotrophins and the extracellular domain of the low‐affinity neurotrophin receptor
Authors: Timm David E. , Ross Alonzo H. , Neet Kenneth E. 
Published: July 2010

AbstractInteractions between the purified recombinant receptor extracellular domain (RED) of the human low‐affinity neurotrophin receptor (LANR) and recombinant human brain‐derived neurotrophic factor, neurotrophin‐3 (NT‐3) and neurotrophin‐4/5 have been studied by chemical crosslinking and circular dichroism. Conformational changes subsequent to binding have been shown by these procedures. First, relative affinities of the neurotrophins for RED were determined by binding competition assays in which radioiodinated nerve growth factor (NGF) from mouse submaxillary gland was crosslinked to RED in the presence of varying amounts of unlabeled neurotrophin competitors. RED bound each of the 3 recombinant human neurotrophins with affinities that were indistinguishable from authentic mouse NGF. These results are the first measurement of binding of the neurotrophin family to their common receptor using purified components. In order to study the effect of binding on the conformation of the proteins, CD measurements were made before and after mixing neurotrophins and RED, as had previously been done with NGF and RED (Timm DE, Vissavajjhala P, Ross AH, Neet KE, 1992, Protein Sci 1:1023‐1031). Similar changes in CD spectra occurred upon combination of each of the neurotrophins and RED, with negative changes near 220‐225 nm and positive changes near 190‐200 nm; however, significant differences existed among the various neurotrophin‐RED difference spectra. The NT‐3/RED complex showed the largest spectral change and NGF the smallest. Thus, specific conformational changes in secondary structure of neurotrophin, RED, or both accompany the binding of each neurotrophin to the extracellular domain of the LANR.
Regeneration of catalytic activity of glutamine synthetase mutants by chemical activation: Exploration of the role of arginines 339 and 359 in activity
Authors: Dhalla Adil M. , Li Bin , Alibhai Murtaza F. , Yost Kenneth J. , Hemmingsen Jens M. , Atkins William M. , Schineller Jeffrey , Villafranca Joseph J. 
Published: July 2010

AbstractIn order to understand the nature of ATP and L‐glutamate binding to glutamine synthetase, and the involvement of Arg 339 and Arg 359 in catalysis, these amino acids were changed to cysteine via site‐directed mutagenesis. Individual mutations (Arg → Cys) at positions 339 and 359 led to a sharp drop in catalytic activity. Additionally, the Km values for the substrates ATP and glutamate were elevated substantially above the values for wild‐type (WT) enzyme. Each cysteine was in turn chemically modified to an arginine “analog” to attempt to “rescue” catalytic activity by covalent modification; 2‐chloroacetamidine (CA) (producing a thioether) and 2,2′‐dithiobis (acetamidine) (DTBA) (producing a disulfide) were the reagents used to effect these chemical transformations. Upon reaction with CA, both R339C and R359C mutants showed a significant regain of catalytic activity (50% and 70% of WT, respectively) and a drop in Km value for ATP close to that for WT enzyme. With DTBA, chemically modified R339C had a greater kcat than WT glutamine synthetase, but chemically modified R359C only regained a small amount of activity. Modification with DTBA was quantitative for each mutant and each modified enzyme had similar Km values for both ATP and glutamate. The high catalytic activity of DTBA‐modified R339C could be reversed to that of unmodified R339C by treatment with dithiothreitol, as expected for a modified enzyme containing a disulfide bond. Modification of each cysteine‐containing mutant to a lysine “analog” was accomplished using 3‐bromopropylamine (BPA). The R339C mutant, upon modification with BPA, had a greater kcat than WT enzyme; however, the R359C mutant did not show significant regeneration of activity with this reagent. The data are consistent with X‐ray crystallographic studies showing Arg 339 and Arg 359 at the active site of glutamine synthetase (Liaw SH, Eisenberg D, 1994, Biochemistry 33:675‐681) interacting with ATP, glutamate, and intermediates along the catalytic pathway. Because enzyme activity could be restored for the R339C mutant by monofunctional (amine) and bifunctional (amidine) reagents, Arg 339 most likely interacts with substrates in a monodentate fashion. Conversely, Arg 359 seems to interact bifunctionally with substrates because covalent modification of R359C with BPA did not lead to a significant regain of catalytic activity.
Self‐organized neural maps of human protein sequences
Authors: Ferrán Edgardo A. , Pflugfelder Bernard , Ferrara Pascual 
Published: July 2010

AbstractWe have recently described a method based on artificial neural networks to cluster protein sequences into families. The network was trained with Kohonen's unsupervised learning algorithm using, as inputs, the matrix patterns derived from the dipeptide composition of the proteins. We present here a large‐scale application of that method to classify the 1,758 human protein sequences stored in the SwissProt database (release 19.0), whose lengths are greater than 50 amino acids. In the final 2‐dimensional topologically ordered map of 15 × 15 neurons, proteins belonging to known families were associated with the same neuron or with neighboring ones. Also, as an attempt to reduce the time‐consuming learning procedure, we compared 2 learning protocols: one of 500 epochs (100 SUN CPU‐hours [CPU‐h]), and another one of 30 epochs (6.7 CPU‐h). A further reduction of learning‐computing time, by a factor of about 3.3, with similar protein clustering results, was achieved using a matrix of 11×11 components to represent the sequences. Although network training is time consuming, the classification of a new protein in the final ordered map is very fast (14.6 CPU‐seconds). We also show a comparison between the artificial neural network approach and conventional methods of biosequence analysis.
A structural role for arginine in proteins: Multiple hydrogen bonds to backbone carbonyl oxygens
Authors: Borders C.L. , Broadwater John A. , Bekeny Paula A. , Salmon Johanna E. , Lee Ann S. , Eldridge Aimee M. , Pett Virginia B. 
Published: July 2010

AbstractWe propose that arginine side chains often play a previously unappreciated general structural role in the maintenance of tertiary structure in proteins, wherein the positively charged guanidinium group forms multiple hydrogen bonds to backbone carbonyl oxygens. Using as a criterion for a “structural” arginine one that forms 4 or more hydrogen bonds to 3 or more backbone carbonyl oxygens, we have used molecular graphics to locate arginines of interest in 4 proteins: Arg 180 in Thermus thermophilus manganese superoxide dismutase, Arg 254 in human carbonic anhydrase II, Arg 31 in Streptomyces rubiginosus xylose isomerase, and Arg 313 in Rhodospirillum rubrum ribulose‐1,5‐bisphosphate carboxylase/oxygenase. Arg 180 helps to mold the active site channel of superoxide dismutase, whereas in each of the other enzymes the structural arginine is buried in the “mantle” (i.e., inside, but near the surface) of the protein interior well removed from the active site, where it makes 5 hydrogen bonds to 4 backbone carbonyl oxygens. Using a more relaxed criterion of 3 or more hydrogen bonds to 2 or more backbone carbonyl oxygens, arginines that play a potentially important structural role were found in yeast enolase, Bacillus stearothermophilus glyceraldehyde‐3‐phosphate dehydrogenase, bacteriophage T4 and human lysozymes, Enteromorpha prolifera plastocyanin, HIV‐1 protease, Trypanosoma brucei brucei and yeast triosephosphate isomerases, and Escherichia coli trp aporepressor (but not trp repressor or the trp repressor/operator complex). In addition to helping form the active site funnel in superoxide dismutase, the structural arginines found in this study play such diverse roles as stapling together 3 strands of backbone from different regions of the primary sequence, and tying α‐helix to α‐helix, βturn to β‐turn, and subunit to subunit.
De novo protein design using pairwise potentials and a genetic algorithm
Authors: Jones David T. 
Published: July 2010

AbstractOne of the major goals of molecular biology is to understand how protein chains fold into a unique 3‐dimensional structure. Given this knowledge, perhaps the most exciting prospect will be the possibility of designing new proteins to perform designated tasks, an application that could prove to be of great importance in medicine and biotechnology. It is possible that effective protein design may be achieved without the requirement for a full understanding of the protein folding process. In this paper a simple method is described for designing an amino acid sequence to fit a given 3‐dimensional structure. The compatibility of a designed sequence with a given fold is assessed by means of a set of statistically determined potentials (including interresidue pairwise and solvation terms), which have been previously applied to the problem of protein fold recognition. In order to generate sequences that best fit the fold, a genetic algorithm is used, whereby the sequence is optimized by a stochastic search in the style of natural selection.
Distribution of distances between the tryptophan and the N‐terminal residue of melittin in its complex with calmodulin, troponin C, and phospholipids
Authors: Lakowicz Joseph R. , Gryczynski Ignacy , Laczko Gabor , Wiczk Wieslaw , Johnson Michael L. 
Published: July 2010

AbstractWe used frequency‐domain measurements of fluorescence resonance energy transfer to measure the distribution of distances between Trp‐19 of melittin and a 1‐dimethylamino‐5‐sulfonylnaphthalene (dansyl) residue on the N‐terminal‐α‐amino group. Distance distributions were obtained for melittin free in solution and when complexed with calmodulin (CaM), troponin C (TnC), or palmitoyloleoyl‐L‐α‐phosphatidylcholine (POPC) vesicles. A wide range of donor (Trp‐19)‐to‐acceptor (dansyl) distances was found for free melittin, which is consistent with that expected for the random coil state, characterized by a Gaussian width (full width at half maxima) of 28.2 Å. In contrast, narrow distance distributions were found for melittin complexed with CaM, 8.2 Å, or with POPC vesicles, 4.9 Å. A somewhat wider distribution was found for the melittin complex with TnC, 12.8 Å, suggesting the presence of heterogeneity in the mode of binding between melittin and TnC. For all the complexes the mean Trp‐19 to dansyl distance was near 20 Å. This value is somewhat smaller than expected for the free α‐helical state of melittin, suggesting that binding with CaM or TnC results in a modest decrease in the length of the melittin molecule.
Thermodynamics of barnase unfolding
Authors: Griko Yuri V. , Makhatadze George I. , Privalov Peter L. , Hartley Robert W. 
Published: July 2010

AbstractThe thermodynamics of barnase denaturation has been studied calorimetrically over a broad range of temperature and pH. It is shown that in acidic solutions the heat denaturation of barnase is well approximated by a 2‐state transition. The heat denaturation of barnase proceeds with a significant increase of heat capacity, which determines the temperature dependencies of the enthalpy and entropy of its denaturation. The partial specific heat capacity of denatured barnase is very close to that expected for the completely unfolded protein. The specific denaturation enthalpy value extrapolated to 130 °C is also close to the value expected for the full unfolding. Therefore, the calorimetrically determined thermodynamic characteristics of barnase denaturation can be considered as characteristics of its complete unfolding and can be correlated with structural features — the number of hydrogen bonds, extent of van der Waals contacts, and the surface areas of polar and nonpolar groups. Using this information and thermodynamic information on transfer of protein groups into water, the contribution of various factors to the stabilization of the native structure of barnase has been estimated. The main contributors to the stabilization of the native state of barnase appear to be intramolecular hydrogen bonds. The contributions of van der Waals interactions between nonpolar groups and those of hydration effects of these groups are not as large if considered separately, but the combination of these 2 factors, known as hydrophobic interactions, is of the same order of magnitude as the contribution of hydrogen bonding.