bluuebunny/crossref_metadata_2025 huggingface dataset:
Helical peptides with three pairs of Asp‐Arg and Glu‐Arg residues in different orientations and spacings
Authors: Huyghues‐Despointes Beatrice M.P. , Martin Scholtz J. , Baldwin Robert L.
Published: July 2010

The helix‐stabilizing effects of repeating pairs of Asp‐Arg and Glu‐Arg residues have been characterized using a peptide system of the same design used earlier to study Glu‐Lys (Marqusee, S. & Baldwin, R.L., 1987, Proc. Natl. Acad. Sci. USA 84, 8898–8902) and Asp‐Lys ion pairs (Marqusee, S. & Baldwin, R.L., 1990, In Protein Folding [Gierasch, L.M. & King, J., Eds.], pp. 85–94, AAAS, Washington, D.C.). The consequences of breaking ion pair and charge‐helix dipole interactions by titration to pH 2 have been compared with the results of screening these interactions with NaCl at pH 7.0 and pH 2.5. The four peptides in each set contain three pairs of acidic (A) and basic (B) residues spaced either i, i + 4 or i, i + 3 apart. In one peptide of each kind the pairwise order of residues is AB, with the charges oriented favorably to the helix macrodipole, and in the other peptide the order is BA.

The results are as follows: (1) Remarkably, both Asp‐Arg and Glu‐Arg peptides show the same pattern of helix stabilization at pH 7.0 found earlier for Glu‐Lys and Asp‐Lys peptides: i + 4 AB > i + 4 BA = i + 3 AB > i + 3 BA. (2) The ion pairs and charge‐helix dipole interactions cannot be cleanly separated, but the results suggest that both interactions make important contributions to helix stability. Because the four peptides of each set have nearly identical compositions, the large differences in helix content within each set cannot be caused by differences in length and sequence. (3) Significant differences in helix content remain at 5 M NaCl, where ion pair interactions are completely screened. The residual interactions may either be nonscreenable H‐bond interactions or remaining charge‐helix dipole interactions.
Increasing nitrogenase catalytic efficiency for MgATP by changing serine 16 of its Fe protein to threonine: Use of Mn2+ to show interaction of serine 16 with Mg2+
Authors: Seefeldt Lance C. , Mortenson Leonard E.
Published: July 2010

MgATP‐binding and hydrolysis are an integral part of the nitrogenase catalytic mechanism. We are exploring the function of MgATP hydrolysis in this reaction by analyzing the properties of the Fe protein (FeP) component of Azotobacter vinelandii nitrogenase altered by site‐directed mutagenesis. We have previously (Seefeldt, L.C., Morgan, T.V., Dean, D.R., & Mortenson, L.E., 1992, J. Biol. Chem. 267, 6680–6688) identified a region near the N‐terminus of FeP that is involved in interaction with MgATP. This region of FeP is homologous to the well‐known nucleotide‐binding motif GXXXXGKS/T. In the present work, we examined the function of the four hydroxyl‐containing amino acids immediately C‐terminal to the conserved lysine 15 that is involved in interaction with the β‐phosphate of MgATP. We have established, by altering independently Thr 17, Thr 18, and Thr 19 to alanine, that a hydroxyl‐containing residue is not needed at these positions for FeP to function. In contrast, an hydroxyl‐containing amino acid at position 16 was found to be critical for FeP function. When the strictly conserved Ser 16 was altered to Ala, Cys, Asp, or Gly, the FeP did not support N2 fixation when expressed in place of the wild‐type FeP in A. vinelandii. Altering Ser 16 to Thr (S16T), however, resulted in the expression of an FeP that was partially active. This S16T FeP was purified to homogeneity, and its biochemical examination allowed us to assign a catalytic function to this hydroxyl group in the nitrogenase mechanism. Of particular importance was the finding that the S16T FeP had a significantly higher affinity for MgATP than the wild‐type FeP, with a measured Km of 20 μM compared to the wild‐type FeP Km of 220 μM. This increased kinetic affinity for MgATP was reflected in a significantly stronger binding of the S16T FeP for MgATP. In contrast, the affinity for MgADP, which binds at the same site as MgATP, was unchanged. The catalytic efficiency (kcat/Km) of S16T FeP was found to be 5.3‐fold higher than for the wild‐type FeP, with the S16T FeP supporting up to 10 times greater nitrogenase activity at low MgATP concentrations. This indicates a role for the hydroxyl group at position 16 in interaction with MgATP but not MgADP. The site of interaction of this residue was further defined by examining the properties of wild‐type and S16T FePs in utilizing MnATP compared with MgATP. The S16T FeP was severely compromised in its interaction with MnATP, suggesting a mechanism where the hydroxyl group of amino acid 16 interacts with the Mg2+ of bound MgATP.
Probing the pyrophosphate‐binding site in potato tuber UDP‐glucose pyrophosphorylase with pyridoxal diphosphate
Authors: Kazuta Yasuaki , Tagaya Mitsuo , Tanizawa Katsuyuki , Fukui Toshio
Published: July 2010

Potato tuber UDP‐glucose pyrophosphorylase (EC 2.7.7.9) catalyzes the reversible uridylyl transfer from UDP‐glucose to MgPPi forming glucose 1‐phosphate and MgUTP, according to an ordered bi‐bi mechanism in which UDP‐glucose and MgPPi bind in this order. To probe the active site of this enzyme, we have applied pyridoxal 5′‐diphosphate, a reactive PPi analogue. The enzyme was rapidly inactivated when incubated with the reagent in the presence of Mg2+ followed by sodium borohydride reduction. The degree of the inactivation was decreased by MgUTP, MgPPi, and glucose 1‐phosphate, but enhanced by UDP‐glucose. The enhancement was prevented by co‐addition of Pi, the competitive inhibitor with respect to PPi. The complete inactivation corresponded to the incorporation of 0.9–1.1 mol of reagent/mol of enzyme monomer. In the presence of UDP‐glucose, labels were almost exclusively incorporated into Lys‐329. Thus, this residue may be located near the bound MgPPi and its modification is promoted, probably through conformational changes, by the binding of UDP‐glucose to the enzyme. The results of the modification by the same reagent of the mutant enzymes in which Lys‐329 and Lys‐263 are individually replaced by Gln suggest the roles of these lysyl residues in the binding of MgPPi and in the UDP‐glucose‐induced conformational changes, respectively.
The binding domain structure of retinoblastoma‐binding proteins
Authors: Figge James , Breese Kimberly , Vajda Sandor , Zhu Qing‐Lin , Eisele Leslie , Andersen Thomas T. , Maccoll Robert , Friedrich Thomas , Smith Temple F.
Published: July 2010

The retinoblastoma gene product (Rb), a cellular growth suppressor, complexes with viral and cellular proteins that contain a specific binding domain incorporating three invariant residues: Leu‐X‐Cys‐X‐Glu, where X denotes a nonconserved residue. Hydrophobic and electrostatic properties are strongly conserved in this segment even though the nonconserved amino acids vary considerably from one Rb‐binding protein to another. In this report, we present a diagnostic computer pattern for a high‐affinity Rb‐binding domain featuring the three conserved residues as well as the conserved physico‐chemical properties. Although the pattern encompasses only 10 residues (with only 4 of these explicitly defined), it exhibits 100% sensitivity and 99.95% specificity in database searches. This implies that a certain pattern of structural and physico‐chemical properties encoded by this short sequence is sufficient to govern specific Rb binding. We also present evidence that the secondary structural conformation through this region is important for effective Rb binding.
Characterization and 2D NMR study of the stable [9–21, 15–27] 2 disulfide intermediate in the folding of the 3 disulfide trypsin inhibitor EETI II
Authors: Le‐Nguyen D. , Heitz A. , Chiche L. , Hajji M. El , Castro B.
Published: July 2010

The three disulfide Ecballium elaterium trypsin inhibitor II (EETI II) reduction with dithiothreitol (DTT) and re‐oxidation of the fully reduced derivative have been examined. A common stable intermediate has been observed for both processes. Isolation and sequencing of carboxymethylated material showed that the intermediate lacks the [2–19] bridge. The NMR study showed a very strong structural conservation as compared to the native EETI II, suggesting that the bridges are the [9–21] and [15–27] native ones. The differences occurred in sections 2–7 (containing the free cysteine 2 and the Arg 4‐Ile 5 active site) and 19–21 (containing the second free cysteine). Distance geometry calculations and restrained molecular dynamics refinements were also in favor of a [9–21, 15–27] arrangement and resulted in a well‐conserved (7–28) segment.
Identification of a nucleic acid‐binding region within the largest subunit ofDrosophila melanogasterRNA polymerase II
Authors: Kontermann Roland E. , Kobor Michael , Bautz Ekkehard K. F.
Published: July 2010

The largest and the second‐largest subunit of the multisubunit eukaryotic RNA polymerases are involved in interaction with the DNA template and the nascent RNA chain. Using Southwestern DNA‐binding techniques and nitrocellulose filter binding assays of bacterially expressed fusion proteins, we have identified a region of the largest, 215‐kDa, subunit ofDrosophilaRNA polymerase II that has the potential to bind nucleic acids nonspecifically. This nucleic acid‐binding region is located between amino acid residues 309–384 and is highly conserved within the largest subunits of eukaryotic and bacterial RNA polymerases. A homology to a region of the DNA‐binding cleft ofEscherichia coliDNA polymerase I involved in binding of the newly synthesized DNA duplex provides indirect evidence that the nucleic acid‐binding region of the largest subunit participates in interaction with double‐stranded nucleic acids during transcription. The nonspecific DNA‐binding behavior of the region is similar to that observed for the native enzyme in nitrocellulose filter binding assays and that of the separated largest subunit in Southwestern assays. A high content of basic amino acid residues is consistent with the electrostatic nature of nonspecific DNA binding by RNA polymerases.
Retroviral nucleocapsid proteins possess potent nucleic acid strand renaturation activity
Authors: Dib‐Hajj Fadia , Khan Raza , Giedroc David P.
Published: July 2010

The nucleocapsid protein (NC) is the major genomic RNA binding protein that plays integral roles in the structure and replication of all animal retroviruses. In this report, select biochemical properties of recombinant Mason‐Pfizer monkey virus (MPMV) and HIV‐1 NCs are compared. Evidence is presented that two types of saturated Zn2 NC‐polynucleotide complexes can be formed under conditions of low [NaCl] that differ in apparent site‐size (n = 8 vs. n = 14). The formation of one or the other complex appears dependent on the molar ratio of NC to RNA nucleotide with the putative low site‐size mode apparently predominating under conditions of protein excess. Both MPMV and HIV‐1 NCs kinetically facilitate the renaturation of two complementary DNA strands, suggesting that this is a general property of retroviral NCs. NC proteins increase the second‐order rate constant for renaturation of a 149‐bp DNA fragment by more than four orders of magnitude over that obtained in the absence of protein at 37°C. The protein‐assisted rate is 100–200‐fold faster than that obtained at 68°C, 1 M NaCl, solution conditions considered to be optimal for strand renaturation. Provided that sufficient NC is present to coat all strands, the presence of 400–1,000‐fold excess nonhomologous DNA does not greatly affect the reaction rate. The HIV‐1 NC‐mediated renaturation reaction functions stoichiometrically, requiring a saturated strand of DNA nucleotide:NC ratio of about 7–8, rather than 14. Under conditions of less protein, the rate acceleration is not realized. The finding of significant nucleic acid strand renaturation activity may have important implications for various events of reverse transcription particularly in initiation and cDNA strand transfer.
Structural analysis based on state‐space modeling
Authors: Stultz Collin M. , White James V. , Smith Temple F.
Published: July 2010

A new method has been developed to compute the probability that each amino acid in a protein sequence is in a particular secondary structural element. Each of these probabilities is computed using the entire sequence and a set of predefined structural class models. This set of structural classes is patterned after Jane Richardson's taxonomy for the domains of globular proteins. For each structural class considered, a mathematical model is constructed to represent constraints on the pattern of secondary structural elements characteristic of that class. These are stochastic models having discrete state spaces (referred to as hidden Markov models by researchers in signal processing and automatic speech recognition). Each model is a mathematical generator of amino acid sequences; the sequence under consideration is modeled as having been generated by one model in the set of candidates. The probability that each model generated the given sequence is computed using a filtering algorithm. The protein is then classified as belonging to the structural class having the most probable model. The secondary structure of the sequence is then analyzed using a “smoothing” algorithm that is optimal for that structural class model. For each residue position in the sequence, the smoother computes the probability that the residue is contained within each of the defined secondary structural elements of the model. This method has two important advantages: (1) the probability of each residue being in each of the modeled secondary structural elements is computed using the totality of the amino acid sequence, and (2) these probabilities are consistent with prior knowledge of realizable domain folds as encoded in each model. As an example of the method's utility, we present its application to flavodoxin, a prototypical α/β protein having a central β‐sheet, and to thioredoxin, which belongs to a similar structural class but shares no significant sequence similarity.
Total synthesis and functional properties of the membrane‐intrinsic protein phospholamban
Authors: Vorherr Thomas , Carafoli Ernesto , Wrzosek Antoni , Chiesi Michele
Published: July 2010

The membrane‐intrinsic protein phospholamban (PLN), the regulatory protein of the sarcoplasmic reticulum (SR) Ca2+‐ATPase, was chemically synthesized. The synthesis was accomplished by double couplings and efficient capping procedures, thus eliminating hydrophobic failure sequences. The crude peptide was purified by high‐performance liquid chromatographic ion exchange and gel permeation chromatography in chloroform–methanol mixtures. Ion spray mass spectroscopy showed that the product had the correct molecular mass. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis runs produced the typical monomer–pentamer structural pattern. A predominantly helical CD spectrum was obtained in 0.075% C12E8 (67.9% helix, 1.8% beta, 12.2% turn, 18.1% random coil). Synthetic PLN was phosphorylated in detergent solutions by protein kinase A with a stoichiometry close to 1:1 (Pi to PLN monomer). Reconstitution of the isolated skeletal muscle SR Ca2+ ATPase in phosphatidylcholine membranes in the presence of PLN using the freezing and thawing technique yielded a preparation with lower Ca2+‐dependent ATPase activity. The inhibition was mainly due to a decrease in the affinity (Km(Ca)) of the ATPase for Ca2+ and was partially reversed by PLN phosphorylation with protein kinase A. By contrast, addition of PLN to diluted intact SR vesicles uncoupled the Ca2+‐transport reaction, suggesting an ionophoric effect of PLN. Because this effect was observed at very high PLN‐to‐SR vesicle ratios and was not influenced by PLN phosphorylation, its biological function is doubtful.
Unnatural amino acid packing mutants of Escherichia coli thioredoxin produced by combined mutagenesis/chemical modification techniques
Authors: Wynn R. , Richards F. M.
Published: July 2010

We have produced several mutants of Escherichia coli thioredoxin (Trx) using a combined mutagenesis/chemical modification technique. The protein C32S, C35S, L78C Trx was produced using standard mutagenesis procedures. After unfolding the protein with guanidine hydrochloride (GdmCl), the normally buried cysteine residue was modified with a series of straight chain aliphatic thiosulfonates, which produced cysteine disulfides to methane, ethane, 1‐n‐propane, 1‐n‐butane, and 1‐n‐pentane thiols. These mutants all show native‐like CD spectra and the ability to activate T7 gene 5 protein DNA polymerase activity. In addition, all mutants show normal unfolding transitions in GdmCl solutions. However, the midpoint of the transition, [GdmCl]1/2, and the free energy of unfolding at zero denaturant concentration, δG, give inverse orders of stability. This effect is due to changes in m, the dependence of δGunfolding0 on the GdmCl concentration. The method described here may be used to produce unnatural amino acids in the hydrophobic cores of proteins.
Crystal structure of human annexin I at 2.5 Å resolution
Authors: Weng Xiangwei , Luecke Hartmut , Song In Sung , Kang Doe Sun , Kim Sung‐Hou , Huber Robert
Published: July 2010

cDNA coding for N‐terminally truncated human annexin I, a member of the family of Ca2+‐dependent phospholipid binding proteins, has been cloned and expressed in Escherichia coli. The expressed protein is biologically active, and has been purified and crystallized in space group P212121 with cell dimensions a = 139.36 Å, b = 67.50 Å, and c = 42.11 Å. The crystal structure has been determined by molecular replacement at 3.0 Å resolution using the annexin V core structure as the search model. The average backbone deviation between these two structures is 2.34 Å. The structure has been refined to an R‐factor of 17.7% at 2.5 Å resolution. Six calcium sites have been identified in the annexin I structure. Each is located in the loop region of the helix–loop–helix motif. Two of the six calcium sites in annexin I are not occupied in the annexin V structure. The superpositions of the corresponding loop regions in the four domains show that the calcium binding loops in annexin I can be divided into two classes: type II and type III. Both classes are different from the well‐known EF‐hand motif (type I).
The escherichia coli adenylyl cyclase complex: Stimulation by GTP and other nucleotides
Authors: Peterkofsky Alan , Gollop Natan
Published: July 2010

Escherichia coli cells permeabilized by treatment with low concentrations of toluene contain an adenylyl cyclase activity that can be stimulated 3.6–7.6‐fold by GTP. The stimulatory effect of GTP is maximal at concentrations of the nucleotide in the physiological range (above 0.7 mM). Studies of the dependence of velocity on substrate (ATP) concentration indicate that the velocity vs. substrate plots are sigmoid in the absence of GTP but hyperbolic in the presence of GTP, suggesting an allosteric regulatory site that can be occupied by either ATP or GTP. Replacement of ATP by AMPPNP as substrate results in velocity vs. substrate plots that are hyperbolic in the absence or presence of GTP, although GTP increases the Vmax by a factor of 2.2; these findings indicate that AMPPNP specifically occupies the substrate site and GTP exclusively occupies the regulatory site. A test of the capacity of other guanine nucleotides to stimulate adenylyl cyclase activity showed that 2′‐deoxy‐GTP was almost as effective as GTP, but that GDP, GMP, ppGpp, and 3′, 5′‐cGMP were not stimulatory effectors; GTP‐γ‐S and GMPPNP stimulated adenylyl cyclase activity but to a lesser degree than did GTP. In addition to the previous indication that ATP can occupy the regulatory site on adenylyl cyclase, it was found that CTP and UTP were potent stimulators. Thus, all the naturally occurring RNA precursor nucleoside triphosphates are capable of stimulating adenylyl cyclase activity. In contrast, PPPi inhibits adenylyl cyclase activity. Additional studies showed that there is only one regulatory site for all the nucleotide stimulators and that the nucleotide occupying the substrate site can influence the properties of the regulatory site. These observations suggest a mechanism by which adenylyl cyclase activity can be regulated by the availability of nucleic acid precursors.
Determination of amide hydrogen exchange by mass spectrometry: A new tool for protein structure elucidation
Authors: Zhang Zhongqi , Smith David L.
Published: July 2010

A new method based on protein fragmentation and directly coupled microbore high‐performance liquid chromatography–fast atom bombardment mass spectrometry (HPLC‐FABMS) is described for determining the rates at which peptide amide hydrogens in proteins undergo isotopic exchange. Horse heart cytochrome c was incubated in D2O as a function of time and temperature to effect isotopic exchange, transferred into slow exchange conditions (pH 2–3, 0 °C), and fragmented with pepsin. The number of peptide amide deuterons present in the proteolytic peptides was deduced from their molecular weights, which were determined following analysis of the digest by HPLC‐FABMS. The present results demonstrate that the exchange rates of amide hydrogens in cytochrome c range from very rapid (k > 140 h−1) to very slow (k < 0.002 h−1). The deuterium content of specific segments of the protein was determined as a function of incubation temperature and used to indicate participation of these segments in conformational changes associated with heating of cytochrome c. For the present HPLC‐FABMS system, approximately 5 nmol of protein were used for each determination. Results of this investigation indicate that the combination of protein fragmentation and HPLC‐FABMS is relatively free of constraints associated with other analytical methods used for this purpose and may be a general method for determining hydrogen exchange rates in specific segments of proteins.
Three‐dimensional structural model of the serine receptor ligand‐binding domain
Authors: Jeffery Constance J. , Koshland D.E.
Published: July 2010

Computer‐based homology modeling techniques were used to construct a three‐dimensional model of the Escherichia coli serine receptor ligand‐binding domain based on the crystal structure of the Salmonella typhimurium aspartate receptor and the sequence homology between the two receptors. Residues that have been found in mutagenesis studies to be necessary for serine binding are located in a proposed serine‐binding site. Several other mutations that affect swimming behavior require relatively small shifts in α‐carbon positions in the model to give a minimized structure, suggesting that small changes in receptor conformation can affect the signaling state of the receptor.
Structural investigation of catalytically modified F120L and F120Y semisynthetic ribonucleases
Authors: Demel V. Srini J. , Doscher Marilynn S. , Glinn Michele A. , Martin Philip D. , Ram Michal L. , Edwards Brian F.P.
Published: July 2010

The structures of two catalytically modified semisynthetic RNases obtained by replacing phenylalanine 120 with leucine and tyrosine have been determined and refined at a resolution of 2.0 Å (R = 0.161 and 0.184, respectively). These structures have been compared with the refined 1.8‐Å structure (R = 0.204) of the fully active phenylalanine‐containing enzyme (Martin PD, Doscher MS, Edwards BFP, 1987, J Biol Chem 262:15930‐15938) and with the catalytically defective D121A (2.0 Å, R = 0.172) and D121N (2.0 Å, R = 0.186) analogs (deMel VSJ, Martin PD, Doscher MS, Edwards BFP, 1992, J Biol Chem 267:247‐256). The movement away from the active site of the loop containing residues 65‐72 is seen in all three catalytically defective analogs‐F120L, D121A, and D121N‐but not in the fully active (or hyperactive) F120Y. The insertion of the phenolic hydroxyl of Tyr 120 into a hydrogen‐bonding network involving the hydroxyl group of Ser 123 and a water molecule in F120Y is the likely basis for the hyperactivity toward uridine 2′,3′‐cyclic phosphate previously found for this analog (Hodges RS, Merrifield RB, 1974, Int J Pept Protein Res 6:397‐405) as well as the threefold increase in KM for cytidine 2′,3′‐cyclic phosphate found for this analog by ourselves.
Crystal structure of the unactivated ribulose 1, 5‐bisphosphate carboxylase/oxygenase complexed with a transition state analog, 2‐carboxy‐D‐arabinitol 1, 5‐bisphosphate
Authors: Zhang Kam Y.J. , Cascio Duilio , Eisenberg David
Published: July 2010

The crystal structure of unactivated ribulose 1, 5‐bisphosphate carboxylase/oxygenase from Nicotiana tabacum complexed with a transition state analog, 2‐carboxy‐D‐arabinitol 1, 5‐bisphosphate, was determined to 2.7 Å resolution by X‐ray crystallography. The transition state analog binds at the active site in an extended conformation. As compared to the binding of the same analog in the activated enzyme, the analog binds in a reverse orientation. The active site Lys 201 is within hydrogen bonding distance of the carboxyl oxygen of the analog. Loop 6 (residues 330‐339) remains open and flexible upon binding of the analog in the unactivated enzyme, in contrast to the closed and ordered loop 6 in the activated enzyme complex. The transition state analog is exposed to solvent due to the open conformation of loop 6.
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

We 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

The 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

Using 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

The 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

Alignment 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

An 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

A 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

Interactions 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

In 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

We 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

We 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

One 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

We 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

The 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.