bluuebunny/crossref_metadata_2025_split huggingface dataset:
Properties of a recombinant human hemoglobin with aspartic acid 99 (β), an important intersubunit contact site, substituted by lysine
Authors: Yanase Hideshi , Cahill Sean , De Llano Jose Javier Martin , Manning Lois R. , Schneider Klaus , Chait Brian T. , Vandegriff Kim D. , Winslow Robert M. , Manning James M. 
Published: February 2009

AbstractSite‐directed mutagenesis of an important subunit contact site, Asp‐99(β), by a Lys residue (D99K(β)) was proven by sequencing the entire β‐globin gene and the mutant tryptic peptide. Oxygen equilibrium curves of the mutant hemoglobin (Hb) (2–15 mM in heme) indicated that it had an increased oxygen affinity and a lowered but significant amount of cooperativity compared to native HbA. However, in contrast to normal HbA, oxygen binding of the recombinant mutant Hb was only marginally affected by the allosteric regulators 2,3‐diphosphoglycerate or inositol hexaphosphate and was not at all responsive to chloride. The efficiency of oxygen binding by HbA in the presence of allosteric regulators was limited by the mutant Hb. At concentrations of 0.2 mM or lower in heme, the mutant D99K(β) Hb was predominantly a dimer as demonstrated by gel filtration, haptoglobin binding, fluorescence quenching, and light scattering. The purified dimeric recombinant Hb mutant exists in 2 forms that are separable on isoelectric focusing by about 0.1 pH unit, in contrast to tetrameric hemoglobin, which shows 1 band. These mutant forms, which were present in a ratio of 60:40, had the same masses for their heme and globin moieties as determined by mass spectrometry. The elution positions of the α‐ and β‐globin subunits on HPLC were identical. Circular dichroism studies showed that one form of the mutant Hb had a negative ellipticity at 410 nm and the other had positive ellipticity at this wavelength. The findings suggest that the 2 D99K(β) recombinant mutant forms have differences in their heme‐protein environments.
Reversible unfolding of fructose 6‐phosphate, 2‐kinase:fructose 2,6‐bisphosphatase
Authors: Tominaga Nobuaki , Jameson David M. , Uyeda Kosaku 
Published: February 2009

AbstractReversible unfolding of rat testis fructose 6‐phosphate, 2‐kinase:fructose 2,6‐bisphosphatase in guanidine hydrochloride was monitored by following enzyme activities as well as by fluorescence methodologies (intensity, emission maximum, polarization, and quenching), using both intrinsic (tryptophan) and extrinsic (5((2‐(iodoacetyl)amino) ethyl)naphthalene‐1‐sulfonic acid) probes. The unfolding reaction is described minimally as a 4‐state transition from folded dimer → partially unfolded dimer → monomer → unfolded monomer. The partially unfolded dimer had a high phosphatase/kinase ratio due to preferential unfolding of the kinase domain. The renaturation reaction proceeded by very rapid conversion (less than 1 s) of unfolded monomer to dimer, devoid of any enzyme activity, followed by slow (over 60 min) formation of the active enzyme. The recovery rates of the kinase and the phosphatase were similar. Thus, the refolding appeared to be a reversal of the unfolding pathway involving different forms of the transient dimeric intermediates. Fluorescence quenching studies using iodide and acrylamide showed that the tryptophans, including Trp‐15 in the N‐terminal peptide, were only slightly accessible to iodide but were much more accessible to acrylamide. Fructose 6‐phosphate, but not ATP or fructose 2,6‐bisphosphate, diminished the iodide quenching, but all these ligands inhibited the acrylamide quenching by 25%. These results suggested that the N‐terminal peptide (containing a tryptophan) was not exposed on the protein surface and may play an important role in shielding other tryptophans from solvent.
Stability and peptide binding affinity of an SH3 domain from the Caenorhabditis elegans signaling protein Sem‐5
Authors: Lim Wendell A. , Fox Robert O. , Richards Frederic M. 
Published: February 2009

AbstractWe have determined the thermodynamic stability and peptide binding affinity of the carboxy‐terminal Src homology 3 (SH3) domain from the Caenorhabditis elegans signal‐transduction protein Sem‐5. Despite its small size (62 residues) and lack of disulfide bonds, this domain is highly stable to thermal denaturation — at pH 7.3, the protein has a Tm of 73.1 °C. Interestingly, the protein is not maximally stable at neutral pH, but reaches a maximum at around pH 4.7 (Tm ≅ 80 °C). Increasing ionic strength also stabilizes the protein, suggesting that 1 or more car‐boxylate ions are involved in a destabilizing electrostatic interaction. By guanidine hydrochloride denaturation, the protein is calculated to have a free energy of unfolding of 4.1 kcal/mol at 25 °C. We have also characterized binding of the domain to 2 different length proline‐rich peptides from the guanine nucleotide exchange factor, Sos, one of Sem‐5′s likely physiological ligands in cytoplasmic signal transduction. Upon binding, these peptides cause about a 2‐fold increase in fluorescence intensity. Both bind with only modest affinities (Kd ≅ 30 μM), lower than some previous estimates for SH3 domains. By fluorescence, the domain also appears to associate with the homopolymer poly‐L‐proline in a similar fashion.
Two‐step selective formation of three disulfide bridges in the synthesis of the C‐terminal epidermal growth factor‐like domain in human blood coagulation factor IX
Authors: Yang Yan , Sweeney William V. , Schneider Klaus , Chait Brian T. , Tam James P. 
Published: February 2009

AbstractThe 45‐residue C‐terminal EGF‐like domain in human blood coagulation factor IX has been synthesized by a 2‐step method to form selectively 3 disulfide bridges. Four out of 6 cysteines are blocked with either trityl or 4‐methyl‐benzyl, and the remaining 2 cysteines are blocked with acetamidomethyl (Acm). In the first step, 4 free cysteinyl thiols are released concurrently with the removal of all protecting groups except Acm and are oxidized to form 1 of the 3 possible isomers containing 2 pairs of disulfides. In the second step, iodine is used to remove the Acm groups to yield the third disulfide bridge. This approach reduces the number of possible disulfide bridging patterns from 15 to 3. To determine the optimal protecting group strategy, 3 peptides are synthesized, each with Acm blocking 1 of the 3 pairs of cysteines involved in disulfide bridges: Cys5 to Cys16 (Cys 1–3), Cys12 to Cys26 (Cys 2–4), or Cys28 to Cys41 (Cys 5–6). Only the peptide having the Cys 2–4 pair blocked with Acm forms the desired disulfide isomer (Cys 1–3/5–6) in high yield after the first step folding, as identified by proteolytic digestion in conjunction with mass spectrometric peptide mapping. Thus, the choice of which pair of cysteines to block with Acm is critically important. In the case of EGF‐like peptides, it is better to place the Acm blocking groups on one of the pairs of cysteines involved in the crossing of disulfide bonds.
A negative electrostatic determinant mediates the association between the Escherichia coli trp repressor and its operator DNA
Authors: Guenot Jeanmarie , Fletterick Robert J. , Kollman Peter A. 
Published: February 2009

AbstractThe electrostatic potential surfaces were characterized for trp repressor models that bind to DNA with sequence specificity, without specificity, and not at all. Comparisons among the surfaces were used to isolate protein surface features likely to be important in DNA binding. Models that differ in protein conformation and tryptophan‐analogue binding consistently showed positive potential associated with the protein surfaces that interact with the DNA major groove. However, negative potential is associated with the trp repressor surface that contacts the DNA minor groove. This negative potential is significantly neutralized in the protein conformation that is bound to DNA. Positive potential is also associated with the tryptophan binding‐site surface, a consequence of the tryptophan‐or tryptophan analogue‐induced allosteric change. This protein region is complementary to the strongest negative potential associated with the DNA phosphate backbone and is also present in the isolated protein structure from the protein‐DNA complex. The effects of charge‐change mutation, pH dependence, and salt dependence on the electrostatic potential surfaces were also examined with regard to their effects on protein‐DNA binding constants. A consistent model is formed that defines a role for long‐range electrostatics early in the protein‐DNA association process and complements previous structural, molecular association, and mutagenesis studies.
The sequence of a subtilisin‐type protease (aerolysin) from the hyperthermophilic archaeum Pyrobaculum aerophilum reveals sites important to thermostability
Authors: Völkl Paul , Markiewicz Peter , Stetter Karl O. , Miller Jeffrey H. 
Published: February 2009

AbstractThe hyperthermophilic archaeum Pyrobaculum aerophilum grows optimally at 100 °C and pH 7.0. Cell homogenates exhibit strong proteolytic activity within a temperature range of 80–130 °C. During an analysis of cDNA and genomic sequence tags, a genomic clone was recovered showing strong sequence homology to alkaline subtilisins of Bacillus sp. The total DNA sequence of the gene encoding the protease (named “aerolysin”) was determined. Multiple sequence alignment with 15 different serine‐type proteases showed greatest homology with subtilisins from gram‐positive bacteria rather than archaeal or eukaryal serine proteases.Models of secondary and tertiary structure based on sequence alignments and the tertiary structures of subtilisin Carlsberg, BPN', thermitase, and protease K were generated for P. aerophilum subtilisin. This allowed identification of sites potentially contributing to the thermostability of the protein. One common transition put alanines at the beginning and end of surface alpha‐helices. Aspartic acids were found at the N‐terminus of several surface helices, possibly increasing stability by interacting with the helix dipole. Several of the substitutions in regions expected to form surface loops were adjacent to each other in the tertiary structure model.
Extending the B7 (CD80) gene family
Authors: Linsley Peter S. , Peach Robert , Gladstone Paul , Bajorath Jürgen 
Published: February 2009

AbstractB7‐1 and B7‐2 are members of the immunoglobulin superfamily (IgSF) and important regulators of T cell‐mediated immune responses. Despite sharing only limited sequence identity, B7‐1 and B7‐2 bind common receptors, CD28 and CTLA‐4, on T cells and have similar functional properties. We have found that the extracellular V(ariable)‐like domains of B7‐1 and B7‐2 share significant sequence similarities with 3 major histocompatibility complex (MHC)‐encoded members of the IgSF: butyrophilin, myelin/oligodendrocyte glycoprotein, and the chicken MHC molecule, B‐G. This raises the question whether there is an evolutionary link between the MHC, which encodes molecules regulating the antigen specificity of T lymphocyte responses, and B7 molecules, which co‐stimulate these responses in antigen‐nonspecific fashion.
Interactions between human defensins and lipid bilayers: Evidence for formation of multimeric pores
Authors: Wimley William C. , Selsted Michael E. , White Stephen H. 
Published: February 2009

AbstractDefensins comprise a family of broad‐spectrum antimicrobial peptides that are stored in the cytoplasmic granules of mammalian neutrophils and Paneth cells of the small intestine. Neutrophil defensins are known to permeabilize cell membranes of susceptible microorganisms, but the mechanism of permeabilization is uncertain. We report here the results of an investigation of the mechanism by which HNP‐2, one of 4 human neutrophil defensins, permeabilizes large unilamellar vesicles formed from the anionic lipid palmitoyloleoylphosphatidylglycerol (POPG). As observed by others, we find that HNP‐2 (net charge = +3) cannot bind to vesicles formed from neutral lipids. The binding of HNP‐2 to vesicles containing varying amounts of POPG and neutral (zwitterionic) palmitoyloleoylphosphatidylcholine (POPC) demonstrates that binding is initiated through electrostatic interactions. Because vesicle aggregation and fusion can confound studies of the interaction of HNP‐2 with vesicles, those processes were explored systematically by varying the concentrations of vesicles and HNP‐2, and the POPG:POPC ratio. Vesicles (300 μM POPG) readily aggregated at HNP‐2 concentrations above 1 μM, but no mixing of vesicle contents could be detected for concentrations as high as 2 μM despite the fact that intervesicular lipid mixing could be demonstrated. This indicates that if fusion of vesicles occurs, it is hemi‐fusion, in which only the outer monolayers mix at bilayer contact sites. Under conditions of limited aggregation and intervesicular lipid mixing, the fractional leakage of small solutes is a sigmoidal function of peptide concentration. For 300 μM POPG vesicles, 50% of entrapped solute is released by 0.7 μM HNP‐2. We introduce a simple method for determining whether leakage from vesicles is graded or all‐or‐none. We show by means of this fluorescence “requenching” method that native HNP‐2 induces vesicle leakage in an all‐or‐none manner, whereas reduced HNP‐2 induces partial, or graded, leakage of vesicle contents. At HNP‐2 concentrations that release 100% of small (∼400 Da) markers, a fluorescent dextran of 4,400 Da is partially retained in the vesicles, and a 18,900‐Da dextran is mostly retained. These results suggest that HNP‐2 can form pores that have a maximum diameter of approximately 25 Å. A speculative multimeric model of the pore is presented based on these results and on the crystal structure of a human defensin.
Human immunodeficiency virus‐1 reverse transcriptase heterodimer stability
Authors: Lebowitz Jacob , Kar Sambit , Braswell Emory , Mcpherson Sylvia , Richard Dean L. 
Published: February 2009

AbstractStructural and biochemical evidence strongly supports a heterodimeric (p66p51) active form for human immunodeficiency virus‐1 reverse transcriptase (RT). Heterodimer stability was examined by sedimentation analysis as a function of temperature and ionic strength. Using NONLIN regression software, monomer‐dimer‐trimer and monomer‐dimer‐tetramer association models gave the best fit to the analytical ultracentrifuge sedimentation equilibrium data. The heterodimer is the predominant form of RT at 5 °C, with a dimerization Ka value of 5.2 × 105 M−1 for both models. Ka values of 2.1 × 105 and 3.8 × 105 M−1 were obtained for the respective association models at 20 °C. RT in 50 and 100 mM Tris, pH 7.0, completely dissociates at 37 °C and behaves as an ideal monomeric species. The dissociation of RT as a function of increasing temperature was also observed by measuring the decrease in sedimentation velocity (sw,20). If the stabilization of the heterodimer was due primarily to hydrophobic interactions we would anticipate an increase in the association from 21 °C to 37 °C. The opposite temperature dependence for the association of RT suggests that electrostatic and hydrogen bond interactions play an important role in stabilizing heterodimers. To examine the effect of ionic strength on p66p51 association we determined the changes in sw,20 as a function of NaCl concentration. There is a sharp decrease in sw,20 between 0.10 and 0.5 M NaCl, leading to apparent complete dissociation. The above results support a major role for electrostatic interactions in the stabilization of the RT heterodimer.
Dimerization of βB2‐crystallin: The role of the linker peptide and the N‐ and C‐terminal extensions
Authors: Trinkl Sabine , Glockshuber Rudi , Jaenicke Rainer 
Published: February 2009

AbstractβB2‐ and γB‐crystallins of vertebrate eye lens are 2‐domain proteins in which each domain consists of 2 Greek key motifs connected by a linker peptide. Although the folding topologies of βB2‐ and γB‐domains are very similar, γB‐crystallin is always monomeric, whereas βB2‐crystallin associates to homodimers. It has been suggested that the linker or the protruding N‐ and C‐terminal arms of βB2‐crystallin (not present in γB) are a necessary requirement for this association. In order to investigate the role of these segments for dimerization, we constructed two βB2 mutants. In the first mutant, the linker peptide was replaced with the one from γB (βB2γL). In the second mutant, the N‐ and C‐terminal arms of 15‐ and 12‐residues length were deleted (βB2ΔNC). The βB2γL mutant is monomeric, whereas the βB2ΔNC mutant forms dimers and tetramers that cannot be interconverted without denaturation. The spectral properties of the βh2 mutants, as well as their stabilities against denaturants, resemble those of wild‐type βB2‐crystallin, thus indicating that the overall peptide fold of the subunits is not changed significantly. We conclude that the peptide linker in βB2‐crystallin is necessary for dimerization, whereas the N‐ and C‐terminal arms appear to be involved in preventing the formation of higher homo‐oligomers.
Equilibrium unfolding of Escherichia coli ribonuclease H: Characterization of a partially folded state
Authors: Dabora Jonathan M. , Marqusee Susan 
Published: February 2009

AbstractWe have examined the equilibrium unfolding of Escherichia coli ribonuclease HI (RNase H), a member of a family of enzymes that cleaves RNA from RNA:DNA hybrids. A completely synthetic gene was constructed that expresses a variant of the wild‐type sequence with all 3 cysteines replaced with alanine. The resulting recombinant protein is active and folds reversibly. Denaturation studies monitored by circular dichroism and tryptophan fluorescence yield coincident curves that suggest the equilibrium unfolding reaction is a 2‐state process. Acid denaturation, however, reveals a cooperative transition at ∼pH 1.8 to a partially folded state. This acid state can be further denatured in a reversible manner by the addition of heat or urea as monitored by either CD or tryptophan fluorescence. Analytical ultracentrifugation studies indicate that the acid state of RNase H is both compact and monomeric. Although compact, the acid state does not resemble the native protein: the acid state displays a near‐UV CD spectrum similar to the unfolded state and binds to and enhances the fluorescence of the dye 1‐anilinonaphthalene, 8‐sulfonate much more than either the native or unfolded states. Therefore, the acid state of E. coli RNase H has the characteristics of a molten globule: it retains a high degree of secondary structure, remains compact, yet does not appear to contain a tightly packed core.
Thermal unfolding of tetrameric melittin: Comparison with the molten globule state of cytochrome c
Authors: Hagihara Yoshihisa , Oobatake Motohisa , Goto Yuji 
Published: February 2009

AbstractWhereas melittin at micromolar concentrations is unfolded under conditions of low salt at neutral pH, it transforms to a tetrameric α‐helical structure under several conditions, such as high peptide concentration, high anion concentration, or alkaline pH. The anion‐ and pH‐dependent stabilization of the tetrameric structure is similar to that of the molten globule state of several acid‐denatured proteins, suggesting that tetrameric melittin might be a state similar to the molten globule state. To test this possibility, we studied the thermal unfolding of tetrameric melittin using far‐UV CD and differential scanning calorimetry. The latter technique revealed a broad but distinct heat absorption peak. The heat absorption curves were consistent with the unfolding transition observed by CD and were explainable by a 2‐state transition mechanism between the tetrameric α‐helical state and the monomelic unfolded state. From the peptide or salt‐concentration dependence of unfolding, the heat capacity change upon unfolding was estimated to be 5 kJ (mol of tetramer)−1 K−1 at 30 °C and decreased with increasing temperature. The observed change in heat capacity was much smaller than that predicted from the crystallographic structure (9.2 kJ (mol of tetramer)−1 K−1), suggesting that the hydrophobic residues of tetrameric melittin in solution are exposed in comparison with the crystallographic structure. However, the results also indicate that the structure is more ordered than that of a typical molten globule state. We consider that the conformation is intermediate between the molten globule state and the native state of globular proteins.
Stabilization of myoglobin by multiple alanine substitutions in helical positions
Authors: Lin Laura , Pinker Rachel J. , Phillips George N. , Kallenbach Neville R. 
Published: February 2009

AbstractWe have carried out a series of multiple Xaa → Ala changes at nonadjacent surface positions in the sequence of sperm whale myoglobin. Although the corresponding single substitutions do not increase the thermal stability of the protein, multiple substitutions enhance the stability of the resulting myoglobins. The effect observed is an increase in the observed Tm (midpoint unfolding temperature) relative to that predicted from assuming additivity of the free energy changes corresponding to single mutations. The stabilization occurs in the presence of urea, as measured by the dependence of the unfolding temperature on urea concentration. The sites that have been altered occur in different helices and are not close in sequence or in the native structure of myoglobin. The observed effect is consistent with a role of multiple alanines in residual interactions in the unfolded state of the mutant proteins.
The chaperonin from the archaeon Sulfolobus solfataricus promotes correct refolding and prevents thermal denaturation in vitro
Authors: Guagliardi Annamaria , Cerchia Laura , Bartolucci Simonetta , Rossi Mosé 
Published: February 2009

AbstractWe have isolated a chaperonin from the hyperthermophilic archaeon Sulfolobus solfataricus based on its ability to inhibit the spontaneous refolding at 50 °C of dimeric S. solfataricus malic enzyme. The chaperonin, a 920‐kDa oligomer of 57‐kDa subunits, displays a potassium‐dependent ATPase activity with an optimum temperature at 80 °C. S. solfataricus chaperonin promotes correct refoldings of several guanidine hydrochloride‐denatured enzymes from thermophilic and mesophilic sources. At a molar ratio of chaperonin oligomer to single polypeptide chain of 1:1, S. solfataricus chaperonin completely inhibits spontaneous refoldings and suppresses aggregation upon dilution of the denaturant; refoldings resume upon ATP hydrolysis, with yields of active molecules and rates of folding notably higher than in spontaneous processes. S. solfataricus chaperonin prevents the irreversible in‐activations at 90 °C of several thermophilic enzymes by the binding of the denaturation intermediate; the time‐courses of inactivations are unaffected and most activity is regained upon hydrolysis of ATP. S. solfataricus chaperonin completely prevents the formation of aggregates during thermal inactivation of chicken egg white lysozyme at 70 °C, without affecting the rate of activity loss; ATP hydrolysis results in the recovery of most lytic activity. Tryptophan fluorescence measurements provide evidence that S. solfataricus chaperonin undergoes a dramatic conformational rearrangement in the presence of ATP/Mg, and that the hydrolysis of ATP is not required for the conformational change. The ATP/Mg‐induced conformation of the chaperonin is fully unable to bind the protein substrates, probably due to disappearance or modification of the substrate binding sites. This is the first archaeal chaperonin whose involvement in protein folding has been demonstrated.