bluuebunny/crossref_metadata_2025_split huggingface dataset:
Helicity of α(404–451) and β(394–445) tubulin C‐terminal recombinant peptides
Authors: Jimenez M. Angeles , Evangelio Juan A. , Aranda Carlos , Lopez‐Brauet Adamari , Andreu David , Rico Manuel , Lagos Rosalba , Andreu Jose M. , Monasterio Octavio 
Published: October 2010

AbstractWe have investigated the solution conformation of the functionally relevant C‐terminal extremes of α‐ and β‐tubulin, employing the model recombinant peptides RL52a3 and RL33b6, which correspond to the amino acid sequences 404‐451(end) and 394–445(end) of the main vertebrate isotypes of α‐ and β‐tubulin, respectively, and synthetic peptides with the α‐tubulin(430–443) and β‐tubulin(412–431) internal sequences. α(404–451) and β(394–445) are monomeric in neutral aqueous solution (as indicated by sedimentation equilibrium), and have circular dichroism (CD) spectra characteristic of nearly disordered conformation, consistent with low scores in peptide helicity prediction. Limited proteolysis of β(394–445) with subtilisin, instead of giving extensive degradation, resulted in main cleavages at positions Thr409‐Glu410 and Tyr422‐Gln423‐Gln424, defining the proteolysis resistant segment 410–422, which corresponds to the central part of the predicted β‐tubulin C‐terminal helix. Both recombinant peptides inhibited microtubule assembly, probably due to sequestration of the microtubule stabilizing associated proteins. Trifluoroethanol (TFE)‐induced markedly helical CD spectra in α(404–451) and β(394–445). A substantial part of the helicity of β(394–445) was found to be in the CD spectrum of the shorter peptide β(412–431) with TFE. Two‐dimensional 1H‐NMR parameters (nonsequential nuclear Overhauser effects (NOE) and conformational CαH shifts) in 30% TFE permitted to conclude that about 25% of α(404–451) and 40% of β(394–451) form well‐defined helices encompassing residues 418–432 and 408–431, respectively, flanked by disordered N‐ and C‐segments. The side chains of β(394–451) residues Leu418, Val419, Ser420, Tyr422, Tyr425, and Gln426 are well defined in structure calculations from the NOE distance constraints. The apolar faces of the helix in both α and β chains share a characteristic sequence of conserved residues Ala,Met(+4),Leu(+7),Tyr(+11). The helical segment of α(404–451) is the same as that described in the electron crystallographic model structure of αβ‐tubulin, while in β(394–451) it extends for nine residues more, supporting the possibility of a functional coil → helix transition at the C‐terminus of β‐tubulin. These peptides may be employed to construct model complexes with microtubule associated protein binding sites.
Thermodynamic stability of ribonuclease A in alkylurea solutions and preferential solvation changes accompanying its thermal denaturation: A calorimetric and spectroscopic study
Authors: Poklar Nataša , Petrovčič Nina , Oblak Miha , Vesnaver Gorazd 
Published: October 2010

AbstractThe effect of methylurea, N,N'‐dimethylurea, ethylurea, and butylurea as well as guanidine hydrochloride (GuHCl), urea and pH on the thermal stability, structural properties, and preferential solvation changes accompanying the thermal unfolding of ribonuclease A (RNase A) has been investigated by differential scanning calorimetry (DSC), UV, and circular dichroism (CD) spectroscopy. The results show that the thermal stability of RNase A decreases with increasing concentration of denaturants and the size of the hydrophobic group substituted on the urea molecule. From CD measurements in the near‐ and far‐UV range, it has been observed that the tertiary structure of RNase A melts at about 3 °C lower temperature than its secondary structure, which means that the hierarchy in structural building blocks exists for RNase A even at conditions at which according to DSC and UV measurements the RNase A unfolding can be interpreted in terms of a two‐state approximation. The far‐UV CD spectra also show that the final denatured states of RNase A at high temperatures in the presence of different denaturants including 4.5 M GuHCl are similar to each other but different from the one obtained in 4.5 M GuHCl at 25 °C. The concentration dependence of the preferential solvation change Δγ23, expressed as the number of cosolvent molecules entering or leaving the solvation shell of the protein upon denaturation and calculated from DSC data, shows the same relative denaturation efficiency of alkylureas as other methods.
De novo design of a monomeric three‐stranded antiparallel β‐sheet
Authors: Alba Eva De , Santoro Jorge , Rico Manuel , Jiménez M. Angeles 
Published: October 2010

AbstractHere we describe the NMR conformational study of a 20‐residue linear peptide designed to fold into a monomeric three‐stranded antiparallel β‐sheet in aqueous solution. Experimental and statistical data on amino acid β‐turn and β‐sheet propensities, cross‐strand side‐chain interactions, solubility criteria, and our previous experience with β‐hairpins were considered for a rational selection of the peptide sequence. Sedimentation equilibrium measurements and NMR dilution experiments provide evidence that the peptide is monomeric. Analysis of 1H and 13C‐NMR parameters of the peptide, in particular NOEs and chemical shifts, and comparison with data obtained for two 12‐residue peptides encompassing the N‐ and C‐segments of the designed sequence indicates that the 20‐residue peptide folds into the expected conformation. Assuming a two‐state model, the exchange kinetics between the β‐sheet and the unfolded peptide molecules is in a suitable range to estimate the folding rate on the basis of the NMR linewidths of several resonances. The time constant for the coil‐β‐sheet transition is of the order of several microseconds in the designed peptide. Future designs based on this peptide system are expected to contribute greatly to our knowledge of the many factors involved in β‐sheet formation and stability.
Selection of antibody probes to correlate protein sequence domains with their structural distribution
Authors: Valle Mikel , Muñoz Manuel , Kremer Leonor , Valpuesta Jose M. , Martínez‐A Carlos , Carrascosa Jose L. , Albar Juan P. 
Published: October 2010

AbstractWe propose a new approach that permits correlation of specific domains defined by their primary sequence with their location in the structure of complex macromolecular aggregates. It is based on the combination of well‐established structural analysis methods that incorporate the use of overlapping peptides on cellulose membranes for the isolation and purification of specific antibodies from a polyclonal antiserum. Monospecific antibodies to the connector protein of bacteriophage ø29 were isolated from polyclonal antisera using a new development of the spotscan method. These antibodies can be purified in quantities that allow antigenicity testing in enzyme‐linked immunosorbent assays, Western blotting and immunoprecipitations, demonstrating the specificity of this isolation procedure. This approach has allowed us to generate direct antibody probes for immunoelectron microscopy mapping of different connector protein domains in a low resolution three‐dimensional epitope map.
Folding of beta‐sandwich proteins: Three‐state transition of a fibronectin type III module
Authors: Cota Ernesto , Clarke Jane 
Published: October 2010

AbstractAn analysis of the folding of the 94 residue tenth fibronectin type III (fnIII) domain of human fibronectin (FNfn10) is presented. Use of guanidine isothiocyanate as a denaturant allows us to obtain equilibrium and kinetic data across a broad range of denaturant concentrations that are unavailable in guanidine hydrochloride. Equilibrium unfolding experiments show that FNfn10 is significantly more stable than has been reported previously. Comparison of equilibrium and kinetic parameters reveals the presence of an intermediate that accumulates at low denaturant concentrations. This is the first demonstration of three‐state folding kinetics for a fnIII domain. We have previously shown that a homologous domain from human tenascin (TNfn3) folds by a two‐state mechanism, but this does not necessarily indicate that the two proteins fold by different folding pathways.
A step toward the prediction of the fluorescence lifetimes of tryptophan residues in proteins based on structural and spectral data
Authors: Sillen Alain , Díaz José Fernando , Engelborghs Yves 
Published: October 2010

AbstractA method is presented that allows the calculation of the lifetimes of tryptophan residues on the basis of spectral and structural data. It is applied to two different proteins. The calcium binding protein from the sarcoplasm of the muscles of the sand worm Nereis diversicolor (NSCP) changes its conformation upon binding of Ca1+ or Mg1+. NSCP contains three tryptophan residues at position 4, 57, and 170, respectively. The fluorescence lifetimes of W57 are investigated in a mutant in which W4 and W170 have been replaced. The time resolved fluorescence properties of W57 are linked to its different microconformations, which were determined by a molecular dynamics simulation map. Together with the determination of the radiative rate constant from the wavelength of maximum intensity of the decay associated spectra, it was possible to determine an exponential relation between the nonradiative rate constant and the distance between the indole CE3 atom and the carbonyl carbon of the peptide bond reflecting a mechanism of electron transfer as the main determinant of the value for the nonradiative rate constant. This result allows the calculation of the fluorescence lifetimes from the protein structure and the spectra. This method was further tested for the tryptophan of Ha‐ras p21 (W32) and for W43 of the Tet repressor, which resulted in acceptable values for the predicted lifetimes.
Phosphorylation and subcellular redistribution of high mobility group proteins 14 and 17, analyzed by mass spectrometry
Authors: Louie Donna R , Gloor Kristen K. , Galasinski Scott C. , Resing Katheryn A. , Ahn Natalie G. 
Published: October 2010

AbstractHigh mobility group (HMG) proteins 14 and 17 are nonhistone nuclear proteins that have been implicated in control of transcription and chromatin structure. To examine the posttranslational modifications of HMG‐14 and ‐17 in vivo, HMG proteins were prepared from nuclear vs. cytosolic fractions of human K562 cells treated with 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) or okadaic acid (OA) and examined by electrospray mass spectrometry. Analysis of full‐length masses demonstrated mono‐, di‐, and triphosphorylation of HMG‐14 and mono‐ and diphosphorylation of HMG‐17 from OA treated cells, whereas HMG‐14 and ‐17 from TPA treated cells were monophosphorylated. Peptide mass and sequence analysis showed major and minor phosphorylation sites, respectively, at Ser24 and Ser28 in HMG‐17, and Ser20 and Ser24 in HMG‐14. These sites were found in the consensus sequence RRSARLSAK, within the nucleosomal binding domain of each protein. A third phosphorylation site in HMG‐14 was located at either Ser6 or Ser7. Interestingly, the proportion of HMG‐14 and ‐17 found in cytosolic pools increased significantly after 1 h of treatment compared to control cells and showed preferential phosphorylation compared with proteins from nuclear fractions. These results suggest that phosphorylation of HMG‐14 and ‐7 interferes with nuclear localization mechanisms in a manner favoring release from nuclei.
Crystal structures of two mutants (K206Q, H207E) of the N‐lobe of human transferrin with increased affinity for iron
Authors: Yang Amy H.‐W. , Macgillivray Ross T.A. , Chen Jie , Luo Yaoguang , Wang Yili , Brayer Gary D. , Mason Anne B. , Woodworth Robert C. , Murphy Michael E.P. 
Published: October 2010

AbstractThe X‐ray crystallographic structures of two mutants (K206Q and H207E) of the N‐lobe of human transferrin (hTF/2N) have been determined to high resolution (1.8 and 2.0 Å, respectively). Both mutant proteins bind iron with greater affinity than native hTF/2N. The structures of the K206Q and H207E mutants show interactions (both H‐bonding and electrostatic) that stabilize the interaction of Lys296 in the closed conformation, thereby stabilizing the iron bound forms.
Conservative mutation met8 → leu affects the folding process and structural stability of squash trypsin inhibitor CMTI‐I
Authors: Zhukov Igor , Bierzynaski Andrzej , Jaroszewski Lukasz 
Published: October 2010

AbstractProtein molecules can accommodate a large number of mutations without noticeable effects on their stability and folding kinetics. On the other hand, some mutations can have quite strong effects on protein conformational properties. Such mutations either destabilize secondary structures, e.g., α‐helices, are incompatible with close packing of protein hydrophobic cores, or lead to disruption of some specific interactions such as disulfide cross links, salt bridges, hydrogen bonds, or aromatic‐aromatic contacts. The Met8 → Leu mutation in CMTI‐I results in significant destabilization of the protein structure. This effect could hardly be expected since the mutation is highly conservative, and the side chain of residue 8 is situated on the protein surface. We show that the protein destabilization is caused by rearrangement of a hydrophobic cluster formed by side chains of residues 8, Ile6, and Leu17 that leads to partial breaking of a hydrogen bond formed by the amide group of Leu17 with water and to a reduction of a hydrophobic surface buried within the cluster. The mutation perturbs also the protein folding. In aerobic conditions the reduced wild‐type protein folds effectively into its native structure, whereas more then 75% of the mutant molecules are trapped in various misfolded species. The main conclusion of this work is that conservative mutations of hydrophobic residues can destabilize a protein structure even if these residues are situated on the protein surface and partially accessible to water. Structural rearrangement of small hydrophobic clusters formed by such residues can lead to local changes in protein hydration, and consequently, can affect considerably protein stability and folding process.
An interdomain distance in cardiac troponin c determined by fluorescence spectroscopy
Authors: Dong Wen‐Ji , Robinson John M. , Xing Jun , Cheung Herbert C. , Umeda Patrick K. 
Published: October 2010

AbstractThe distance between Ca2+‐binding site III in the C‐terminal domain and Cys35 in the N‐terminal domain in cardiac muscle troponin C (cTnC) was determined with a single‐tryptophan mutant using bound Tb3+ as the energy donor and iodoacetamidotetramethylrhodamine linked to the cysteine residue as energy acceptor. The luminescence of bound Tb3+ was generated through sensitization by the tryptophan located in the 12‐residue binding loop of site III upon irradiation at 295 nm, and this sensitized luminescence was the donor signal transferred to the acceptor. In the absence of bound cation at site II, the mean interdomain distance was found to be 48–49 Å regardless of whether the cTnC was unbound or bound to cardiac troponin I, or reconstituted into cardiac troponin. These results suggest that cTnC retains its overall length in the presence of bound target proteins. The distribution of the distances was wide (half‐width >9 Å) and suggests considerable interdomain flexibility in isolated cTnC, but the distributions became narrower for cTnC in the complexes with the other subunits. In the presence of bound cation at the regulatory site II, the interdomain distance was shortened by 6 Å for cTnC, but without an effect on the half‐width. The decrease in the mean distance was much smaller or negligible when cTnC was complexed with cTnI or cTnI and cTnT under the same conditions. Although free cTnC has considerable interdomain flexibility, this dynamics is slightly reduced in troponin. These results indicate that the transition from the relaxed state to an activated state in cardiac muscle is not accompanied by a gross alteration of the cTnC conformation in cardiac troponin.
Microscopic stability of cold shock protein a examined by NMR native state hydrogen exchange as a function of urea and trimethylamine N‐oxide
Authors: Jaravine Victor A. , Rathgeb‐Szabo Klara , Alexandrescu Andrei T. 
Published: October 2010

AbstractNative state hydrogen exchange of cold shock protein A (CspA) has been characterized as a function of the denaturant urea and of the stabilizing agent trimethylamine N‐oxide (TMAO). The structure of CspA has five strands of β‐sheet. Strands β1‐β4 have strongly protected amide protons that, based on experiments as a function of urea, exchange through a simple all‐or‐none global unfolding mechanism. By contrast, the protection of amide protons from strand β5 is too weak to measure in water. Strand β5 is hydrogen bonded to strands β3 and β4, both of which afford strong protection from solvent exchange. Gaussian network model (GNM) simulations, which assume that the degree of protection depends on tertiary contact density in the native structure, accurately predict the strong protection observed in strands β1‐β4 but fail to account for the weak protection in strand β5. The most conspicuous feature of strand β5 is its low sequence hydrophobicity. In the presence of TMAO, there is an increase in the protection of strands b̊1‐β4, and protection extends to amide protons in more hydrophilic segments of the protein, including strand β5 and the loops connecting the β‐strands. TMAO stabilizes proteins by raising the free energy of the denatured state, due to highly unfavorable interactions between TMAO and the exposed peptide backbone. As such, the stabilizing effects of TMAO are expected to be relatively independent of sequence hydrophobicity. The present results suggest that the magnitude of solvent exchange protection depends more on solvent accessibility in the ensemble of exchange susceptible conformations than on the strength of hydrogen‐bonding interactions in the native structure.
Resolution of ligand positions by site‐directed tryptophan fluorescence in tear lipocalin
Authors: Gasymov Oktay K. , Abduragimov Adil R. , Yusifov Taleh N. , Glasgow Ben J. 
Published: October 2010

AbstractThe lipocalin superfamily of proteins functions in the binding and transport of a variety of important hydrophobic molecules. Tear lipocalin is a promiscuous lipid binding member of the family and serves as a paradigm to study the molecular determinants of ligand binding. Conserved regions in the lipocalins, such as the G strand and the F‐G loop, may play an important role in ligand binding and delivery. We studied structural changes in the G strand of holo‐ and apo‐tear lipocalin using spectroscopic methods including circular dichroism analysis and site‐directed tryptophan fluorescence. Apo‐tear lipocalin shows the same general structural characteristics as holo‐tear lipocalin including alternating periodicity of a β‐strand, orientation of amino acid residues 105, 103, 101, and 99 facing the cavity, and progressive depth in the cavity from residues 105 to 99. For amino acid residues facing the internal aspect of cavity, the presence of a ligand is associated with blue shifted spectra. The collisional rate constants indicate that these residues are not less exposed to solvent in holo‐tear lipocalin than in apo‐tear lipocalin. Rather the spectral blue shifts may be accounted for by a ligand induced rigidity in holo‐TL. Amino acid residues 94 and 95 are consistent with positions in the F‐G loop and show greater exposure to solvent in the holo‐ than the apo‐proteins. These findings are consistent with the general hypothesis that the F‐G loop in the holo‐proteins of the lipocalin family is available for receptor interactions and delivery of ligands to specific targets. Site‐directed tryptophan fluorescence was used in combination with a nitroxide spin labeled fatty acid analog to elucidate dynamic ligand interactions with specific amino acid residues. Collisional quenching constants of the nitroxide spin label provide evidence that at least three amino acids of the G strand residues interact with the ligand. Stern‐Volmer plots are inconsistent with a ligand that is held in a static position in the calyx, but rather suggest that the ligand is in motion. The combination of site‐directed tryptophan fluorescence with quenching by nitroxide labeled species has broad applicability in probing specific interactions in the solution structure of proteins and provides dynamic information that is not attainable by X‐ray crystallography.
Role of the switch II region in the conformational transition of activation of Ha‐ras‐p21
Authors: Díaz José Fernando , Escalona María Milagrosa , Kuppens Steven , Engelborghs Yves 
Published: October 2010

AbstractThe role of the switch II region in the conformational transition of activation of Ha‐ras‐p21 has been investigated by mutating residues predicted to act as hinges for the conformational transition of this loop (Ala59, Gly60, and Gly75) (Diaz JF, Wroblowski B, Schlitter J, Engelborghs Y, 1997, Proteins 28:434–451), as well as mutating the catalytic residue Gln61. The proposed mutations of the hinge residues decrease the rate of the conformational transition of activation as measured by the binding of BeF3‐ to the GDP‐p21 complex. Also, the thermodynamic parameters of the binding reaction are altered by a factor between three and five, depending on the temperature. (Due to changes in activation and reaction enthalpies, partially compensated by entropy changes.) The control mutation Q61H in which only the catalytic residue is changed has only a limited effect on the kinetic rate constants of the conformational transition and on the thermodynamic parameters of the reaction. The fact that mutations of the hinge residues of the switch II region affect both the binding of the phosphate analog and the conformational transition of activation indicates that the switch II is implicated both in the early and the late states of the transition.
Cleaved antitrypsin polymers at atomic resolution
Authors: Dunstone Michelle A. , Rossjohn Jamie , Feil Susanne C. , Parker Michael W. , Dai Weiwen , Whisstock James C. , Pike Robert N. , Bottomley Stephen P. , Le Bonniec Bernard F. 
Published: October 2010

Abstractα1‐Antitrypsin deficiency, which can lead to both emphysema and liver disease, is a result of the accumulation of α1 antitrypsin polymers within the hepatocyte. A wealth of biochemical and biophysical data suggests that α1‐antitrypsin polymers form via insertion of residues from the reactive center loop of one molecule into the β‐sheet of another. However, this long‐standing hypothesis has not been confirmed by direct structural evidence. Here, we describe the first crystallographic evidence of a β‐strand linked polymer form of α1‐antitrypsin: the crystal structure of a cleaved α1‐antitrypsin polymer.