bluuebunny/crossref_metadata_2025_split huggingface dataset:
Crystal structure of deoxygenated limulus polyphemus subunit II hemocyanin at 2.18 Å resolution: Clues for a mechanism for allosteric regulation
Authors: Hazes Bart , Kalk Kor H. , Hol WIM G.J. , Magnus Karen A. , Bonaventura Celia , Bonaventura Joseph , Dauter Zbigniew 
Published: July 2010

AbstractThe crystal structure of Limulus polyphemus subunit type II hemocyanin in the deoxygenated state has been determined to a resolution of 2.18 Å. Phase information for this first structure of a cheliceratan hemocyanin was obtained by molecular replacement using the crustacean hemocyanin structure of Panulirus interruptus. The most striking observation in the Limulus structure is the unexpectedly large distance of 4.6 Å between both copper ions in the oxygen‐binding site. Each copper has approximate trigonal planar coordination by three histidine Nε atoms. No bridging ligand between the copper ions could be detected. Other important new discoveries are (1) the presence of a cis‐peptide bond between Glu 309 and Ser 310, with the carbonyl oxygen of the peptide plane hydrogen bonded to the Nδ atom of the copper B ligand His 324; (2) localization of a chloride‐binding site in the interface between the first and second domain; (3) localization of a putative calcium‐binding site in the third domain. Furthermore, comparison of Limulus versus Panulirus hemocyanin revealed considerable tertiary and quaternary rigid body movements, although the overall folds are similar. Within the subunit, the first domain is rotated by about 7.5° with respect to the other two domains, whereas within the hexamer the major movement is a 3.1° rotation of the trimers with respect to each other. The rigid body rotation of the first domain suggests a structural mechanism for the allosteric regulation by chloride ions and probably causes the cooperative transition of the hexamer between low and high oxygen affinity states. In this postulated mechanism, the fully conserved Phe 49 is the key residue that couples conformational changes of the dinuclear copper site into movements of the first domain.
A model for the calmodulin—peptide complex based on the troponin C crystal packing and its similarity to the NMR structure of the calmodulin—myosin light chain kinase peptide complex
Authors: Sekharudu C.Y. , Sundaralingam M. 
Published: July 2010

AbstractIn the crystal structure of troponin C, the holo C‐domain is bound in a head‐to‐tail fashion to the A‐helix of the apo N‐domain of a symmetry‐related molecule. Using this interaction, we have proposed a model for the calmodulin‐peptide complex. We find that the interaction of the C‐domain with the A‐helix is similar to that observed in the NMR structure of the calmodulin‐myosin light chain kinase (MLCK) peptide complex. This similarity in binding has enabled us to make a precise sequence alignment of the target peptides in the calmodulin‐binding cleft and to rationalize the amino acid sequence‐dependent binding strengths of various peptides. Our model differs from that proposed by Strynadka and James (Proteins Struct. Funct. Genet. 7, 234–248, 1990) in that the peptides are rotated by 100° in the calmodulin binding cleft.
Investigations of the thermostability of rubredoxin models using molecular dynamics simulations
Authors: Bradley Elizabeth A. , Adams MICHAEL W.W. , Wampler John E. , Stewart David E. 
Published: July 2010

AbstractThe affects of differences in amino acid sequence on the temperature stability of the three‐dimensional structure of the small β‐sheet protein, rubredoxin (Rd), was revealed when a set of homology models was subjected to molecular dynamics simulations at relatively high temperatures. Models of Rd from the hyperthermophile, Pyrococcus furiosus (Pf), an organism that grows optimally at 100°C, were compared to three mesophilic Rds of known X‐ray crystal structure. Simulations covering the limits of known Rd thermostabilities were carried out at temperatures of 300 K, 343 K, 373 K, and 413 K. They suggest that Rd stability is correlated with structural dynamics. Because the dynamic behavior of three Pf Rd models was consistently different from the dynamic behavior of the three mesophilic Rd structures, detailed analysis of the temperature‐dependent dynamic behavior was carried out. The major differences between the models of the protein from the hyperthermophile and the others were: (1) an obvious temperature‐dependent transition in the mesophilic structures not seen with the Pf Rd models, (2) consistent AMBER energy for the Pf Rd due to differences in nonbonded interaction terms, (3) less variation in the average conformations for the Pf Rd models with temperature, and (4) the presence of more extensive secondary structure for the Pf Rd models. These unsolvated dynamics simulations support a simple, general hypothesis to explain the hyperthermostability of Pf Rd. Its structure simplifies the conformational space to give a single minimum accessible over an extreme range of temperatures, whereas the mesophilic proteins sample a more complex conformational space with two or more minima over the same temperature range.
Hydrogen bond interactions of G proteins with the guanine ring moiety of guanine nucleotides
Authors: Weng G. , Chen C.X. , Balogh‐Nair V. , Callender R. , Manor D. 
Published: July 2010

AbstractWe have utilized Raman difference spectroscopy to investigate hydrogen bonding interactions of the guanine moiety in guanine nucleotides with the binding site of two G proteins, EF‐Tu (elongation factor Tu from Escherichia coli) and the c‐Harvey ras protein, p21 (the gene product of the human c‐H‐rus proto‐oncogene). Raman spectra of proteins complexed with GDP (guanosine 5′ diphosphate), IDP (inosine 5′ diphosphate), 6‐thio‐GDP, and 6‐18O‐GDP were measured, and the various difference spectra were determined. These were compared to the difference spectra obtained in solution, revealing vibrational features of the nucleotide that are altered upon binding. Specifically, we observed significant frequency shifts in the vibrational modes associated with the 6‐keto and 2‐amino positions of the guanine group of GDP and IDP that result from hydrogen bonding interactions between these groups and the two proteins. These shifts are interpreted as being proportional to the local energy of interaction (δH) between the two groups and protein residues at the nucleotide binding site. Consistent with the tight binding between the nucleotides and the two proteins, the shifts indicate that the enthalpic interactions are stronger between these two polar groups and protein than with water. In general, the spectral shifts provide a rationale for the stronger binding of GDP and IDP with p21 compared to EF‐Tu. Despite the structural similarity of the binding sites of EF‐Tu and p21, the strengths of the observed hydrogen bonds at the 6‐keto and 2‐amino positions vary substantially, by up to a factor of 2. The hydrogen bond between the protein and the 6‐keto group is stronger for GDP in EF‐Tu compared to p21, but this comparison is reversed for bound IDP. Thus, the removal of the 2‐amino group from GDP to form IDP affects binding properties distal to the amino site.
Structure of the membrane channel porin from Rhodopseudomonas blastica at 2.0 Å resolution
Authors: Kreusch A. , Neubüser A. , Schiltz E. , Weckesser J. , Schulz G.E. 
Published: July 2010

AbstractThe crystal structure of a membrane channel, homotrimeric porin from Rhodopseudomonas blastica has been determined at 2.0 Å resolution by multiple isomorphous replacement and structural refinement. The current model has an R‐factor of 16.5% and consists of 289 amino acids, 238 water molecules, and 3 detergent molecules per subunit. The partial protein sequence and subsequently the complete DNA sequence were determined. The general architecture is similar to those of the structurally known porins. As a particular feature there are 3 adjacent binding sites for n‐alkyl chains at the molecular 3‐fold axis. The side chain arrangement in the channel indicates a transverse electric field across each of the 3 pore eyelets, which may explain the discrimination against nonpolar solutes. Moreover, there are 2 significantly ordered girdles of aromatic residues at the nonpolar/polar borderlines of the interface between protein and membrane. Possibly, these residues shield the polypeptide conformation against adverse membrane fluctuations.
Solution structure of villin 14T, a domain conserved among actin‐severing proteins
Authors: Markus Michelle A. , Nakayama Tomoko , Matsudaira Paul , Wagner Gerhard 
Published: July 2010

AbstractThe solution structure of the N‐terminal domain of the actin‐severing protein villin has been determined by multidimensional heteronuclear resonance spectroscopy. Villin is a member of a family of actin‐severing proteins that regulate the organization of actin in the eukaryotic cytoskeleton. Members of this family are built from 3 or 6 homologous repeats of a structural domain of approximately 130 amino acids that is unrelated to any previously known structure. The N‐terminal domain of villin (14T) contains a central β‐sheet with 4 antiparallel strands and a fifth parallel strand at one edge. This sheet is sandwiched between 2 helices on one side and a 2‐stranded parallel β‐sheet with another helix on the other side. The strongly conserved sequence characteristic of the protein family corresponds to internal hydrophobic residues. Calcium titration experiments suggest that there are 2 binding sites for Ca2+, a stronger site near the N‐terminal end of the longest helix, with a Kd of 1.8 ± 0.4 mM, and a weaker site near the C‐terminal end of the same helix, with a Kd of 11 ± 2 mM. Mutational and biochemical studies of this domain in several members of the family suggest that the actin monomer binding site is near the parallel strand at the edge of the central β‐sheet.
Two conformational states of Candida rugosa lipase
Authors: Grochulski Pawel , Li Yunge , Schrag Joseph D. , Cygler Miroslaw 
Published: July 2010

AbstractThe structure of Candida rugosa lipase in a new crystal form has been determined and refined at 2.1 Å resolution. The lipase molecule was found in an inactive conformation, with the active site shielded from the solvent by a part of the polypeptide chain — the flap. Comparison of this structure with the previously determined “open” form of this lipase, in which the active site is accessible to the solvent and presumably the substrate, shows that the transition between these 2 states requires only movement of the flap. The backbone NH groups forming the putative oxyanion hole do not change position during this rearrangement, indicating that this feature is preformed in the inactive state. The 2 lipase conformations probably correspond to states at opposite ends of the pathway of interfacial activation. Quantitative analysis indicates a large increase of the hydrophobic surface in the vicinity of the active site. The flap undergoes a flexible rearrangement during which some of its secondary structure refolds. The interactions of the flap with the rest of the protein change from mostly hydrophobic in the inactive form to largely hydrophilic in the “open” conformation. Although the flap movement cannot be described as a rigid body motion, it has very definite hinge points at Glu 66 and at Pro 92. The rearrangement is accompanied by a cis‐trans isomerization of this proline, which likely increases the energy required for the transition between the 2 states, and may play a role in the stabilization of the active conformation at the water/lipid interface. Carbohydrate attached at Asn 351 also provides stabilization for the open conformation of the flap.
Positions of His‐64 and a bound water in human carbonic anhydrase II upon binding three structurally related inhibitors
Authors: Smith Graham M. , Alexander Richard S , Christianson David W. , Mckeever Brian M. , Ponticello Gerald S. , Springer James P. , Randall William C. , Baldwin John J. , Habecker Charles N. 
Published: July 2010

AbstractThe 3‐dimensional structure of human carbonic anhydrase II (HCAII; EC 4.2.1.1) complexed with 3 structurally related inhibitors, la, lb, and lc, has been determined by X‐ray crystallographic methods. The 3 inhibitors (la = C8H12N2O4S3) vary only in the length of the substituent on the 4‐amino group: la, proton; lb, methyl; and lc, ethyl. The binding constants (Ki's) for la, Ib, and lc to HCAII are 1.52, 1.88, and 0.37 nM, respectively. These structures were solved to learn if any structural cause could be found for the difference in binding. In the complex with inhibitors la and lb, electron density can be observed for His‐64 and a bound water molecule in the native positions. When inhibitor lc is bound, the side chain attached to the 4‐amino group is positioned so that His‐64 can only occupy the alternate position and the bound water is absent. While a variety of factors contribute to the observed binding constants, the major reason lc binds tighter to HCAII than does la or lb appears to be entropy: the increase in entropy when the bound water molecule is released contributes to the increase in binding and overcomes the small penalty for putting the His‐64 side chain in a higher energy state.
Studies on the specificity of acetylaminoacylpeptide hydrolase
Authors: Sokolik Charles W. , Liang T. Chyau , Wold Finn 
Published: July 2010

AbstractIn a continuing attempt to explore the types of specificity determinants that may affect protein‐protein (peptide) interactions, a number of short (2‐5 residues) acetylated peptides have been compared as substrates for the enzyme acetylaminoacyl‐peptide hydrolase (EC 3.4.19.1). The reference substrate was Ac‐AAAA, and most of the other substrates were derived from this basic structure by single amino acid substitutions. The Km and kcat, for the different substrates were determined by standard steady‐state kinetics, and the corresponding δδGT D̊x value derived from kcat/Km was used for the comparison, setting δδGT D̊ for Ac‐AAAA equal to 0. The best substrates were found to be those containing negative charges (Asp > Glu) or aromatic residues in positions 1′, 2′, or 3′ (δδGT D̊ values of 2‐5 kJ); the negative charge provided by the C‐terminus of the substrate also appears to be important, since the amide and O‐Me ester derivatives caused a change in δδGT D̊ values of ‐7 to ‐8 kJ from the reference peptide. The stimulating effect of the negative charges is consistent with the inhibitory effect of positive charges in similar peptides (Krishna RG, Wold F, 1992, Protein Sci 1:582‐589), and the proposed active site model incorporates subsites for both charge‐charge and hydrophobic interactions. In assessing all the data, it is clear that the properties of the individual substrates reflect the total make‐up of each peptide and not only the effect of a single residue in a given position. Thus, while the peptides with single Asp or Phe substitutions in 1′, 2′, and 3′ gave δδGT D̊ values of 3‐5 kJ, the peptide containing all 3 modifications, Ac‐ADDF, gave only 1 kJ. Similarly, Ac‐TAAA was a poor substrate and Ac‐GAAA was not cleaved at all in this study, while in the past other peptides such as Ac‐TGG and Ac‐GGG have been found to be excellent and reasonably good substrates, respectively. Although the rate differences observed in this work are minor, they nevertheless appear to reflect the kind of structural detail that is involved in determining the specificity of protein‐protein (peptide) interactions.
The native state of apomyoglobin described by proton NMR spectroscopy: Interaction with the paramagnetic probe HyTEMPO and the fluorescent dye ANS
Authors: Cocco Melanie J. , Lecomte Juliette T.J. 
Published: July 2010

AbstractProton NMR experiments were carried out on apomyoglobin from sperm whale and horse skeletal muscle. Two small molecules, the paramagnetic relaxation agent 4‐hydroxy‐2,2,6,6‐tetramethylpiperidinyl‐l‐oxy (HyTEMPO) and the fluorescent dye 8‐anilino‐1‐naphthalenesulfonic acid (ANS), were used to alter and simplify the spectrum. Both were shown to bind in the heme pocket by docking onto the hydrophobic residues lining the distal side. Only 1 extensive region of the apoprotein structure, composed of hydrophobic residues, is not affected by HyTEMPO. It includes the 2 tryptophans (located in the A helix), other nonpolar residues of the A helix and side chains from the E, G, and GH helices. The spectral perturbations induced by ANS allowed assignment of the distal histidine (His‐64) in horse apomyoglobin. This residue was previously reported to titrate with a pKa below 5 and tentatively labeled as His‐82 on the basis of this value (Cocco MJ, Kao YH, Phillips AT, Lecomte JTJ, 1992, Biochemistry 31:6481‐6491). The packing of the side chains and the low pKa of His‐64 reinforce the idea that the distal side of the binding site is folded in a manner closely related to that in the holoprotein. ANS was found to sharpen the protein signals and the improvement of the spectral resolution facilitated the assignment of backbone amide resonances. Secondary structure, as manifested in characteristic inter‐amide proton NOEs, was detected in the A, B, C, E, G, and H helices. The combined information on the hydrophobic cores and the secondary structure composes an improved representation of the native state of apomyoglobin.
Enzyme IIB bcellobiose of the phosphoenol‐pyruvate‐dependent phosphotransferase system of Escherichia coli: Backbone assignment and secondary structure determined by three‐dimensional NMR spectroscopy
Authors: Ab Eiso , Schuurman‐Wolters Gea K. , Saier Milton H. , Reizer Jonathan , Jacuinod Michel , Roepstorff Peter , Dijkstra Klaas , Scheek Ruud M. , Robillard George T. 
Published: July 2010

AbstractThe assignment of backbone resonances and the secondary structure determination of the Cys 10 Ser mutant of enzyme IIBcellobiose of the Escherichia coli cellobiose‐specific phosphoenol‐pyruvate‐dependent phosphotransferase system are presented. The backbone resonances were assigned using 4 triple resonance experiments, the HNCA and HN(CO)CA experiments, correlating backbone 1H, 15N, and 13Cα resonances, and the HN(CA)CO and HNCO experiments, correlating backbone 1H,15N and 13CO resonances. Heteronuclear 1H‐NOE 1H‐15N single quantum coherence (15N‐NOESY‐HSQC) spectroscopy and heteronuclear 1H total correlation 1H‐15N single quantum coherence (15N‐TOCSY‐HSQC) spectroscopy were used to resolve ambiguities arising from overlapping 13Cα and 13CO frequencies and to check the assignments from the triple resonance experiments. This procedure, together with a 3‐dimensional 1Ha‐13Cα‐13CO experiment (COCAH), yielded the assignment for all observed backbone resonances. The secondary structure was determined using information both from the deviation of observed 1Hα and 13Cα chemical shifts from their random coil values and 1H‐NOE information from the 15N‐NOESY‐HSQC. These data show that enzyme IIBcellobiose consists of a 4‐stranded parallel β‐sheet and 5 α‐helices. In the wild‐type enzyme IIBcellobiose, the catalytic residue appears to be located at the end of a β‐strand.
Characterization of a truncated form of arrestin isolated from bovine rod outer segments
Authors: Palczewski Krzysztof , Buczylko Janina , Ohguro Hiroshi , Annan Roland S. , Carr Steven A. , Crabb John W. , Kaplan Michael W. , Johnson Richard S. , Walsh Kenneth A. 
Published: July 2010

AbstractThe inactivation of photolyzed rhodopsin requires phosphorylation of the receptor and binding of a 48‐kDa regulatory protein, arrestin. By binding to phosphorylated photolyzed rhodopsin, arrestin inhibits G protein (Gt) activation and blocks premature dephosphorylation, thereby preventing the reentry of photolyzed rhodopsin into the phototransduction pathway. In this study, we isolated a 44‐kDa form of arrestin, called p44, from fresh bovine rod outer segments and characterized its structure and function. A partial primary structure of p44 was established by a combination of mass spectrometry and automated Edman degradation of proteolytic peptides. The amino acid sequence was found to be identical with arrestin, except that the C‐terminal 35 residues (positions 370‐404) are replaced by a single alanine. p44 appeared to be generated by alternative mRNA splicing, because intron 15 interrupts within the nucleotide codon for 369Ser in the arrestin gene. Functionally, p44 binds avidly to photolyzed or phosphorylated and photolyzed rhodopsin. As a consequence of its relatively high affinity for bleached rhodopsin, p44 blocks Gt activation. The binding characteristics of p44 set it apart from tryptic forms of arrestin (truncated at the N‐ and C‐termini), which require phosphorylation of rhodopsin for tight binding. We propose that p44 is a novel splice variant of arrestin that could be involved in the regulation of Gt activation.
Sequence relationships between integral inner membrane proteins of binding protein‐dependent transport systems: Evolution by recurrent gene duplications
Authors: Saurin W. , Dassa E. 
Published: July 2010

AbstractPeriplasmic binding protein‐dependent transport systems are composed of a periplasmic substrate‐binding protein, a set of 2 (sometimes 1) very hydrophobic integral membrane proteins, and 1 (sometimes 2) hydrophilic peripheral membrane protein that binds and hydrolyzes ATP. These systems are members of the superfamily of ABC transporters. We performed a molecular phylogenetic analysis of the sequences of 70 hydrophobic membrane proteins of these transport systems in order to investigate their evolutionary history. Proteins were grouped into 8 clusters. Within each cluster, protein sequences displayed significant similarities, suggesting that they derive from a common ancestor. Most clusters contained proteins from systems transporting analogous substrates such as monosaccharides, oligopeptides, or hydrophobic amino acids, but this was not a general rule. Proteins from diverse bacteria are found within each cluster, suggesting that the ancestors of current clusters were present before the divergence of bacterial groups. The phylogenetic trees computed for hydrophobic membrane proteins of these per‐meases are similar to those described for the periplasmic substrate‐binding proteins. This result suggests that the genetic regions encoding binding protein‐dependent permeases evolved as whole units. Based on the results of the classification of the proteins and on the reconstructed phylogenetic trees, we propose an evolutionary scheme for periplasmic permeases. According to this model, it is probable that these transport systems derive from an ancestral system having only 1 hydrophobic membrane protein. None of the proteins considered in this study display detectable sequence similarity to hydrophobic membrane proteins or domains from other ABC transporters such as bacterial polysaccharide export systems, bacterial toxin proteins exporters, and eukaryotic ABC proteins. It is likely that they constitute a specific subfamily within the superfamily of ABC transporters.