bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural changes in α‐synuclein affect its chaperone‐like activity in vitro
Authors: Kim Thomas D. , Paik Seung R. , Yang Chul‐Hak , Kim Jongsun 
Published: February 2009

Abstractα‐Synuclein, a major constituent of Lewy bodies (LBs) in Parkinson's disease (PD), has been implicated to play a critical role in synaptic events, such as neuronal plasticity during development, learning, and degeneration under pathological conditions, although the physiological function of α‐synuclein has not yet been established. We here present biochemical evidence that recombinant α‐synuclein has a chaperone‐like function against thermal and chemical stress in vitro. In our experiments, α‐synuclein protected glutathione S‐transferase (GST) and aldolase from heat‐induced precipitation, and α‐lactalbumin and bovine serum albumin from dithiothreitol (DTT)‐induced precipitation like other molecular chaperones. Moreover, preheating of α‐synuclein, which is believed to reorganize the molecular surface of α‐synuclein, increased the chaperone‐like activity. Interestingly, in organic solvents, which promotes the formation of secondary structure, α‐synuclein aggregated more easily than in its native condition, which eventually might abrogate the chaperone‐like function of the protein. In addition, α‐synuclien was also rapidly and significantly precipitated by heat in the presence of Zn2+in vitro, whereas it was not affected by the presence of Ca2+or Mg2+. Circular dichroism spectra confirmed that α‐synuclein underwent conformational change in the presence of Zn2+. Taken together, our data suggest that α‐synuclein could act as a molecular chaperone, and that the conformational change of the α‐synuclein could explain the aggregation kinetics of α‐synuclein, which may be related to the abolishment of the chaperonic‐like activity.
Methylene as a possible universal footprinting reagent that will include hydrophobic surface areas: Overview and feasibility: Properties of diazirine as a precursor
Authors: Richards Frederic M. , Lamed Raphael , Wynn Richard , Patel Darshan , Olack Gerard 
Published: February 2009

AbstractMethylene is one of, if not the, most reactive organic chemical known. It has a very low specificity, which makes it essentially useless for synthesis, but suggests a possible role in protein footprinting with special importance in labeling solvent accessible nonpolar areas, identifying ligand binding sites, and outlining interaction areas on protomers that form homo or hetero oligomers in cellular assemblies. The singlet species is easily and conveniently formed by photolysis of diazirine. The reactions of interest are insertion into C‐H bonds and addition to multiple bonds, both forming strong covalent bonds and stable compounds. Reaction with proteins and peptides is reported even in aqueous solutions where the vast majority of the reagent is used up in forming methanol. Species containing up to 5 to 10 extra : CH2 groups are easily detected by electrospray mass spectroscopy. In a mixture of a 14 Kd protein and a noninteracting 1.7 Kd peptide, the distribution of mass peaks in the electrospray spectra was close to that expected from random modification of the estimated solvent accessible area for the two molecules. For analysis at the single residue level, quantitation at labeling levels of one 13CH2 group per 10 to 20 kDa of protein appears to be possible with isotope ratio mass spectroscopy. In the absence of reactive solvents, photolysis of diazirine produces oily polymeric species that contain one or two nitrogen atoms, but not more, and are water soluble.
An improved tripod amphiphile for membrane protein solubilization
Authors: Yu Seungju M. , McQuade D. Tyler , Quinn Mariah A. , Hackenberger Christian P.R. , Gellman Samuel H. , Krebs Mark P. , Polans Arthur S. 
Published: February 2009

AbstractIntrinsic membrane proteins represent a large fraction of the proteins produced by living organisms and perform many crucial functions. Structural and functional characterization of membrane proteins generally requires that they be extracted from the native lipid bilayer and solubilized with a small synthetic amphiphile, for example, a detergent. We describe the development of a small molecule with a distinctive amphiphilic architecture, a “tripod amphiphile,” that solubilizes both bacteriorhodopsin (BR) and bovine rhodopsin (Rho). The polar portion of this amphiphile contains an amide and an amine‐oxide; small variations in this polar segment are found to have profound effects on protein solubilization properties. The optimal tripod amphiphile extracts both BR and Rho from the native membrane environments and maintains each protein in a monomeric native‐like form for several weeks after delipidation. Tripod amphiphiles are designed to display greater conformational rigidity than conventional detergents, with the long‐range goal of promoting membrane protein crystallization. The results reported here represent an important step toward that ultimate goal.
Ab initio molecular dynamics studies on HIV‐1 reverse transcriptase triphosphate binding site: Implications for nucleoside‐analog drug resistance
Authors: Alber Frank , Carloni Paolo 
Published: February 2009

AbstractQuantum‐chemical methods are used to shed light on the functional role of residues involved in the resistance of HIV‐1 reverse transcriptase against nucleoside‐analog drugs. Ab initio molecular dynamics simulations are carried out for models representing the adduct between the triphosphate substrate and the nucleoside binding site. The triphosphate is considered either deprotonated or protonated at the γ‐position. Although the protonated form already experiences large rearrangements in the ps time scale, the fully deprotonated state exhibits a previously unrecognized low‐barrier hydrogen bond between Lys65 and γ‐phosphate. Absence of this interaction in Lys65→Arg HIV‐1 RT might play a prominent role in the resistance of this mutant for nucleoside analogs (Gu Z et al., 1994b, Antimicrob Agents Chemother 38:275–281; Zhang D et al., 1994, Antimicrob Agents Chemother 38:282–287). Water molecules present in the active site, not detected in the X‐ray structure, form a complex H‐bond network. Among these waters, one may be crucial for substrate recognition as it bridges Gln151 and Arg72 with the β‐phosphate. Absence of this stabilizing interaction in Gln151→Met HIV‐1 RT mutant may be a key factor for the known drug resistance of this mutant toward dideoxy‐type drugs and AZT (Shirasaka T et al., 1995, Proc Natl Acad Sci USA 92:2398–2402; Iversen AK et al., 1996, J Virol 70:1086–1090).
N‐terminal mutations in the anti‐estradiol Fab 57‐2 modify its hapten binding properties
Authors: Saviranta Petri , Jauria Piitu , Lamminmäki Urpo , Eriksson Susann , Lövgren Timo , Hellman Jukka 
Published: February 2009

AbstractRecombinant antibodies often contain N‐terminal mutations arising from the use of degenerate cloning primer sets and/or the introduction of restriction sites in the framework 1 regions. We studied the effects of such mutations in a recombinant anti‐estradiol Fab fragment derived from the hybridoma cell line 57‐2. The 5′ ends of the heavy and light chain genes were originally modified to introduce the restriction sites XhoI and SacI, respectively, for cloning purposes. However, the affinity and specificity of the recombinant Fab were lowered compared to the proteolytic Fab' fragment of the parental hybridoma IgG. Replacing the mutated sites with authentic amino acid coding sequences restored the binding properties as well as increased the bacterial production levels fivefold and 10‐fold at 30 and 37°C, respectively. Local changes in the antigen binding site were probed by determining the affinity constants (Ka) for estradiol and four related steroids. It was found that the mutated heavy chain amino terminus specifically increased the Ka for testosterone whereas the mutated light chain amino terminus decreased the Ka for all of the steroids to the same extent; the heavy and light chain effects were additive. Analysis of a newly determined crystal structure of the authentic Fab 57‐2 in complex with estradiol suggests that mutations in the residue 2 in VH, and 2 and 4 in the VL domain were those responsible for the observed effects. Their general roles as structure‐determining residues for the CDR3 loops imply that similar effects can occur with other recombinant antibodies as well.
For the record: A single amino acid substitution affects substrate specificity in cysteine proteinases from Fasciola hepatica
Authors: Smooker Peter M. , Whisstock James C. , Irving James A. , Siyaguna Sugeeshwara , Spithill Terry W. , Pike Robert N. 
Published: February 2009

AbstractThe trematode Fasciola hepatica secretes a number of cathepsin L‐like proteases that are proposed to be involved in feeding, migration, and immune evasion by the parasite. To date, six full cDNA sequences encoding cathepsin L preproproteins have been identified. Previous studies have demonstrated that one of these cathepsins (L2) is unusual in that it is able to cleave substrates with a proline in the P2 position, translating into an unusual ability (for a cysteine proteinase) to clot fibrinogen. In this study, we report the sequence of a novel cathepsin (L5) and compare the substrate specificity of a recombinant enzyme with that of recombinant cathepsin L2. Despite sharing 80% sequence identity with cathepsin L2, cathepsin L5 does not exhibit substantial catalytic activity against substrates containing proline in the P2 position. Molecular modeling studies suggested that a single amino acid change (L69Y) in the mature proteinases may account for the difference in specificity at the S2 subsite. Recombinant cathepsin L5/L69Y was expressed in yeast and a substantial increase in the ability of this variant to accommodate substrates with a proline residue in the P2 position was observed. Thus, we have identified a single amino acid substitution that can substantially influence the architecture of the S2 subsite of F. hepatica cathepsin L proteases.
For the record: Fluorescence and 19F NMR evidence that phenylalanine, 3‐L‐fluorophenylalanine and 4‐L‐fluorophenylalanine bind to the L‐leucine specific receptor of Escherichia coli
Authors: Luck Linda A. , Johnson Colin 
Published: February 2009

AbstractThe binding capacity of the L‐leucine receptor from Escherichia coli was measured with L‐phenylalanine and 4‐fluoro‐L‐phenylalanine as substrates by fluorescence. The apparent dissociation constants (KD) for L‐leucine, L‐phenylalanine, and 4‐fluoro‐L‐phenylalanine are 0.40, 0.18, and 0.26 respectively. 19F NMR data show protein‐induced shifts for the 4‐fluoro‐L‐phenylalanine peak and 3‐fluoro‐L‐phenylalanine when receptor is present. Evidence points to the binding of only the L‐isomers of these fluorine analogs.
Post‐translational modification is essential for catalytic activity of nitrile hydratase
Authors: Murakami Taku , Nojiri Masaki , Nakayama Hiroshi , Dohmae Naoshi , Takio Koji , Odaka Masafumi , Endo Isao , Nagamune Teruyuki , Yohda Masafumi 
Published: February 2009

AbstractNitrile hydratase from Rhodococcus sp. N‐771 is an αβ heterodimer with a nonheme ferric iron in the catalytic center. In the catalytic center, αCys112 and αCys114 are modified to a cysteine sulfinic acid (Cys‐SO2H) and a cysteine sulfenic acid (Cys‐SOH), respectively. To understand the function and the biogenic mechanism of these modified residues, we reconstituted the nitrile hydratase from recombinant unmodified subunits. The αβ complex reconstituted under argon exhibited no activity. However, it gradually gained the enzymatic activity through aerobic incubation. ESI‐LC/MS analysis showed that the anaerobically reconstituted αβ complex did not have the modification of αCys112‐SO2H and aerobic incubation induced the modification. The activity of the reconstituted αβ complex correlated with the amount of αCys112‐SO2H. Furthermore, ESI‐LC/MS analyses of the tryptic digest of the reconstituted complex, removed of ferric iron at low pH and carboxamidomethylated without reduction, suggested that αCys114 is modified to Cys‐SOH together with the sulfinic acid modification of αCys112. These results suggest that αCys112 and αCys114 are spontaneously oxidized to Cys‐SO2H and Cys‐SOH, respectively, and αCys112‐SO2H is responsible for the catalytic activity solely or in combination with αCys114‐SOH.
A model for the sickle hemoglobin fiber using both mutation sites
Authors: Roufberg Adam , Ferrone Frank A. 
Published: February 2009

AbstractThe standard molecular model of the fiber of the sickle hemoglobin (Hbs: β6 Glu → Val) has been revised to allow both β6 mutation sites to participate in intermolecular contacts, rather than only one β6 site as previously thought, for four molecules per 14‐molecule fiber cross section. This structure accurately predicts the copolymerization of hybridized mixtures of HbS with HbA or HbC (β6 Glu → Lys), which could not be reconciled with prior models in which only half the β6 sites were required for assembly. This model suggests new contacts within the fiber and raises the question of whether these cross‐linked double strands could possess added stability important in such processes as nucleation.
Binding of Zn2+ to a Ca2+ loop allosterically attenuates the activity of factor VIIa and reduces its affinity for tissue factor
Authors: Petersen Lars C. , Ård Per‐Ola Freskg , Persson Egon , Olsen Ole H. , Nielsen Lars S. 
Published: February 2009

AbstractThe protease domain of coagulation factor VIIa (FVIIa) is homologous to trypsin with a similar active site architecture. The catalytic function of FVIIa is regulated by allosteric modulations induced by binding of divalent metal ions and the cofactor tissue factor (TF). To further elucidate the mechanisms behind these transformations, the effects of Zn2+ binding to FVIIa in the free form and in complex with TF were investigated. Equilibrium dialysis suggested that two Zn2+ bind with high affinity to FVIIa outside the N‐terminal y‐carboxyglutamic acid (Gla) domain. Binding of Zn2+ to FVIIa, which was influenced by the presence of Ca2+, resulted in decreased amidolytic activity and slightly reduced affinity for TF. After binding to TF, FVIIa was less susceptible to zinc inhibition. Alanine substitutions for either of two histidine residues unique for FVIIa, His216, and His257, produced FVIIa variants with decreased sensitivity to Zn2+ inhibition. A search for putative Zn2+ binding sites in the crystal structure of the FVIIa protease domain was performed by Grid calculations. We identified a pair of Zn2+ binding sites in the Glu210–Glu220 Ca2+ binding loop adjacent to the so‐called activation domain canonical to serine proteases. Based on our results, we propose a model that describes the conformational changes underlying the Zn2+‐mediated allosteric down‐regulation of FVIIa's activity.
Conformational transitions and fibrillation mechanism of human calcitonin as studied by high‐resolution solid‐state 13C NMR
Authors: Kamihira Miya , Naito Akira , Tuzi Satoru , Saitô Hazime , Nosaka Atsuko Y. 
Published: February 2009

AbstractConformational transitions of human calcitonin (hCT) during fibril formation in the acidic and neutral conditions were investigated by high‐resolution solid‐state 13C NMR spectroscopy. In aqueous acetic acid solution (pH 3.3), a local α‐helical form is present around Gly10, whereas a random coil form is dominant as viewed from Phe22, Ala26, and Ala31 in the monomer form on the basis of the 13C chemical shifts. On the other hand, a local β‐sheet form as viewed from Gly10 and Phe22, and both β‐sheet and random coil as viewed from Ala26 and Ala31 were detected in the fibril at pH 3.3. The results indicate that conformational transitions from α‐helix to β‐sheet, and from random coil to β‐sheet forms occurred in the central and C‐terminus regions, respectively, during the fibril formation. The increased 13C resonance intensities of fibrils after a certain delay time suggests that the fibrillation can be explained by a two‐step reaction mechanism in which the first step is a homogeneous association to form a nucleus, and the second step is an autocatalytic heterogeneous fibrillation. In contrast to the fibril at pH 3.3, the fibril at pH 7.5 formed a local β‐sheet conformation at the central region and exhibited a random coil at the C‐terminus region. Not only a hydrophobic interaction among the amphiphilic α‐helices, but also an electrostatic interaction between charged side chains can play an important role for the fibril formation at pH 7.5 and 3.3 acting as electrostatically favorable and unfavorable interactions, respectively. These results suggest that hCT fibrils are formed by stacking antiparallel β‐sheets at pH 7.5 and a mixture of antiparallel and parallel β‐sheets at pH 3.3.
Ser45 plays an important role in managing both the equilibrium and transition state energetics of the streptavidin—biotin system
Authors: Hyre David E. , Stayton Patrick S. , Trong Isolde Le , Freitag Stefanie , Stenkamp Ronald E. 
Published: February 2009

AbstractThe contribution of the Ser45 hydrogen bond to biotin binding activation and equilibrium thermodynamics was investigated by biophysical and X‐ray crystallographic studies. The S45A mutant exhibits a 1,700‐fold greater disso‐ciation rate and 907‐fold lower equilibrium affinity for biotin relative to wild‐type streptavidin at 37dGC, indicating a crucial role in binding energetics. The crystal structure of the biotin‐bound mutant reveals only small changes from the wild‐type bound structure, and the remaining hydrogen bonds to biotin retain approximately the same lengths. No additional water molecules are observed to replace the missing hydroxyl, in contrast to the previously studied D128A mutant. The equilibrium ΔG°, ΔH°, ΔS°, ΔCP°, and activation ΔG‡ of S45A at 37°C are ‐13.7 ± 0.1 kcal/mol, ‐21.1 ± 0.5 kcal/mol, ‐23.7 ± 1.8 cal/molK, ‐223 ± 12 cal/mol K, and 20.0 ± 2.5 kcal/mol, respectively. Eyring analysis of the large temperature dependence of the S45A off‐rate resolves the ΔH‡ and ΔS‡ of dissociation, 25.8 ± 1.2 kcal/mol and 18.7 ± 4.3 cal/mol K. The large increases of ΔH‡ and ΔS‡ in the mutant, relative to wild‐type, indicate that Ser45 could form a hydrogen bond with biotin in the wild‐type dissociation transition state, enthalpically stabilizing it, and constraining the transition state entropically. The postulated existence of a Ser45‐mediated hydrogen bond in the wild‐type streptavidin transition state is consistent with potential of mean force simulations of the dissociation pathway and with molecular dynamics simulations of biotin pullout, where Ser45 is seen to form a hydrogen bond with the ureido oxygen as biotin slips past this residue after breaking the native hydrogen bonds.
Conformational flexibility in the apolipoprotein E amino‐terminal domain structure determined from three new crystal forms: Implications for lipid binding
Authors: Segelke Brent W. , Forstner Michael , Knapp Mark , Trakhanov Sergei D. , Parkin Sean , Newhouse Yvonne M. , Bellamy Henry D. , Weisgraber Karl H. , Rupp Bernhard 
Published: February 2009

AbstractAn amino‐terminal fragment of human apolipoprotein E3 (residues 1‐165) has been expressed and crystallized in three different crystal forms under similar crystallization conditions. One crystal form has nearly identical cell dimensions to the previously reported orthorhombic (P212121) crystal form of the amino‐terminal 22 kDa fragment of apolipoprotein E (residues 1‐191). A second orthorhombic crystal form (P212121 with cell dimensions differing from the first form) and a trigonal (P3121) crystal form were also characterized. The structures of the first orthorhombic and the trigonal form were determined by seleno‐methionine multiwavelength anomalous dispersion, and the structure of the second ortho‐rhombic form was determined by molecular replacement using the structure from the trigonal form as a search model. A combination of modern experimental and computational techniques provided high‐quality electron‐density maps, which revealed new features of the apolipoprotein E structure, including an unambiguously traced loop connecting helices 2 and 3 in the four‐helix bundle and a number of multiconformation side chains. The three crystal forms contain a common intermolecular, antiparallel packing arrangement. The electrostatic complimentarity observed in this antiparallel packing resembles the interaction of apolipoprotein E with the monoclonal antibody 2E8 and the low density lipoprotein receptor. Superposition of the model structures from all three crystal forms reveals flexibility and pronounced kinks in helices near one end of the four‐helix bundle. This mobility at one end of the molecule provides new insights into the structural changes in apolipoprotein E that occur with lipid association.
Alternate modes of binding in two crystal structures of alkaline phosphatase‐inhibitor complexes
Authors: Holtz Kathleen M. , Kantrowitz Evan R. , Stec Boguslaw , Myers Jason K. , Antonelli Stephen M. , Widlanski Theodore S. 
Published: February 2009

AbstractTwo high resolution crystal structures of Escherichia coli alkaline phosphatase (AP) in the presence of phosphonate inhibitors are reported. The phosphonate compounds, phosphonoacetic acid (PAA) and mercaptomethylphosphonic acid (MMP), bind competitively to AP with dissociation constants of 5.5 and 0.6 mM, respectively. The structures of the complexes of AP with PAA and MMP were refined at high resolution to crystallographic R‐values of 19.0 and 17.5%, respectively. Refinement of the AP‐inhibitor complexes was carried out using X‐PLOR. The final round of refinement was done using SHELXL‐97. Crystallographic analyses of the inhibitor complexes reveal different binding modes for the two phosphonate compounds. The significant difference in binding constants can be attributed to these alternative binding modes observed in the high resolution X‐ray structures. The phosphinyl group of PAA coordinates to the active site zinc ions in a manner similar to the competitive inhibitor and product inorganic phosphate. In contrast, MMP binds with its phosphonate moiety directed toward solvent. Both enzyme‐inhibitor complexes exhibit close contacts, one of which has the chemical and geometrical potential to be considered an unconventional hydrogen bond of the type C‐H…X.