bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of a specific tyrosine residue in Bryodin 1 distinct from the active site but required for full catalytic and cytotoxic activity
Authors: Fryxell Daniel K. , Gawlak Susan L. , Dodge Robert W. , Siegall Clay B. 
Published: July 2010

AbstractBryodin 1 (BD1) is a type I ribosome‐inactivating protein (RIP) with low inherent animal toxicity. It has been cloned recently and the recombinant protein (rBDl) has been produced and crystallized. To gain insight into the relationship of rBDl structure and function, we investigated the role of sequences in a region (residues 128‐156) that exhibits homology with membrane interactive sequences and is not part of the enzymatically defined active site. Progressive deletions representing α‐helical turns within these residues were generated; mutant rBDl proteins were expressed in Escherichia coli and demonstrated increasing losses of enzymatic activity. Point mutations were also generated within this region to replace Y140, Y141, and Y142 with either alanine or lysine. Mutants at position 140 or 142 retained full enzymatic activity, whereas A141 and K141 mutants were > 19‐fold less potent. In cytotoxicity assays, the rBDl point mutants at Y141 were > 80‐fold less potent than either rBDl or mutants at residues 140 or 142. However, when introduced into the anti‐CD40 single‐chain immunotoxin rBDl‐G28‐5 sFv, the A140 and A141 point mutations led to decreased cytotoxicity toward CD40 positive cell lines. These data indicate that Y141 plays an important role in the enzymatic activity of BD1 and that Y140, although not essential for catalytic activity, is required for full BD1 function. Because residues 140 and 141 are distinct from residues implicit in the active site, they may be involved in ribosomal and/or membrane interactions or in intracellular trafficking of the toxin and immunotoxin.
Interhelical contacts are required for the helix bundle fold of apolipophorin III and its ability to interact with lipoproteins
Authors: Wang Jianjun , Narayanaswami Vasanthy , Ryan Robert O. , Sykes Brian D. 
Published: July 2010

AbstractApolipophorin‐III (apoLp‐III) from the insect, Manduca sexta, is a 166‐residue exchangeable apolipoprotein that plays a critical role in the dynamics of plasma lipoprotein interconversions. Our previous work indicated that a 36‐residue C‐terminal peptide fragment, generated by cyanogen bromide digestion of apoLp‐III, was unable to bind to lipid surfaces (Narayanaswami V, Kay CM, Oikawa K, Ryan RO, 1994, Biochemistry 33:13312‐13320), and showed no secondary structure in aqueous solution. In this paper, we have performed structural studies of this peptide (E131‐Q166) complexed with SDS detergent micelles, or in the presence of the helix‐inducing solvent trifluoroethanol (TFE), by two‐dimensional 1H NMR spectroscopy. The peptide adopts an α‐helical structure in the presence of both SDS and 50% TFE. The lipid‐bound structure of the peptide, generated from the NMR NOE data, showed an elongated, slightly curved α‐helix. Despite its high α‐helix forming propensity, the peptide requires a helix‐promoting environment to adopt an α‐helical structure. This indicates the importance of the surrounding chemical environment and implies that, in the absence of lipid, tertiary contacts in the folded protein play a role in maintaining its structural integrity. Furthermore, the data suggest that the amphipathic helix bundle organization serves as a prerequisite structural motif for the reversible lipoprotein‐binding activity of M. sexta apoLp‐III.
Solid‐state NMR studies of the membrane‐bound closed state of the colicin E1 channel domain in lipid bilayers
Authors: Kim Yongae , Valentine Kathleen , Opella Stanley J. , Schendel Sharon L. , Cramer William A. 
Published: July 2010

AbstractThe colicin El channel polypeptide was shown to be organized anisotropically in membranes by solid‐state NMR analysis of samples of uniformly 15N‐labeled protein in oriented planar phospholipid bilayers. The 190 residue C‐terminal colicin E1 channel domain is the largest polypeptide to have been characterized by 15N solid‐state NMR spectroscopy in oriented membrane bilayers. The 15N‐NMR spectra of the colicin E1 show that: (1) the structure and dynamics are independent of anionic lipid content in both oriented and unoriented samples; (2) assuming the secondary structure of the polypeptide is helical, there are both trans‐membrane and in‐plane helical segments; (3) trans‐membrane helices account for approximately 20‐25% of the channel polypeptide, which is equivalent to 38‐48 residues of the 190‐residue polypeptide. The results of the two‐dimensional PISEMA spectrum are interpreted in terms of a single trans‐membrane helical hairpin inserted into the bilayer from each channel molecule. These data are also consistent with this helical hairpin being derived from the 38‐residue hydrophobic segment near the C‐terminus of the colicin E1 channel polypeptide.
Three solutions of the protein solubility problem
Authors: Terry Jenkins W. 
Published: July 2010

AbstractThree simple equations are presented, which describe the variation of protein solubility (S) with changes in salt concentration, in terms of either the salt molality (M), the salt activity (ax), or the water activity (aw). Each equation yields, essentially independent, estimates of the numbers of salt ions (Δvx) and water molecules (Δvw) involved in the dissolution of a mol of the protein. The equations can be used to elucidate the physical significance of the parameters in other empirical equations for protein solubility.
15N backbone dynamics of the S‐peptide from ribonuclease A in its free and S‐protein bound forms: Toward a site‐specific analysis of entropy changes upon folding
Authors: Alexandrescu Andrei T. , Rathgeb‐Szabo Klara , Jahnke Wolfgang , Schulthess Therese , Kammerer Richard A. , Rumpel Klaus 
Published: July 2010

AbstractBackbone 15N relaxation parameters (Rl, R2, 1H‐15N NOE) have been measured for a 22‐residue recombinant variant of the S‐peptide in its free and S‐protein bound forms. NMR relaxation data were analyzed using the “model‐free” approach (Lipari & Szabo, 1982). Order parameters obtained from “model‐free” simulations were used to calculate 1H‐15N bond vector entropies using a recently described method (Yang & Kay, 1996), in which the form of the probability density function for bond vector fluctuations is derived from a diffusion‐in‐a‐cone motional model. The average change in 1H‐15N bond vector entropies for residues T3‐S15, which become ordered upon binding of the S‐peptide to the S‐protein, is ‐12.6 ± 1.4 J/mol.residue.K. 15N relaxation data suggest a gradient of decreasing entropy values moving from the termini toward the center of the free peptide. The difference between the entropies of the terminal and central residues is about ‐ 12 J/mol.residue.K, a value comparable to that of the average entropy change per residue upon complex formation. Similar entropy gradients are evident in NMR relaxation studies of other denatured proteins. Taken together, these observations suggest denatured proteins may contain entropic contributions from non‐local interactions. Consequently, calculations that model the entropy of a residue in a denatured protein as that of a residue in a di ‐ or tri‐peptide, might over‐estimate the magnitude of entropy changes upon folding.
Chemical synthesis and structural characterization of the RGD‐protein decorsin: A potent inhibitor of platelet aggregation
Authors: Laureto Patrizia Polverino De , Scaramella Elena , Filippis Vincezo De , Marin Oriano , Doni Maria Gabriella , Fontana Angelo 
Published: July 2010

AbstractDecorsin is a 39‐residue RGD‐protein crosslinked by three disulfide bridges isolated from the leech Macrobdella decora belonging to the family of GPIIb‐IIIa antagonists and acting as a potent inhibitor of platelet aggregation. Here we report the solid‐phase synthesis of decorsin using the Fmoc strategy. The crude polypeptide was purified by reverse‐phase HPLC in its reduced form and allowed to refold in the presence of glutathione. The homogeneity of the synthetic oxidized decorsin was established by reverse‐phase HPLC and capillary zone electrophoresis. The results of amino acid analysis after acid hydrolysis of the synthetic protein, NH2‐terminal sequencing and mass determination (4,377 Da) by electrospray mass spectrometry were in full agreement with this theory. The correct pairing of the three disulfide bridges in synthetic decorsin was determined by a combined approach of both peptide mapping using proteolytic enzymes and analysis of the disulfide chirality by CD spectroscopy in the near‐UV region. Synthetic decorsin inhibited human platelet aggregation with an IC50 of 0.1μM, a figure quite similar to that determined utilizing decorsin from natural source. In particular, the synthetic protein was 2,000‐fold more potent than a model RGD‐peptide (e.g., Arg‐Gly‐Asp‐Ser) in inhibiting platelet aggregation. Thermal denaturation experiments of synthetic decorsin, monitored by CD spectroscopy, revealed its high thermal stability (Tm 74 °C). The features of the oxidative refolding process of reduced decorsin, as well as the thermal stability of the oxidized species, were compared with those previously determined for the NH2‐terminal core domain fragment 1‐41 or 1‐43 from hirudin. This fragment shows similarity in size, pairing of the three be disulfides and three‐dimensional structure with those of decorsin, even if very low sequence similarity. It is suggested that the less efficient oxidative folding and the enhanced thermal stability of decorsin in respect to those of hirudin core domain likely can be ascribed to the presence of the six Pro residues in the decorsin chain, whereas none is present in the hirudin domain. The results of this study indicate that decorsin can be obtained by solid‐phase methodology in purity and quantities suitable for structural and functional studies and thus open the way to prepare by chemical methods novel decorsin derivatives containing unusual amino acids or even non‐peptidic moieties.
Stability and folding properties of a model β‐sheet protein, Escherichia coli CspA
Authors: Reid Katherine L. , Rodriguez Hector M. , Hillier Brian J. , Gregoret Lydia M. 
Published: July 2010

AbstractAlthough β‐sheets represent a sizable fraction of the secondary structure found in proteins, the forces guiding the formation of β‐sheets are still not well understood. Here we examine the folding of a small, all β‐sheet protein, the E. coli major cold shock protein CspA, using both equilibrium and kinetic methods. The equilibrium denaturation of CspA is reversible and displays a single transition between folded and unfolded states. The kinetic traces of the unfolding and refolding of CspA studied by stopped‐flow fluorescence spectroscopy are monoexponential and thus also consistent with a two‐state model. In the absence of denaturant, CspA refolds very fast with a time constant of 5 ms. The unfolding of CspA is also rapid, and at urea concentrations above the denaturation midpoint, the rate of unfolding is largely independent of urea concentration. This suggests that the transition state ensemble more closely resembles the native state in terms of solvent accessibility than the denatured state. Based on the model of a compact transition state and on an unusual structural feature of CspA, a solvent‐exposed cluster of aromatic side chains, we propose a novel folding mechanism for CspA. We have also investigated the possible complications that may arise from attaching polyhistidine affinity tags to the carboxy and amino termini of CspA.
The importance of dynamic light scattering in obtaining multiple crystal forms of trypanosoma brucei PGK
Authors: Bernstein Bradley E. , Michels Paul A. M. , Kim Hidong , Petra Philip H. , Hol Wim G. J. 
Published: July 2010

AbstractPhosphoglycerate kinase (PGK) catalyzes the phosphoryl transfer between 1,3 bis‐phosphoglycerate and ADP to form 3‐phosphoglycerate and ATP, undergoing significant conformational changes during catalysis. To more precisely document this reaction and the corresponding conformational changes, we have crystallized Trypanosoma brucei PGK in several crystal forms: (1) in the presence of 3‐phosphoglycerate and MgADP, PGK crystallizes with four molecules in the asymmetric unit; (2) in the presence of the ATP analog, AMP‐PNP, PGK crystallizes in a similar form; (3) in the presence of the bisubstrate analog, adenylyl 1,1,5,5‐tetrafluoropentane‐l,5‐bisphosphonate, PGK crystals grow with one molecule in the asymmetric unit. Large scale expression and purification of T. brucei PGK from an E. coli overexpression system was required to obtain sufficient enzyme yields. Results from dynamic light scattering experiments allowed us to identify substrates and analogs which were amenable for crystallization. Ease of crystal growth and diffraction quality for a particular PGK‐ligand complex is highly consistent with the apparent monodispersity of the complex in solution as judged by dynamic light scattering. The three‐dimensional structures of the various enzyme‐ligand complexes are currently being exploited to obtain a better understanding of PGK catalysis, as well as for structure based design of enzyme inhibitors to be used in the development of anti‐trypanosomal agents.
Mechanism and evolution of protein dimerization
Authors: Nussinov Ruth , Xu Dong , Tsai Chung‐Jung 
Published: July 2010

AbstractWe have investigated the mechanism and the evolutionary pathway of protein dimerization through analysis of experimental structures of dimers. We propose that the evolution of dimers may have multiple pathways, including (1) formation of a functional dimer directly without going through an ancestor monomer, (2) formation of a stable monomer as an intermediate followed by mutations of its surface residues, and (3), a domain swapping mechanism, replacing one segment in a monomer by an equivalent segment from an identical chain in the dimer. Some of the dimers which are governed by a domain swapping mechanism may have evolved at an earlier stage of evolution via the second mechanism. Here, we follow the theory that the kinetic pathway reflects the evolutionary pathway. We analyze the structure‐kinetics‐evolution relationship for a collection of symmetric homodimers classified into three groups: (1)14 dimers, which were referred to as domain swapping dimers in the literature; (2) nine 2‐state dimers, which have no measurable intermediates in equilibrium denaturation; and (3), eight 3‐state dimers, which have stable intermediates in equilibrium denaturation. The analysis consists of the following stages: (i) The dimer is divided into two structural units, which have twofold symmetry. Each unit contains a contiguous segment from one polypeptide chain of the dimer, and its complementary contiguous segment from the other chain, (ii) The division is repeated progressively, with different combinations of the two segments in each unit. (iii) The coefficient of compactness is calculated for the units in all divisions. The coefficients obtained for different cuttings of a dimer form a compactness profile. The profile probes the structural organization of the two chains in a dimer and the stability of the monomelic state. We describe the features of the compactness profiles in each of the three dimer groups. The profiles identify the swapping segments in domain swapping dimers, and can usually predict whether a dimer has domain swapping. The kinetics of dimerization indicates that some dimers which have been assigned in the literature as domain swapping cases, dimerize through the 2‐state kinetics, rather than through swapping segments of performed monomers. The compactness profiles indicate a wide spectrum in the kinetics of dimerization: dimers having no intermediate stable monomers; dimers having an intermediate with a stable monomer structure; and dimers having an intermediate with a stable structure in part of the monomer. These correspond to the multiple evolutionary pathways for dimer formation. The evolutionary mechanisms proposed here for dimers are applicable to other oligomers as well.
Crystallization of phycoerythrin 545 of Rhodomonas lens using detergents and unusual additives
Authors: Becker Michael , Stubbs Milton T. , Huber Robert 
Published: July 2010

AbstractPhycoerythrin 545 from the cryptomonad alga, Rhodomonas lens, has been crystallized under a wide variety of conditions. Although this type of photosynthetic light‐harvesting protein is water soluble, detergents were always required for crystallization. The crystals were typically poorly ordered, or ordered in only two dimensions. However, crystals that were well‐ordered in three dimensions could be obtained under two different conditions. Both used polyethylene glycol as precipitant and the detergent lauryldimethylaminoxide, but the additives that were critical for obtaining well‐ordered crystals were propionamide in one case and Cs+/Br− in the other. Crystals obtained in the presence of propionamide have the space group P212121, with cell constants of a = 85.6 Å, b = 108.2 Å, and c = 131.0 Å, and contain two dimers [i.e., 2 × (α2β2)] in the asymmetric unit. They show diffraction to at least 3.0 Å resolution. The crystals grown with Cs+/Br− are nearly isomorphous. Both types of crystals show intense, strongly polarized fluorescence, suggesting that energy transfer in the crystals is highly efficient. This should provide a basis for quantitative investigation of the role of exciton interactions in energy transfer in cryptomonad phycobiliproteins.
A proposed architecture for lecithin cholesterol acyl transferase (LCAT): Identification of the catalytic triad and molecular modeling
Authors: Peelman F. , Vinaimont N. , Vanloo B. , Labeur C. , Rosseneu M. , Verhee A. , Verschelde J‐L. , Vanderckove J. , Tavernier J. , Seguret‐Mace S. , Duverger N. , Hutchinson G. 
Published: July 2010

AbstractThe enzyme cholesterol lecithin acyl transferase (LCAT) shares the Ser/Asp‐Glu/His triad with lipases, esterases and proteases, but the low level of sequence homology between LCAT and these enzymes did not allow for the LCAT fold to be identified yet. We, therefore, relied upon structural homology calculations using threading methods based on alignment of the sequence against a library of solved three‐dimensional protein structures, for prediction of the LCAT fold. We propose that LCAT, like lipases, belongs to the α/β hydrolase fold family, and that the central domain of LCAT consists of seven conserved parallel beta‐strands connected by four α‐helices and separated by loops. We used the conserved features of this protein fold for the prediction of functional domains in LCAT, and carried out site‐directed mutagenesis for the localization of the active site residues. The wild‐type enzyme and mutants were expressed in Cos‐1 cells. LCAT mass was measured by ELISA, and enzymatic activity was measured on recombinant HDL, on LDL and on a monomelic substrate. We identified D345 and H377 as the catalytic residues of LCAT, together with F103 and L182 as the oxyanion hole residues. In analogy with lipases, we further propose that a potential “lid” domain at residues 50‐74 of LCAT might be involved in the enzyme‐substrate interaction. Molecular modeling of human LCAT was carried out using human pancreatic and Candida antarctica lipases as templates. The three‐dimensional model proposed here is compatible with the position of natural mutants for either LCAT deficiency or Fish‐eye disease. It enables moreover prediction of the LCAT domains involved in the interaction with the phospholipid and cholesterol substrates.
Comparisons between the structures of HCV and Rep helicases reveal structural similarities between SF1 and SF2 super‐families of helicases
Authors: Korolev Sergey , Lohman Timothy M. , Waksman Gabriel , Yao Nanhua , Weber Patricia C. 
Published: July 2010

AbstractThree helicase structures have been determined recently: that of the DNA helicase PcrA, that of the hepatitis C virus RNA helicase, and that of the Escherichia coli DNA helicase Rep. PcrA and Rep belong to the same super‐family of helicases (SF1) and are structurally very similar. In contrast, the HCV helicase belongs to a different super‐family of helicases, SF2, and shows little sequence homology with the PcrA/Rep helicases. Yet, the HCV helicase is structurally similar to Rep/PcrA, suggesting preservation of structural scaffolds and relationships between helicase motifs across these two super‐families. The comparison study presented here also reveals the existence of a new helicase motif in the SF1 family of helicases.
Spider minor ampullate silk proteins contain new repetitive sequences and highly conserved non‐silk‐like “spacer regions”
Authors: Colgin Mark A. , Lewis Randolph V. 
Published: July 2010

AbstractSpider minor ampullate silk is a strong non‐elastic deformably stretchable silk used in web formation. This silk from Nephila clavipes is composed of two proteins, MiSp 1 and 2, whose transcripts are 9.5 and 7.5 kb, respectively, as determined by northern blots. Both MiSp proteins are organized into a predominantly repetitive region and a small nonrepetitive carboxy terminal region. These highly repetitive regions are composed mainly of glycine and alanine, but also contain tyrosine, glutamine, and arginine. The sequences are mainly GGX and GA repeats. The repetitive regions are interrupted by nonrepetitive serine‐rich spacer regions. Although the sequences of the spacer regions differ from the repetitive regions, sequences of the spacers from different regions of the proteins are nearly identical. The sequence differences between major and minor ampullate silks may explain the differing mechanical properties of the fibers.
Multiple catalytic roles of His 287 of Rhodospirillum rubrum ribulose 1,5‐bisphosphate carboxylase/oxygenase
Authors: Harpel Mark R. , Larimer Frank W. , Hartman Fred C. 
Published: July 2010

AbstractActive‐site His 287 of Rhodospirillum rubrum ribulose 1,5‐bisphosphate (RuBP) carboxylase/oxygenase interacts with the C3‐hydroxyl of bound substrate or reaction‐intermediate analogue (CABP), water molecules, and ligands for the activator metal‐ion (Andersson I, 1996, J Mol Biol 259:160‐174; Taylor TC, Andersson I, 1997, J Mol Biol 265:432‐444). To test structure‐based postulates of catalytic functionality, His 287 was replaced with Asn or Gln. The mutants are not affected adversely in subunit assembly, activation (binding of Mg2+ and carbamylation of Lys 191), or recognition of phosphorylated ligands; they bind CABP with even greater tenacity than does wild‐type enzyme. H287N and H287Q are severely impaired in catalyzing overall carboxylation (∼103‐fold and >105‐fold, respectively) and enolization (each mutant below threshold for detection) of RuBP. H287N preferentially catalyzes decarboxylation of carboxylated reaction intermediate instead of forward processing to phosphoglycerate. Analysis of RuBP turnover that occurs at high concentrations of mutants over extended time periods reveal > 10‐fold reduced CO2/O2 specificities, elevated misprotonation of the enediol intermediate, and misprocessing of the oxygenated intermediate of the oxygenase pathway. These results are consistent with multifaceted roles for His 287 in promoting enediol formation, enediol tautomerization, and forward‐processing of carboxylated intermediate.