bluuebunny/crossref_metadata_2025_split huggingface dataset:
Anatomy of protein pockets and cavities: Measurement of binding site geometry and implications for ligand design
Authors: Liang Jie , Woodward Clare , Edelsbrunner Herbert 
Published: February 2009

AbstractIdentification and size characterization of surface pockets and occluded cavities are initial steps in protein structure‐based ligand design. A new program, CAST, for automatically locating and measuring protein pockets and cavities, is based on precise computational geometry methods, including alpha shape and discrete flow theory. CAST identifies and measures pockets and pocket mouth openings, as well as cavities. The program specifies the atoms lining pockets, pocket openings, and buried cavities; the volume and area of pockets and cavities; and the area and circumference of mouth openings. CAST analysis of over 100 proteins has been carried out; proteins examined include a set of 51 monomeric enzyme‐ligand structures, several elastase‐inhibitor complexes, the FK506 binding protein, 30 HIV‐1 protease‐inhibitor complexes, and a number of small and large protein inhibitors. Medium‐sized globular proteins typically have 10‐20 pockets/cavities. Most often, binding sites are pockets with 1‐2 mouth openings; much less frequently they are cavities. Ligand binding pockets vary widely in size, most within the range 102‐103Å3. Statistical analysis reveals that the number of pockets and cavities is correlated with protein size, but there is no correlation between the size of the protein and the size of binding sites. Most frequently, the largest pocket/cavity is the active site, but there are a number of instructive exceptions. Ligand volume and binding site volume are somewhat correlated when binding site volume is < 700 Å3, but the ligand seldom occupies the entire site. Auxiliary pockets near the active site have been suggested as additional binding surface for designed ligands (Mattos C et al., 1994,Nat Struct Bid 1:55‐58). Analysis of elastase‐inhibitor complexes suggests that CAST can identify ancillary pockets suitable for recruitment in ligand design strategies. Analysis of the FK506 binding protein, and of compounds developed in SAR by NMR (Shuker SB et al., 1996,Science274:1531‐1534), indicates that CAST pocket computation may provide a priori identification of target proteins for linked‐fragment design. CAST analysis of 30 HIV‐1 protease‐inhibitor complexes shows that the flexible active site pocket can vary over a range of 853‐1,566 Å3, and that there are two pockets near or adjoining the active site that may be recruited for ligand design.
Uclacyanins, stellacyanins, and plantacyanins are distinct subfamilies of phytocyanins: Plant‐specific mononuclear blue copper proteins
Authors: Nersissian Aram M. , Valentine Joan Selverstone , Immoos Chad , Hill Michael G. , Hart P. John , Williams Gregory , Herrmann Reinhold G. 
Published: February 2009

AbstractThe cDNAs encoding plantacyanin from spinach were isolated and characterized. In addition, four new cDNA sequences from Arubidopsis ESTs were identified that encode polypeptides resembling phytocyanins, plant‐specific proteins constituting a distinct family of mononuclear blue copper proteins. One of them encodes plantacyanin from Arubidopsis. while three others, designated as uclacyanin 1, 2, and 3, encode protein precursors that are closely related to precursors of stellacyanins and a blue copper protein from pea pods. Comparative analyses with known phytocyanins allow further classification of these proteins into three distinct subfamilies designated as uclacyanins, stellacyanins, and plantacyanins. This specification is based on (1) their spectroscopic properties, (2) their glycosylation state, (3) the domain organization of their precursors, and (4) their copper‐binding amino acids. The recombinant copper binding domain of Arubidopsis uclacyanin I was expressed, purified, and shown to bind a copper atom in a fashion known as “blue” or type 1. The mutant of cucumber stellacyanin in which the glutamine axial ligand was substituted by a methionine (Q99M) was purified and shown to possess spectroscopic properties similar to uclacyanin I rather than to plantacyanins. Its redox potential was determined by cyclic voltammetry to be +420 mV, a value that is significantly higher than that determined for the wild‐type protein (+260 mV). The available structural data suggest that stellacyanins (and possibly other phytocyanins) might not be diffusible electron‐transfer proteins participating in long‐range electron‐transfer processes. Conceivably, they are involved in redox reactions occurring during primary defense responses in plants and/or in lignin formation.
Lysine‐50 is a likely site for anchoring the plasminogen N‐terminal peptide to lysine‐binding kringles
Authors: An Seong Soo A. , Marti Daniel N. , Llinas Miguel , Carreño Cristina , Albericio Fernando , Schaller Johann 
Published: February 2009

AbstractInteractions between the kringle 4 (K4) domain of human plasminogen (Pgn) and segments of the N‐terminal Glul‐Lys77 peptide (NTP) have been investigated via 1H‐NMR at 500 MHz. NTP peptide stretches devoid of Lys residues but carrying an internal Arg residue show negligible affinity toward K4 (equilibrium association constant Ka < 0.05 mM−1). In contrast, while most fragments containing an internal Lys residue exhibit affinities comparable to that shown by the blocked Lys derivative Nα‐acetyl‐L‐lysine‐methyl ester (Ka ˜ 0.2 mM−1), peptides encompassing Lys50 consistently show higher Ka values. Among the investigated linear peptides, Nα‐acetyl‐Ala‐Phe‐Tyr‐His‐Ser‐Ser‐Lys50‐Glu‐Gln‐NH2 (AcAFYHSKSOEQ‐NH2) exhibits the strongest interaction with K4 (Ka ˜ 1.4 mM−1), followed by AcYHSKSOEQ‐NH2 (Ka ˜ 0.9 mM−1). Relative to the wild‐type sequence, mutated hexapeptides exhibit lesser affinity for K4. When a Lys50 → Ser mutation was introduced (⇒ AcYHSSSOEQ‐NH2), binding was abolished.The Ile27‐Ile56 construct (L‐NTP) contains the Lys50 site within a loop constrained by two cystine bridges. The propensity of recombinant Pgn K1 (rKI) and K2 (rK2) modules, and of Pgn fragments encompassing the intact K4 and K5 domains, for binding L‐NTP, was investigated. We find that L‐NTP interacts with rK1, rK2, K4, and K5 — all lysine‐binding kringles — in a fashion that closely mimics what has been observed for the Glul‐HSer57 N‐terminal fragment of Pgn (CB‐NTP). Thus, both the constellation of kringle lysine binding site (LBS) aromatic residues that are perturbed upon complexation of L‐NTP and magnitudes of kringle‐L‐NTP binding affinities (rK1, K2 ˜ 4.3 mM −1; rK2, Ka ˜ 3.7 mM−1; K4, Ka ˜ 6.4 mM−1; and K5, Ka ˜ 2.1 mM−1) are essentially the same as for the corresponding kringle‐CB‐NTP pairs. Molecular modeling studies suggest that the GIu39‐Lys50 stretch in NTP generates an area that complements, both topologically and electrostatically, the solvent‐exposed kringle LBS surface.
A test of the relationship between sequence and structure in proteins: Excision of the heme binding site in apocytochrome b5
Authors: Constans Aileen J. , Mayer Michael R. , Sukits Steven E , Lecomte Juliette T.J. 
Published: February 2009

AbstractThe water‐soluble domain of rat hepatic holocytochrome b5 is anαβ protein containing elements of secondary structure in the sequence βl ‐α l ‐β4‐β3‐α2‐α3‐β5‐α4‐α5‐β2‐α6. The heme group is enclosed by four helices,α2,α3,α4, andα5. To test the hypothesis that a small b hemoprotein can be constructed in two parts, one forming the heme site, the other an organizing scaffold, a protein fragment corresponding to βl‐αl‐β4‐β3‐Δ‐β2‐α6 was prepared, where Δ is a seven‐residue linker bypassing the heme binding site. The fragment (“abridged b5”) was found to containα and β secondary structure by circular dichroism spectroscopy and tertiary structure by Trp fluorescence emission spectroscopy. NMR data revealed a species with spectral properties similar to those of the full‐length apoprotein. This folded form is in slow equilibrium on the chemical shift time scale with other less folded species. Thermal denaturation, as monitored by circular dichroism, absorption, and fluorescence spectroscopy, as well as size‐exclusion chromatography‐fast protein liquid chromatography (SEC‐FPLC), confirmed the coexistence of at least two distinct conformational ensembles. It was concluded that the protein fragment is capable of adopting a specific fold likely related to that of cytochrome b5, but does not achieve high thermodynamic stability and cooperativity. Abridged b5 demonstrates that the spliced sequence contains the information necessary to fold the protein. It suggests that the dominating influence to restrict the conformational space searched by the chain is structural propensities at a local level rather than internal packing. The sequence also holds the properties necessary to generate a barrier to unfolding.
Amide proton exchange measurements as a probe of the stability and dynamics of the n‐terminal domain of the ribosomal protein L9: Comparison with the intact protein
Authors: Vugmeyster Liliya , Kuhlman Brian , Raleigh Daniel P. 
Published: February 2009

AbstractAmide H/D exchange rates have been measured for the N‐terminal domain of the ribosomal protein L9, residues 1‐56. The rates were measured at pD 3.91, 5.03, and 5.37. At pD 5.37, 18 amides exchange slowly enough to give reliable rate measurements. At pD 3.91, seven additional residues could be followed. The exchange is shown to occur by the EX2 mechanism for all conditions studied. The rates for the N‐terminal domain are very similar to those previously measured for the corresponding region in the full‐length protein (Lillemoen J et al., 1997, J Mol Biol 268:482‐493). In particular, the rates for the residues that we have shown to exchange via global unfolding in the N‐terminal domain agree within the experimental error with the rates measured by Hoffman and coworkers, suggesting that the structure of the domain is preserved in isolation and that the stability of the isolated domain is comparable to the stability of this domain in intact L9.
Distance geometry generates native‐like folds for small helical proteins using the consensus distances of predicted protein structures
Authors: Huang Enoch S. , Ponder Jay W. , Samudrala Ram 
Published: February 2009

AbstractFor successful ab initio protein structure prediction, a method is needed to identify native‐like structures from a set containing both native and non‐native protein‐like conformations. In this regard, the use of distance geometry has shown promise when accurate inter‐residue distances are available. We describe a method by which distance geometry restraints are culled from sets of 500 protein‐like conformations for four small helical proteins generated by the method of Simons et al. (1997). A consensus‐based approach was applied in which every inter‐Cα distance was measured, and the most frequently occurring distances were used as input restraints for distance geometry. For each protein, a structure with lower coordinate root‐mean‐square (RMS) error than the mean of the original set was constructed; in three cases the topology of the fold resembled that of the native protein. When the fold sets were filtered for the best scoring conformations with respect to an all‐atom knowledge‐based scoring function, the remaining subset of 50 structures yielded restraints of higher accuracy. A second round of distance geometry using these restraints resulted in an average coordinate RMS error of 4.38 Å.
Electrostatic coupling to pH‐titrating sites as a source of cooperativity in protein‐ligand binding
Authors: Spassov Velin , Bashford Donald 
Published: February 2009

AbstractThis paper describes an alternative mechanism for the cooperative binding of charged ligands to proteins. The ligand‐binding sites are electrostatically coupled to protein side chains that can undergo protonation and deprotonation. The binding of one ligand alters the protein's protonation equilibrium in a manner that makes the the binding of the second ligand more favorable. This mechanism requires no conformational change to produce a cooperative effect, although it is not exclusive of conformational change. We present a theoretical description of the mechanism, and calculations on three kinds of systems: A model system containing one protonation site and two ligand‐binding sites; a model system containing two protonation sites and two ligand‐binding sites; and calbindin D9k, which contains two Ca2+‐binding sites and 30 protonation sites. For the one‐protonation‐site model, it is shown that the influence of the protonation site can only be cooperative. The competition of this effect with the anticooperative effect of ligand‐ligand repulsion is studied in detail. For the two‐protonation site model, the effect can be either cooperative or, in special cases, anticooperative. For calbindin D9k, the calculations predict that six protonation sites in or near the ligand‐binding sites make a cooperative contribution that approximately cancels the anticooperative effect of Ca2+‐Ca2+ repulsion, accounting for more than half of the total cooperative effect that is needed to overcome repulsion and produce the net cooperativity observed experimentally. We argue that cooperative mechanisms of the kind described here are likely when there is more than one ligand‐binding site in a protein domain.
Characterization of recombinant human cathepsin B expressed at high levels in baculovirus
Authors: Steed Paul M. , Lasala Daniel , Liebman Jeffrey , Wigg Andrew , Clark Kirk , Knap Ania K. 
Published: February 2009

AbstractThe lysosomal cysteine protease cathepsin B has been studied intensely for many years because of its unique characteristics and its potential involvement in disease states. A reproducible, high yield expression system for active recombinant protein is key to biochemical and biophysical studies as well as rational drug design. Although several microbial and mammalian expression systems for recombinant human cathepsin B have been described, these have been limited by low or variable yields. Further, in some of these systems hyper‐glycosylation of the enzyme near the active site affects its activity. We describe a baculovirus expression system and purification scheme that solve all of these problems. Yields of active, protected enzyme were reproducibly in excess of 25 mg/L. Since this protein was not hyper‐glycosylated, it had greater activity than cathepsin B produced in yeast systems as indicated by a threefold increase in Kcat. In addition, the biophysical properties of the baculovirus‐expressed cathepsin B, as measured by dynamic light scattering, were more amenable to crystallographic study since the data indicated proteins of more uniform size. Therefore, this system for the production of recombinant human cathepsin B constitutes a major improvement in both quantity and quality over those previously reported. Further, we demonstrate that the manner of expression and purification of this enzyme has profound effects on its kinetic and physical parameters.
Crystal structure of spinach plastocyanin at 1.7 Å resolution
Authors: Xue Yafeng , Ökvist Mats , Hansson Örjan , Young Simon 
Published: February 2009

AbstractThe crystal structure of plastocyanin from spinach has been determined using molecular replacement, with the structure of plastocyanin from poplar as a search model. Successful crystallization was facilitated by site‐directed mutagenesis in which residue Gly8 was substituted with Asp. The region around residue 8 was believed to be too mobile for the wild‐type protein to form crystals despite extensive screening. The current structure represents the oxidized plastocyanin, copper (II), at low pH (≈4.4). In contrast to the similarity in the core region as compared to its poplar counterpart, the structure shows some significant differences in loop regions. The most notable is the large shift of the 59‐61 loop where the largest shift is 3.0 Å for the Cα atom of Glu59. This results in different patterns of electrostatic potential around the acidic patches for the two proteins.
Structure of betaine aldehyde dehydrogenase at 2.1 Å resolution
Authors: Johansson Kenth , Ramaswamy S. , Eklund Hans , El‐Ahmad Mustapha , Hjelmqvist Lars , Jörnvall Hans 
Published: February 2009

AbstractThe three‐dimensional structure of betaine aldehyde dehydrogenase, the most abundant aldehyde dehydrogenase (ALDH) of cod liver, has been determined at 2.1 Å resolution by the X‐ray crysallographic method of molecular replacement. This enzyme represents a novel structure of the highly multiple ALDH, with at least 12 distinct classes in humans. This betaine ALDH of class 9 is different from the two recently determined ALDH structures (classes 2 and 3). Like these, the betaine ALDH structure has three domains, one coenzyme binding domain, one catalytic domain, and one oligomerization domain. Crystals grown in the presence or absence of NAD+ have very similar structures and no significant conformational change occurs upon coenzyme binding. This is probably due to the tight interactions between domains within the subunit and between subunits in the tetramer. The oligomerization domains link the catalytic domains together into two 20‐stranded pleated sheet structures. The overall structure is similar to that of the tetrameric bovine class 2 and dimeric rat class 3 ALDH, but the coenzyme binding with the nicotinamide in anti conformation, resembles that of class 2 rather than of class 3.
Effects of proline cis‐trans isomerization on TB domain secondary structure
Authors: Yuan Xuemei , Werner Jorn M. , Campbell Iain D. , Downing A. Kristina , Knott Vron , Handford Penny A. 
Published: February 2009

AbstractThe transforming growth factor β (TGF‐β) binding protein‐like (TB) domain is found principally in proteins localized to extracellular matrix fibrils, including human fibrillin‐1, the defective protein in the Marfan syndrome. Analysis of the nuclear magnetic resonance (NMR) data for the sixth TB module from human fibrillin‐1 has revealed the existence of two stable conformers that differ in the isomerization states of two proline residues. Unusually, the two isoforms do not readily interconvert and are stable on the time scale of milliseconds. We have computed independent structures of the major and minor conformers of TB6 to assess how the domain fold adjusts to incorporate alternatively cis‐ or trans‐ prolines. Based on previous observations, it has been suggested that multiple conformers can only be accommodated in flexible regions of protein structure. In contrast, P22, which exists in trans in the major form and cis in the minor form of TB6, is in a rigid region of the domain, which is confirmed by backbone dynamics measurements. Overall, the structures of the major and minor conformers are similar. However, the secondary structure topologies of the two forms differ as a direct consequence of the changes in proline conformation.
Characterization of a folding intermediate from HIV‐1 ribonuclease H
Authors: Kern Gunther , Handel Tracy , Marqusee Susan 
Published: February 2009

AbstractThe RNase H domain from HIV‐1 (HIV RNase H) encodes an essential retroviral activity. Refolding of the isolated HIV RNase H domain shows a kinetic intermediate detectable by stopped‐flow far UV circular dichroism and pulse‐labeling H/D exchange. In this intermediate, strands 1, 4, and 5 as well as helices A and D appear to be structured. Compared to its homolog fromEscherichia coli, the rate limiting step in refolding of HIV RNase H appears closer to the native state. We have modeled this kinetic intermediate using a C‐terminal deletion fragment lacking helix E. Like the kinetic intermediate, this variant folds rapidly and shows a decrease in stability. We propose that inhibition of the docking of helix E to this folding intermediate may present a novel strategy for anti HIV‐1 therapy.
Aggregation states of mitochondrial malate dehydrogenase
Authors: Sánchez Susana A. , Brunet Juan E. , Hazlett Theodore L. , Jameson David M. 
Published: February 2009

AbstractThe oligomeric state of fluorescein‐labeled mitochondrial malate dehydrogenase (L‐malate NAD+ oxidoreductase; mMDH; EC 1.1.1.37), as a function of protein concentration, has been examined using steady‐state and dynamic polarization methodologies. A “global” rotational relaxation time of 103 ± 7 ns was found for micromolar concentrations of mMDH‐fluorescein, which is consistent with the reported size and shape of mMDH. Dilution of the mMDH‐fluorescein conjugates, prepared using a phosphate buffer protocol, to nanomolar concentrations had no significant effect on the rotational relaxation time of the adduct, indicating that the dimer‐monomer dissociation constant for mMDH is below 10−9 M. In contrast to reports in the literature suggesting a pH‐dependent dissociation of mMDH, the oligomeric state of this mMDH‐fluorescein preparation remained unchanged between pH 5.0 and 8.0. Application of hydrostatic pressure up to 2.5 kilobars was ineffective in dissociating the mMDH dimer. However, the mMDH dimer was completely dissociated in 1.5 M guanidinium hydrochloride. Dilution of a mMDH‐fluorescein conjugate, prepared using a Tris buffer protocol, did show dissociation, which can be attributed to aggregates present in these preparations. These results are considered in light of the disparities in the literature concerning the properties of the mMDH dimer‐monomer equilibrium.
Analysis of the interactions between streptokinase domains and human plasminogen
Authors: Conejero‐Lara Francisco , Parrad Juan , Azuaga Ana I. , Dobson Christopher M. , Ponting Chris P. 
Published: February 2009

AbstractThe contrasting roles of streptokinase (SK) domains in binding human Glu l‐plasminogen (Plg) have been studied using a set of proteolytic fragments, each of which encompasses one or more of SK's three structural domains (A, B, C). Direct binding experiments have been performed using gel filtration chromatography and surface plasmon resonance. The latter technique has allowed estimation of association and dissociation rate constants for interactions between Plg and intact SK or SK fragments. Each of the SK fragments that contains domain B (fragments A2‐B‐C, A2‐B, B‐C, and B) binds Plg with similar affinity, at a level approximately 100‐ to 1,000‐fold lower than intact SK. Experiments using 10 mM 6‐aminohexanoic acid or 50 mM benzamidine demonstrate that either of these two lysine analogues abolishes interaction of domain B with Plg. Isolated domain C does not show detectable binding to Plg. Moreover, the additional presence of domain C within other SK fragments (B‐C and A2‐B‐C) does not alter significantly their affinities for Plg. In addition, Plg‐binding by a noncovalent complex of two SK fragments that contains domains A and B is similar to that of domain B. By contrast, species containing domain B and both domains A and C (intact SK and the two‐chain complex A1·A2‐B‐C) show a significantly higher affinity for Plg, which could not be completely inhibited by saturating amounts of 6‐AHA. These results show that SK domain B interacts with Plg in a lysine‐dependent manner and that although domains A and C do not appear independently to possess affinity for Plg, they function cooperatively to establish the additional interactions with Plg to form an efficient native‐like Plg activator complex.