bluuebunny/crossref_metadata_2025_split huggingface dataset:
Water‐protein interactions in the molten‐globule state of carbonic anhydrase b: An NMR spin‐diffusion study
Authors: Kutyshenko Victor P. , Cortijo Manuel 
Published: February 2009

AbstractWe have used the homonuclear Overhauser effect (NOE) to characterize a model protein: carbonic anhydrase B. We have obtained NOE difference spectra for this protein, centering the on‐resonance signals either at the methyl‐proton or at the water‐proton signals. The spin‐diffusion spectra obtained as a function of protein concentration and temperature provide direct evidence of much greater protein–water interaction in the molten‐globule state than in the native and denatured states. Furthermore, although the protein loses its gross tertiary structure in both the molten‐globule and denatured states, it remains almost as compact in its molten‐globule state as it is in the native state. The spin‐diffusion spectra, obtained as a function of a variable delay time after the saturation pulse, allowed us to measure the relaxation times of several types of proton in the solution. These spectra contain enough information to distinguish between those water molecules solvating the protein and the free ones present as bulk water.
Static light scattering studies of OmpF porin: Implications for integral membrane protein crystallization
Authors: Hitscherich Carl , Allaman Margaret , Wiencek John , Kaplan Jeffrey , Loll Patrick J. 
Published: February 2009

AbstractIntegral membrane proteins carry out some of the most important functions of living cells, yet relatively few details are known about their structures. This is due, in large part, to the difficulties associated with preparing membrane protein crystals suitable for X‐ray diffraction analysis. Mechanistic studies of membrane protein crystallization may provide insights that will aid in determining future membrane protein structures. Accordingly, the solution behavior of the bacterial outer membrane protein OmpF porin was studied by static light scattering under conditions favorable for crystal growth. The second osmotic virial coefficient (B22) was found to be a predictor of the crystallization behavior of porin, as has previously been found for soluble proteins. Both tetragonal and trigonal porin crystals were found to form only within a narrow window of B22 values located at approximately −0.5 to −2 × 10‐4 mol mL g‐2, which is similar to the “crystallization slot” observed for soluble proteins. The B22 behavior of protein‐free detergent micelles proved very similar to that of porin‐detergent complexes, suggesting that the detergent's contribution dominates the behavior of protein‐detergent complexes under crystallizing conditions. This observation implies that, for any given detergent, it may be possible to construct membrane protein crystallization screens of general utility by manipulating the solution properties so as to drive detergent B22 values into the crystallization slot. Such screens would limit the screening effort to the detergent systems most likely to yield crystals, thereby minimizing protein requirements and improving productivity.
Use of residue pairs in protein sequence‐sequence and sequence‐structure alignments
Authors: Jung Jongsun , Lee Byungkook 
Published: February 2009

AbstractTwo new sets of scoring matrices are introduced: H2 for the protein sequence comparison and T2 for the protein sequence–structure correlation. Each element of H2 or T2 measures the frequency with which a pair of amino acid types in one protein, k‐residues apart in the sequence, is aligned with another pair of residues, of given amino acid types (for H2) or in given structural states (for T2), in other structurally homologous proteins. There are four types, corresponding to the k‐values of 1 to 4, for both H2 and T2. These matrices were set up using a large number of structurally homologous protein pairs, with little sequence homology between the pair, that were recently generated using the structure comparison program SHEBA.The two scoring matrices were incorporated into the main body of the sequence alignment program SSEARCH in the FASTA package and tested in a fold recognition setting in which a set of 107 test sequences were aligned to each of a panel of 3,539 domains that represent all known protein structures. Six procedures were tested; the straight Smith‐Waterman (SW) and FASTA procedures, which used the Blosum62 single residue type substitution matrix; BLAST and PSI‐BLAST procedures, which also used the Blosum62 matrix; PASH, which used Blosum62 and H2 matrices; and PASSC, which used Blosum62, H2, and T2 matrices. All procedures gave similar results when the probe and target sequences had greater than 30% sequence identity. However, when the sequence identity was below 30%, a similar structure could be found for more sequences using PASSC than using any other procedure. PASH and PSI‐BLAST gave the next best results.
The L6 membrane proteins—A new four‐transmembrane superfamily
Authors: Wright Mark D. , Rudy George B. , Ni Jian 
Published: February 2009

AbstractL6, IL‐TMP, and TM4SF5 are cell surface proteins predicted to have four transmembrane domains. Previous sequence analysis led to their assignment as members of the tetraspanin superfamily. In this paper, we identify a new sequence (L6D) that is strikingly similar to L6, IL‐TMP, and TM4SF5. Analyses of these four sequences indicate that they are not significantly related to genuine tetraspanins, but instead constitute their own L6 superfamily.
The failure of simple empirical relationships to predict the viscosity of mixed aqueous solutions of guanidine hydrochloride and glucose has important implications for the study of protein folding
Authors: Sato Satoshi , Sayid Cosim J. , Raleigh Daniel P. , Raleigh Daniel P. 
Published: February 2009

AbstractViscosities of aqueous solutions of guanidine hydrochloride (GuHCl) were measured in the presence of varying amounts of glucose. At high concentrations of glucose or GuHCl, the measured viscosities showed significant deviation from the values computed using a method proposed by Tanford (1966, J Biol Chem 241:3228–3232). This method was originally derived to allow the calculation of the effects of buffer or low concentrations of salts and other additives on the density and viscosity of aqueous solutions of urea or GuHCl. Recently it has been used to estimate the viscosity of denaturant solutions that contain high concentrations of viscogens. Our results show that the extrapolation of this approach to solutions of highly concentrated viscous co‐solutes leads to significant errors. The implications for experimental studies of the viscosity dependence of conformational transitions in proteins is discussed.
Systematic mutational analysis of the active‐site threonine of HIV‐1 proteinase: Rethinking the “fireman's grip” hypothesis
Authors: Strisovsky Kvido , Tessmer Uwe , Langner Josmar , Konvalinka Jan , Kräusslich Hans‐Georg 
Published: February 2009

AbstractAspartic proteinases share a conserved network of hydrogen bonds (termed “fireman's grip”), which involves the hydroxyl groups of two threonine residues in the active site Asp‐Thr‐Gly triplets (Thr26 in the case of human immunodeficiency virus type 1 (HIV‐1) PR). In the case of retroviral proteinases (PRs), which are active as symmetrical homodimers, these interactions occur at the dimer interface. For a systematic analysis of the “fireman's grip”), which involves the hydroxyl groups of two threonine residues in the active site Asp‐Thr‐Gly triplets (Thr26 in the case of human immunodeficiency virus type 1 (HIV‐1) PR). In the case of retroviral proteinases (PRs), which are active as symmetrical homodimers, these interactions occur at the dimer interface. For a systematic analysis of the “fireman's grip”), which involves the hydroxyl groups of two threonine residues in the active site Asp‐Thr‐Gly triplets (Thr26 in the case of human immunodeficiency virus type 1 (HIV‐1) PR). In the case of retroviral proteinases (PRs), which are active as symmetrical homodimers, these interactions occur at the dimer interface. For a systematic analysis of the “fireman's grip”), which involves the hydroxyl groups of two threonine residues in the active site Asp‐Thr‐Gly triplets (Thr26 in the case of human immunodeficiency virus type 1 (HIV‐1) PR). In the case of retroviral proteinases (PRs), which are active as symmetrical homodimers, these interactions occur at the dimer interface. For a systematic analysis of the “fireman's grip,” Thr26 of HIV‐1 PR was changed to either Ser, Cys, or Ala. The variant enzymes were tested for cleavage of HIV‐1 derived peptide and polyprotein substrates. PR(T26S) and PR(T26C) showed similar or slightly reduced activity compared to wild‐type HIV‐1 PR, indicating that the sulfhydryl group of cysteine can substitute for the hydroxyl of the conserved threonine in this position. PR(T26A), which lacks the “fireman's grip” interaction, was virtually inactive and was monomeric in solution at conditions where wild‐type PR exhibited a monomer‐dimer equilibrium. All three mutations had little effect when introduced into only one chain of a linked dimer of HIV‐1 PR. In this case, even changing both Thr residues to Ala yielded residual activity suggesting that the “fireman's grip” is not essential for activity but contributes significantly to dimer formation. Taken together, these results indicate that the “fireman's grip” is crucial for stabilization of the retroviral PR dimer and for overall stability of the enzyme.
Understanding the sequence determinants of conformational switching using protein design
Authors: Dalal Seema , Regan Lynne 
Published: February 2009

AbstractAn important goal of protein design is to understand the forces that stabilize a particular fold in preference to alternative folds. Here, we describe an extension of earlier studies in which we successfully designed a stable, native‐like helical protein that is 50% identical in sequence to a predominantly β‐sheet protein, the B1 domain of Streptococcal IgG‐binding protein G. We report the characteristics of a series of variants of our original design that have even higher sequence identity to the B1 domain. Their properties illustrate the extent to which protein stability and conformation can be modulated through careful manipulation of key amino acid residues. Our results have implications for understanding conformational change phenomena of central biological importance and in probing the malleability of the sequence/structure relationship.
Immucillin‐H binding to purine nucleoside phosphorylase reduces dynamic solvent exchange
Authors: Wang Fang , Miles Robert W. , Kicska Gregory , Nieves Edward , Schramm Vern L. , Angeletti Ruth Hogue 
Published: February 2009

AbstractThe rate and extent of hydrogen/deuterium (H/D) exchange into purine nucleoside phosphorylase (PNP) was monitored by electrospray ionization mass spectrometry (ESI‐MS) to probe protein conformational and dynamic changes induced by a substrate analogue, products, and a transition state analogue. The genetic deficiency of PNP in humans is associated with severe T‐cell immunodeficiency, while B‐cell immunity remains functional. Inhibitors of PNP have been proposed for treatment of T‐cell leukemia, to suppress the graft‐vs.‐host response, or to counter type IV autoimmune diseases without destroying humoral immunity. Calf spleen PNP is a homotrimer of polypeptide chains with 284 amino residues, molecular weight 31,541. Immucillin‐H inhibits PNP with a Kd of 23 pM when only one of the three catalytic sites is occupied. Deuterium exchange occurs at 167 slow‐exchange sites in 2 h when no catalytic site ligands are present. The substrate analogue and product prevented H/D exchange at 10 of the sites. Immucillin‐H protected 32 protons from exchange at full saturation. When one of the three subunits of the homotrimer is filled with immucillin‐H, and 27 protons are protected from exchange in all three subunits. Deuterium incorporation in peptides from residues 132–152 decreased in all complexes of PNP. The rate and/or extent of deuterium incorporation in peptides from residues 29–49, 50–70, 81–98, and 112–124 decreased only in the complex with the transition state analogue. The peptide‐specific H/D exchange demonstrates that (1) the enzyme is most compact in the complex with immucillin‐H, and (2) filling a single catalytic site of the trimer reduces H/D exchange in the same peptides in adjacent subunits. The peptides most highly influenced by the inhibitor surround the catalytic site, providing evidence for reduced protein dynamic motion caused by the transition state analogue.
Analysis of the internal motion of free and ligand‐bound human lysozyme by use of 15N NMR relaxation measurement: A comparison with those of hen lysozyme
Authors: Mine Shouhei , Ueda Tadashi , Hashimoto Yoshio , Imoto Taiji 
Published: February 2009

AbstractHuman lysozyme has a structure similar to that of hen lysozyme and differs in amino acid sequence by 51 out of 129 residues with one insertion at the position between 47 and 48 in hen lysozyme. The backbone dynamics of free or (NAG)3‐bound human lysozyme has been determined by measurements of 15N nuclear relaxation. The relaxation data were analyzed using the Lipari‐Szabo formalism and were compared with those of hen lysozyme, which was already reported (Mine S et al., 1999, J Mol Biol 286:1547–1565). In this paper, it was found that the backbone dynamics of free human and hen lysozymes showed very similar behavior except for some residues, indicating that the difference in amino acid sequence did not affect the behavior of entire backbone dynamics, but the folded pattern was the major determinant of the internal motion of lysozymes. On the other hand, it was also found that the number of residues in (NAG)3‐bound human and hen lysozymes showed an increase or decrease in the order parameters at or near active sites on the binding of (NAG)3, indicating the increase in picosecond to nanosecond. These results suggested that the immobilization of residues upon binding (NAG)3 resulted in an entropy penalty and that this penalty was compensated by mobilizing other residues. However, compared with the internal motions between both ligand‐bound human and hen lysozymes, differences in dynamic behavior between them were found at substrate binding sites, reflecting a subtle difference in the substrate‐binding mode or efficiency of activity between them.
High resolution refinement of β‐galactosidase in a new crystal form reveals multiple metal‐binding sites and provides a structural basis for α‐complementation
Authors: Juers Douglas H. , Jacobson Raymond H. , Wigley Dale , Zhang Xue‐Jun , Huber Reuben E. , Tronrud Dale E. , Matthews Brian W. 
Published: February 2009

AbstractThe unrefined fold of Escherichia coli β‐galactosidase based on a monoclinic crystal form with four independent tetramers has been reported previously. Here, we describe a new, orthorhombic form with one tetramer per asymmetric unit that has permitted refinement of the structure at 1.7 Å resolution. This high‐resolution analysis has confirmed the original description of the structure and revealed new details. An essential magnesium ion, identified at the active site in the monoclinic crystals, is also seen in the orthorhombic form. Additional putative magnesium binding sites are also seen. Sodium ions are also known to affect catalysis, and five putative binding sites have been identified, one close to the active site. In a crevice on the protein surface, five linked five‐membered solvent rings form a partial clathrate‐like structure. Some other unusual aspects of the structure include seven apparent cis‐peptide bonds, four of which are proline, and several internal salt‐bridge networks. Deep solvent‐filled channels and tunnels extend across the surface of the molecule and pass through the center of the tetramer. Because of these departures from a compact globular shape, the molecule is not well characterized by prior empirical relationships between the mass and surface area of proteins. The 50 or so residues at the amino terminus have a largely extended conformation and mostly lie across the surface of the protein. At the same time, however, segment 13–21 contributes to a subunit interface, and residues 29–33 pass through a “tunnel” formed by a domain interface. Taken together, the overall arrangement provides a structural basis for the phenomenon of α‐complementation.
Tryptophanyl fluorescence lifetime distribution of hyperthermophilic β‐glycosidase from molecular dynamics simulation: A comparison with the experimental data
Authors: Bismuto Ettore , Martelli Pier Luigi , Casadio Rita , Irace Gaetano 
Published: February 2009

AbstractA molecular dynamics simulation approach has been utilized to understand the unusual fluorescence emission decay observed for β‐glycosidase from the hyperthermophilic bacterium Solfolobus sulfataricus (Sβgly), a tetrameric enzyme containing 17 tryptophanyl residues for each subunit. The tryptophanyl emission decay of (Sβgly) results from a bimodal distribution of fluorescence lifetimes with a short‐lived component centered at 2.5 ns and a long‐lived one at 7.4 ns Bismuto E, Nucci R, Rossi M, Irace G, 1999, Proteins 27:71–79). From the examination of the trajectories of the side chains capable of causing intramolecular quenching for each tryptophan microenvironment and using a modified Stern–Volmer model for the emission quenching processes, we calculated the fluorescence lifetime for each tryptophanyl residue of Sβgly at two different temperatures, i.e., 300 and 365 K. The highest temperature was chosen because in this condition Sβlgy evidences a maximum in its catalytic activity and is stable for a very long time. The calculated lifetime distributions overlap those experimentally determined. Moreover, the majority of trytptophanyl residues having longer lifetimes correspond to those originally identified by inspection of the crystallographic structure. The tryptophanyl lifetimes appear to be a complex function of several variables, such as microenvironment viscosity, solvent accessibility, the chemical structure of quencher side chains, and side‐chain dynamics. The lifetime calculation by MD simulation can be used to validate a predicted structure by comparing the theoretical data with the experimental fluorescence decay results.
The oxidation produced by hydrogen peroxide on Ca‐ATP‐G‐actin
Authors: Milzani Aldo , Rossi Ranieri , Simplicio Paolo Di , Giustarini Daniela , Colombo Roberto , Dalledonne Isabella 
Published: February 2009

AbstractWe report here that in vitro exposure of monomeric actin to hydrogen peroxide leads to a conversion of 6 of the 16 methionine residues to methionine sulfoxide residues. Although the initial effect of H2O2 on actin is the oxidation of Cys374, we have found that Met44, Met47, Met176, Met190, Met269, and Met355 are the other sites of the oxidative modification. Met44 and Met47 are the methionyl sites first oxidized. The methionine residues that are oxidized are not simply related to their accessibility to the external medium and are found in all four subdomains of actin. The conformations of subdomain 1, a region critical for the functional binding of different actin‐binding proteins, and subdomain 2, which plays important roles in the polymerization process and stabilization of the actin filament, are changed upon oxidation. The conformational changes are deduced from the increased exposure of hydrophobic residues, which correlates with methionine sulfoxide formation, from the perturbations in tryptophan fluorescence, and from the decreased susceptibility to limited proteolysis of oxidized actin.
Polypeptide stimulators of the Ms‐Lon protease
Authors: Rudyak Stanislav G. , Shrader Thomas E. 
Published: February 2009

AbstractBoth the peptidase activity against small fluorescent peptide substrates and the ATPase activity of Lon (La) proteases are stimulated by unstructured proteins such as α‐casein. This stimulation reveals the simultaneous interaction of Lon with two proteolytic substrates—α‐casein and the peptide substrate. To understand the cellular function of this stimulation, it is important to determine the physical properties of Lon stimulators. The abilities of compositionally simple random copolymers of amino acids (rcAAs) to stimulate the peptidase and ATPase activities of the Lon protease from Mycobacterium smegmatis (Ms‐Lon) and its N‐terminal truncation mutant (N‐E226) were determined. We report that cationic but not anionic rcAAs stimulated Ms‐Lon's peptidase activity but were themselves poor substrates for the enzyme. Peptidase stimulation by rcAAs correlated approximately with the degree of hydrophobicity of these polypeptides and reached levels >10‐fold higher than observed previously for Ms‐Lon stimulators such as α‐casein. In contrast to α‐casein, which stimulates Ms‐Lon's peptidase activity by 40% and ATPase activity by 150%, rcAAs stimulated peptidase activity without concomitant stimulation of ATPase activity. Active site labeling experiments suggested that both rcAAs and ATP increased peptidase activity by increasing accessibility to the peptidase active site. Peptidase activity assays in the presence of both α‐casein and rcAAs revealed that interactions of rcAAs and α‐casein with Ms‐Lon are extremely complex and not mutually exclusive. Specifically, (1) additions of low concentrations of α‐casein (<50 μg/mL) caused a further stimulation of Ms‐Lon's rcAA‐stimulated peptidase activity; (2) additions of higher concentrations of α‐casein inhibited Ms‐Lon's rcAA‐stimulated peptidase activity; (3) additions of all concentrations of α‐casein inhibited N‐E226′s rcAA‐stimulated peptidase activity. We conclude the Ms‐Lon can interact with an rcAA, α‐casein, and a substrate peptide simultaneously, and that formation of this quaternary complex requires the N‐terminal domain of Ms‐Lon. These data support models of Ms‐Lon that include two allosteric polypeptide binding sites distinct from the catalytic peptidase site.
Pharmacotherapy in sickle cell disease – state of the art and future prospects
Authors: Hankins Jane , Aygun Banu 
Published: February 2009

SummaryIn the last decade, the care of patients with sickle cell disease (SCD) has undergone important advances with better understanding of disease pathophysiology and improvement in standards of care, especially among paediatric patients. Although many new drugs are currently being investigated and are at different stages of development, the pace of drug discovery and utilization has been slow and suboptimal. Hydroxycarbamide (hydroxyurea) has been investigated and utilized for at least two decades. Hydroxycarbamide’s efficacy has been demonstrated, albeit with different levels of evidence, in paediatric and adult populations, and yet clinician and patient acceptance and use have been far from ideal. In this review we discuss the current usage of hydroxycarbamide and its possible future indications in SCD, as well as the use of new compounds that have very different mechanisms of action, which may prove safe and efficacious when used alone or in combination in patients with SCD.