bluuebunny/crossref_metadata_2025_split huggingface dataset:
Conformational changes in the NS3 protease from hepatitis C virus strain Bk monitored by limited proteolysis and mass spectrometry
Authors: Orrù Stefania , Piaz Fabrizio Dal , Casbarra Annarita , Biasiol Gabriella , Francesco Raffaele De , Steinkühler Christian , Pucci Piero 
Published: February 2009

AbstractConformational changes occurring within the NS3 protease domain from the hepatitis C virus Bk strain (NS31–180) under different physico‐chemical conditions either in the absence or in the presence of its cofactor Pep4A were investigated by limited proteolysis experiments. Because the surface accessibility of the protein is affected by conformational changes, when comparative experiments were carried out on NS31–180 either at different glycerol concentrations or in the presence of Pep4A, differential peptide maps were obtained from which protein regions involved in the structural changes could be inferred. The surface topology of isolated NS31–180 in solution was essentially consistent with the crystal structure of the protein with the N‐terminal segment showing a high conformational flexibility. At higher glycerol concentration, the protease assumed a more compact structure showing a decrease in the accessibility of the N‐terminal segment that either was forced to interact with the protein or originate intermolecular interactions with neighboring molecules. Binding of the cofactor Pep4A caused the displacement of the N‐terminal arm from the protein moiety, leading this segment to again adopt an open and flexible conformation, thus suggesting that the N‐terminus of the protease contributes only marginally to the stability of the complex. The observed conformational changes might be directly correlated with the activation mechanism of the protease by either the cosolvent or the cofactor peptide because they lead to tighter packing of the substrate binding site.
X‐ray structure of human stromelysin catalytic domain complexed with nonpeptide inhibitors: Implications for inhibitor selectivity
Authors: Pavlovsky Alexander G. , Williams Mark G. , Ye Qi‐Zhuang , Ortwine Daniel F. , Purchase Claude F. , White Andrew D. , Dhanaraj V. , Roth Bruce D. , Johnson Linda L. , Hupe Donald , Humblet Christine , Blundell Tom L. 
Published: February 2009

AbstractEffective inhibitors of matrix metalloproteinases (MMPs), a family of connective tissue‐degrading enzymes, could be useful for the treatment of diseases such as cancer, multiple sclerosis, and arthritis. Many of the known MMP inhibitors are derived from peptide substrates, with high potency in vitro but little selectivity among MMPs and poor bioavail‐ability. We have discovered nonpeptidic MMP inhibitors with improved properties, and report here the crystal structures of human stromelysin‐1 catalytic domain (SCD) complexed with four of these inhibitors. The structures were determined and refined at resolutions ranging from 1.64 to 2.0 Å. Each inhibitor binds in the active site of SCD such that a bulky diphenyl piperidine moiety penetrates a deep, predominantly hydrophobic S1' pocket. The active site structure of the SCD is similar in all four inhibitor complexes, but differs substantially from the peptide hydroxamate complex, which has a smaller side chain bound in the S1' pocket. The largest differences occur in the loop forming the “top” of this pocket. The occupation of these nonpeptidic inhibitors in the S1' pocket provides a structural basis to explain their selectivity among MMPs. An analysis of the unique binding mode predicts structural modifications to design improved MMP inhibitors.
A new protein folding screen: Application to the ligand binding domains of a glutamate and kainate receptor and to lysozyme and carbonic anhydrase
Authors: Armstrong Neali , Lencastre Alexandre De , Gouaux Eric 
Published: February 2009

AbstractProduction of folded and biologically active protein from Escherichia coli derived inclusion bodies can only be accomplished if a scheme exists for in vitro naturation. Motivated by the need for a rapid and statistically meaningful method of determining and evaluating protein folding conditions, we have designed a new fractional factorial protein folding screen. The screen includes 12 factors shown by previous experiments to enhance protein folding and it incorporates the 12 factors into 16 different folding conditions. By examining a 10256th fraction of the full factorial, multiple folding conditions were determined for the ligand binding domains from glutamate and kainate receptors, and for lysozyme and carbonic anhydrase B. The impact of each factor on the formation of biologically active material was estimated by calculating factor main effects. Factors and corresponding levels such as pH (8.5) and L‐ginine (0.5 M) consistently had a positive effect on protein folding, whereas detergent (0.3 mM lauryl maltoside) and nonpolar additive (0.4 M sucrose) were detrimental to the folding of these four proteins. One of the 16 conditions yielded the most folded material for three out of the four proteins. Our results suggest that this protein folding screen will be generally useful in determining whether other proteins will fold in vitro and, if so, what factors are important. Furthermore, fractional factorial folding screens are well suited to the evaluation of previously untested factors on protein folding.
Conformational properties of native sperm whale apomyoglobin in solution
Authors: Lecomte JULIETTE T.J. , Sukits Steven F. , Bhattacharya Shibani , Falzone Christopher J. 
Published: February 2009

AbstractApomyoglobin from sperm whale is often used for studies of ligand binding, protein folding, and protein stability. In an effort to describe its conformational properties in solution, homonuclear and heteronuclear (13C and 15N) NMR methods were applied to the protein in its native state. Assignments were confirmed for nuclear Overhauser effects (NOEs) involving side chain and backbone protons in the folded regions of the structure. These NOEs were used to derive distance restraints. The shifts induced by the hydrophobic dye 8‐anilino‐1‐naphthalenesulfonic acid (ANS) were inspected in the regions remote from its binding site and served as an indicator of conformational flexibility. 3JαH‐NH values were obtained to assess dihedral angle averaging and to provide additional restraints. A family of structures was calculated with X‐PLOR and an ab initio simulated annealing protocol using holomyoglobin as a template. Where the structure appeared well defined by chemical shift, line width, ANS perturbation, and density of NOEs, the low resolution model of apomyoglobin provides a valid approximation for the structure. The new model offers an improved representation of the folded regions of the protein, which encompass the A, B, E, helices as well as parts of the G and H helices. Regions that are less well defined at this stage of calculations include the CD corner and the end of the H‐helix. The EF‐F‐FG segment remains uncharacterized.
Positional preference of proline in α‐helices
Authors: Kim Mee Kyoung , Kang Young Kee 
Published: February 2009

AbstractConformational free energy calculations have been carried out for proline‐containing alanine‐based pentadecapeptides with the sequence Ac‐(Ala)n‐Pro‐(Ala)m‐NHMe, where n + m = 14, to figure out the positional preference of proline in α‐helices. The relative free energy of each peptide was calculated by subtracting the free energy of the extended conformation from that of the α‐helical one, which is used here as a measure of preference. The highest propensity is found for the peptide with proline at the N‐terminus (i.e., Ncap + 1 position), and the next propensities are found at Ncap, N' (Ncap — 1), and C' (Ccap + 1) positions. These computed results are reasonably consistent with the positional propensities estimated from X‐ray structures of proteins. The breaking in hydrogen bonds around proline is found to play a role in destabilizing α‐helical conformations, which, however, provides the favored hydration of the corresponding N‐H and C=O groups. The highest preference of proline at the beginning of α‐helix appears to be due to the favored electrostatic and nonbonded energies between two residues preceding proline and the intrinsic stability of α‐helical conformation of the proline residue itself as well as no disturbance in hydrogen bonds of α‐helix by proline. The average free energy change for the substitution of Ala by Pro in a α‐helix is computed to be 4.6 kcal/mol, which is in good agreement with the experimental value of ∼4 kcal/mol estimated for an oligopeptide dimer and proteins of barnase and T4 lysozyme.
Heat capacity change for ribonuclease A folding
Authors: Pace C. Nick , Grimsley Gerald R. , Thomas Susan T. , Makhatadze George I. 
Published: February 2009

AbstractThe change in heat capacity ΔCp for the folding of ribonuclease A was determined using differential scanning calorimetry and thermal denaturation curves. The methods gave equivalent results, ΔCp = 1.15 ± 0.08 kcal mol–1 K–1. Estimates of the conformational stability of ribonuclease A based on these results from thermal unfolding are in good agreement with estimates from urea unfolding analyzed using the linear extrapolation method.
Structural characterization of the membrane‐associated regulatory subunit of type I cAMP‐dependent protein kinase by mass spectrometry: Identification of Ser81 as the in vivo phosphorylation site of RIα
Authors: Boeshans Karen M. , Resing Katheryn A. , Hunt John B. , Ahn Natalie G. , Shabb John B. 
Published: February 2009

AbstractThe mechanism by which the type Iα regulatory subunit (RIα) of cAMP‐dependent protein kinase is localized to cell membranes is unknown. To determine if structural modification of RIα is important for membrane association, both beef skeletal muscle cytosolic RI and beef heart membrane‐associated RI were characterized by electrospray ionization mass spectrometry. Total sequence coverage was 98% for both the membrane‐associated and cytosolic forms of RI after digestion with AspN protease or trypsin. Sequence data indicated that membrane‐associated and cytosolic forms of RI were the same RIα gene product. A single RIα phosphorylation site was identified at Ser81 located near the autoinhibitory domain of both membrane‐associated and cytosolic RIα. Because both R subunit preparations were 30–40% phosphorylated, this post‐translational modification could not be responsible for the membrane compartmentation of the majority of RIα. Mass spectrometry also indicated that membrane‐associated RIα had a higher extent of disulfide bond formation in the amino‐terminal dimerization domain. No other structural differences between cytosolic and membrane‐associated RIα were detected. Consistent with these data, masses of the intact proteins were identical by LCQ mass spectrometry. Lack of detectable structural differences between membrane‐associated and cytosolic RIα strongly suggests an interaction between RIα and anchoring proteins or membrane lipids as more likely mechanisms for explaining RIα membrane association in the heart.
TRIADs: A new class of proteins with a novel cysteine‐rich signature
Authors: Van Der Reijden Bert A. , Erpelinck‐Verschueren Claudia A.J. , BOB LÖWENBERG  , Jansen Joop H. 
Published: February 2009

AbstractTriad1 was recently identified as a nuclear RING finger protein, which is up‐regulated during retinoic acid induced granulocytic differentiation of acute leukemia cells. Here we show that a cysteine‐rich domain (C6HC), present in Triad1, is conserved in at least 24 proteins encoded by various eukaryotes. The C6HC consensus pattern C‐x(4)‐C‐x(14–30)‐C‐x(1–4)‐C‐x(4)‐C‐x(2)‐C‐x(4)‐H‐x(4)‐C defines this structure as the fourth family member of the zinc‐binding RING, LIM, and LAP/PHD fingers. Strikingly, in 22 of 24 proteins the C6HC domain is flanked by two RING finger structures. We have termed the novel C6HC motif DRIL (double RING finger linked). The strong conservation of the larger tripartite TRIAD (two RING fingers and DRIL) structure indicates that the three subdomains are functionally linked and identifies a novel class of proteins.
Distinguishing between sequential and nonsequentially folded proteins: Implications for folding and misfolding
Authors: Tsai Chung‐Jung , Maizel Jacob V , Nussinov Ruth 
Published: February 2009

AbstractWe describe here an algorithm for distinguishing sequential from nonsequentially folding proteins. Several experiments have recently suggested that most of the proteins that are synthesized in the eukaryotic cell may fold sequentially. This proposed folding mechanism in vivo is particularly advantageous to the organism. In the absence of chaperones, the probability that a sequentially folding protein will misfold is reduced significantly. The problem we address here is devising a procedure that would differentiate between the two types of folding patterns. Footprints of sequential folding may be found in structures where consecutive fragments of the chain interact with each other. In such cases, the folding complexity may be viewed as being lower. On the other hand, higher folding complexity suggests that at least a portion of the polypeptide backbone folds back upon itself to form three‐dimensional (3D) interactions with noncontiguous portion(s) of the chain. Hence, we look at the mechanism of folding of the molecule via analysis of its complexity, that is, through the 3D interactions formed by contiguous segments on the polypeptide chain. To computationally splice the structure into consecutively interacting fragments, we either cut it into compact hydrophobic folding units or into a set of hypothetical, transient, highly populated, contiguous fragments (“building blocks” of the structure). In sequential folding, successive building blocks interact with each other from the amino to the carboxy terminus of the polypeptide chain. Consequently, the results of the parsing differentiate between sequentially vs. nonsequentially folded chains. The automated assessment of the folding complexity provides insight into both the likelihood of misfolding and the kinetic folding rate of the given protein. In terms of the funnel free energy landscape theory, a protein that truly follows the mechanism of sequential folding, in principle, encounters smoother free energy barriers. A simple sequentially folded protein should, therefore, be less error prone and fold faster than a protein with a complex folding pattern.
The progressive development of structure and stability during the equilibrium folding of the α subunit of tryptophan synthase from Escherichia coli
Authors: Gualfetti Peter J. , Bilsel Osman , Matthews C. Robert 
Published: February 2009

AbstractThe urea‐induced equilibrium unfolding of the α subunit of tryptophan synthase (aTS), a single domain α/β barrel protein, displays a stable intermediate at ˜3.2 M urea when monitored by absorbance and circular dichroism (CD) spectroscopy (Matthews CR, Crisanti MM, 1981, Biochemistry 20:784–792). The same experiment, monitored by one‐dimensional proton NMR, shows another cooperative process between 5 and 9 M urea that involves His92 (Saab‐Rincón G et al., 1993, Biochemistry 52:13981–13990). To further test and quantify the implied four‐state model, N ⇄ I1 ⇄ I2 ⇄ U, the urea‐induced equilibrium unfolding process was followed by tyrosine fluorescence total intensity, tyrosine fluorescence anisotropy and far‐UV CD. All three techniques resolve the four stable states, and the transitions between them when the FL total intensity and CD spectroscopy data were analyzed by the singular value decomposition method. Relative to U, the stabilities of the N, I1, and I2 states are 15.4, 9.4, and 4.9 kcal mol−1, respectively. I2 partially buries one or more of the seven tyrosines with a noticeable restriction of their motion; it also recovers ∼6% of the native CD signal. This intermediate, which is known to be stabilized by the hydrophobic effect, appears to reflect the early coalescence of nonpolar side chains without significant organization of the backbone. I1 recovers an additional 43% of the CD signal, further sequesters tyrosine residues in nonpolar environments, and restricts their motion to an extent similar to N. The progressive development of a higher order structure as the denaturant concentration decreases implies a monotonic contraction in the ensemble of conformations that represent the U, I2, I1, and N states of αTS.
Structure and dynamics of the fatty acid binding cavity in apo rat intestinal fatty acid binding protein
Authors: Likić Vladimir A. , Prendergast Franklyn G. 
Published: February 2009

AbstractThe structure and dynamics of the fatty acid binding cavity in I‐FABP (rat intestinal fatty acid binding protein) were analyzed. In the crystal structure of apo I‐FABP, the probe occupied cavity volume and surface are 539 ± 8 Å3 and 428 Å2, respectively (1.4 Å probe). A total of 31 residues contact the cavity with their side chains. The side‐chain cavity surface is partitioned according to the residue type as follows: 36–39% hydrophobic, 21–25% hydrophilic, and 37–43% neutral or ambivalent. Thus, the cavity surface is neither like a typical protein interior core, nor is like a typical protein external surface. All hydrophilic residues that contact the cavity—with the exception of Asp74—are clustered on the one side of the cavity. The cavity appears to expand its hydrophobic surface upon fatty acid binding on the side opposite to this hydrophilic patch. In holo I‐FABP the fatty acid chain interactions with the hydrophilic side chains are mediated by water molecules. Molecular dynamics (MD) simulation of fully solvated apo I‐FABP showed global conformational changes of I‐FABP, which resulted in a large, but seemingly transient, exposure of the cavity to the external solvent. The packing density of the side chains lining the cavity, studied by Voronoi volumes, showed the presence of two distinctive small hydrophobic cores. The MD simulation predicts significant structural perturbations of the cavity on the subnano‐second time scale, which are capable of facilitating exchange of I‐FABP internal water.
The α‐subunit of protein prenyltransferases is a member of the tetratricopeptide repeat family
Authors: Zhang Hong , Grishin Nick V 
Published: February 2009

AbstractLipidation catalyzed by protein prenyltransferases is essential for the biological function of a number of eukaryotic proteins, many of which are involved in signal transduction and vesicular traffic regulation. Sequence similarity searches reveal that the α‐subunit of protein prenyltransferases (PTα) is a member of the tetratricopeptide repeat (TPR) super‐family. This finding makes the three‐dimensional structure of the rat protein farnesyltransferase the first structural model of a TPR protein interacting with its protein partner. Structural comparison of the two TPR domains in protein farnesyltransferase and protein phosphatase 5 indicates that variation in TPR consensus residues may affect protein binding specificity through altering the overall shape of the TPR superhelix. A general approach to evolutionary analysis of proteins with repetitive sequence motifs has been developed and applied to the protein prenyltransferases and other TPR proteins. The results suggest that all members in PTα family originated from a common multirepeat ancestor, while the common ancestor of PTα and other members of TPR superfamily is likely to be a single repeat protein.
Escherichia coli maltose‐binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused
Authors: Kapust Rachel B. , Waugh David S. 
Published: February 2009

AbstractAlthough it is usually possible to achieve a favorable yield of a recombinant protein in Escherichia coli, obtaining the protein in a soluble, biologically active form continues to be a major challenge. Sometimes this problem can be overcome by fusing an aggregation‐prone polypeptide to a highly soluble partner. To study this phenomenon in greater detail, we compared the ability of three soluble fusion partners—maltose‐binding protein (MBP), glutathione S‐transferase (GST), and thioredoxin (TRX)—to inhibit the aggregation of six diverse proteins that normally accumulate in an insoluble form. Remarkably, we found that MBP is a far more effective solubilizing agent than the other two fusion partners. Moreover, we demonstrated that in some cases fusion to MBP can promote the proper folding of the attached protein into its biologically active conformation. Thus, MBP seems to be capable of functioning as a general molecular chaperone in the context of a fusion protein. A model is proposed to explain how MBP promotes the solubility and influences the folding of its fusion partners.
Effects of N‐butyldeoxynojirimycin and the Lec3.2.8.1 mutant phenotype on N‐glycan processing in Chinese hamster ovary cells: Application to glycoprotein crystallization
Authors: Butters Terry D. , Sparks Lisa M. , Harlos Karl , Ikemizu Shinji , Stuart David I. , Jones E. Yvonne , Davis Simon J. 
Published: February 2009

AbstractHeterologous gene expression in either (1) the glycosylation‐defective, mutant Chinese hamster ovary cell line, Lec3.2.8.1, or (2) the presence of the α‐glucosidase inhibitor, N‐butyldeoxynojirimycin facilitates the trimming of N‐linked glycans of glycoproteins to single N‐acetylglucosamine (GlcNAc) residues with endoglycosidase H (endo H). Both approaches are somewhat inefficient, however, with as little as 12% of the total protein being rendered fully endo H‐sensitive under these conditions. It is shown here that the combined effects of these approaches on the restriction of oligosaccharide processing are essentially additive, thereby allowing the production of glycoproteins that are essentially completely endo H‐sensitive. The preparation of a soluble chimeric form of CD58, the ligand of the human T‐cell surface recognition molecule CD2, illustrates the usefulness of the combined approach when expression levels are low or the deglycosylated protein is unstable at low pH. The endo H‐treated chimera produced crystals of space group P3121 or P3221, and unit cell dimensions a = b = 116.4 Å, c = 51.4 Å a = β = 90°, γ = 120°, that diffract to a maximum resolution of 1.8 Å.
Drug resistance mutations can affect dimer stability of HIV‐1 protease at neutral pH
Authors: Xie Dong , Gulnik Sergei , Gustchina Elena , Yu Betty , Shao Wei , Qoronfleh Walid , Nathan Anand , Erickson John W. 
Published: February 2009

AbstractThe monomer‐dimer equilibrium for the human immunodeficiency virus type 1 (HIV‐1) protease has been investigated under physiological conditions. Dimer dissociation at pH 7.0 was correlated with a loss in β‐sheet structure and a lower degree of ANS binding. An autolysis‐resistant mutant, Q7K/L33I/L63I, was used to facilitate sedimentation equilibrium studies at neutral pH where the wild‐type enzyme is typically unstable in the absence of bound inhibitor. The dimer dissociation constant (KD) of the triple mutant was 5.8 μM at pH 7.0 and was below the limit of measurement (˜100 nM) at pH 4.5. Similar studies using the catalytically inactive D25N mutant yielded a KD value of 1.0 μMat pH 7.0. These values differ significantly from a previously reported value of 23 nM obtained indirectly from inhibitor binding measurements (Darke et al., 1994). We show that the discrepancy may result from the thermodynamic linkage between the monomer‐dimer and inhibitor binding equilibria. Under conditions where a significant degree of monomer is present, both substrates and competitive inhibitors will shift the equilibrium toward the dimer, resulting in apparent increases in dimer stability and decreases in ligand binding affinity. Sedimentation equilibrium studies were also carried out on several drug‐resistant HIV‐1 protease mutants: V82F, V82F/I84V, V82T/I84V, and L90M. All four mutants exhibited reduced dimer stability relative to the autolysis‐resistant mutant at pH 7.0. Our results indicate that reductions in drug affinity may be due to the combined effects of mutations on both dimer stability and inhibitor binding.