bluuebunny/crossref_metadata_2025 huggingface dataset:
Recombinant human erythropoietin (rHuEPO): Cross‐linking with disuccinimidyl esters and identification of the interfacing domains in EPO
Authors: Haniu Mitsuru , Narhi Linda O. , Arakawa Tsutomu , Elliott Steve , Rohde Michael F.
Published: February 2009

Several amino groups of recombinant human erythropoietin are selectively cross‐linked by specific cross‐linkers including disuccinimidyl suberate or dithiobis(succinimidyl propionate). Intramolecular cross‐linkings are obtained without significant change of the protein conformation using appropriate concentrations (0.2 mM) of the cross‐linkers, which possess an 11–12‐Å length of a spacer between two reacting groups. Intramolecularly cross‐linked peptides obtained suggest that several amino groups in erythropoietin (EPO) are positioned at a distance of near 12 Å in the solution state. These interfacing amino groups include Lys 20–Lys 154, Lys 45–Lys 140, Lys 52–Lys 154, Lys 52–Lys 140, and Ala 1–Lys 116. A comparison of the cross‐linking results between nonglycosylated EPO and glycosylated EPO suggests that both proteins retain high similarity regarding protein conformation. These results fit a structural model similar to that of human growth hormone, in which four α‐helical bundles and a long stretch of β‐sheet structure are involved in the active protein.
Verification of protein structures: Patterns of nonbonded atomic interactions
Authors: Colovos Chris , Yeates Todd O.
Published: February 2009

A novel method for differentiating between correctly and incorrectly determined regions of protein structures based on characteristic atomic interactions is described. Different types of atoms are distributed nonrandomly with respect to each other in proteins. Errors in model building lead to more randomized distributions of the different atom types, which can be distinguished from correct distributions by statistical methods.

Atoms are classified in one of three categories: carbon (C), nitrogen (N), and oxygen (O). This leads to six different combinations of pairwise noncovalently bonded interactions (CC, CN, CO, NN, NO, and OO). A quadratic error function is used to characterize the set of pairwise interactions from nine‐residue sliding windows in a database of 96 reliable protein structures. Regions of candidate protein structures that are mistraced or misregistered can then be identified by analysis of the pattern of nonbonded interactions from each window.
Rapid protein separation and diffusion coefficient measurement by frit inlet flow field‐flow fractionation
Authors: Liu Min‐Kuang , Li Ping , Giddings J. Calvin
Published: February 2009

In this study three flow field‐flow fractionation (flow FFF) channels are utilized for the separation of proteins and for the simultaneous measurement of their translational diffusion coefficients, D. One channel has a traditional sample inlet, whereas the other two incorporate a frit inlet design that permits more convenient and rapid sample introduction. The dependence of retention time on D, which leads to differential elution and the opportunity to measure D for protein peaks purified by the flow FFF process, is described theoretically and examined experimentally. Factors affecting band broadening, resolution, and optimization are also examined. The separation of proteins is achieved in the time range 4–20 min. Partial resolution is achieved in multiple runs requiring 2 min each. Values of D calculated from retention times are reported for 15 proteins. These include two protein dimers (bovine serum albumin and γ‐globulin) not ordinarily accessible to measurement. The D values from the three channels are compared with one another and with literature data. Reasonable consistency (within 3–4%) is found. High‐speed repetitive runs can be used to acquire multiple values of D in time intervals as short as 1 min.
Disulfide bonds and the stability of globular proteins
Authors: Betz Stephen F.
Published: February 2009

An understanding of the forces that contribute to stability is pivotal in solving the protein‐folding problem. Classical theory suggests that disulfide bonds stabilize proteins by reducing the entropy of the denatured state. More recent theories have attempted to expand this idea, suggesting that in addition to configurational entropic effects, enthalpic and native‐state effects occur and cannot be neglected. Experimental thermodynamic evidence is examined from two sources: (1) the disruption of naturally occurring disulfides, and (2) the insertion of novel disulfides. The data confirm that enthalpic and native‐state effects are often significant. The experimental changes in free energy are compared to those predicted by different theories. The differences between theory and experiment are large near 300 K and do not lend support to any of the current theories regarding the stabilization of proteins by disulfide bonds. This observation is a result of not only deficiencies in the theoretical models but also from difficulties in determining the effects of disulfide bonds on protein stability against the backdrop of numerous subtle stabilizing factors (in both the native and denatured states), which they may also affect.
Stabilization of creatinase from Pseudomonas putida by random mutagenesis
Authors: Schumann Judith , Böhm Gerald , Jaenicke Rainer , Schumacher Günter , Rudolph Rainer
Published: February 2009

Creatinase (creatine amidinohydrolase, EC 3.5.3.3) from Pseudomonas putida is a homodimer of 45 kDa subunit molecular mass, the three‐dimensional structure of which is known at 1.9 Å resolution. Three point mutants, A109V, V355M, and V182I, as well as one double mutant combining A109V and V355M, and the triple mutant with all three replacements, were compared with wild‐type creatinase regarding their physical and enzymological properties. High‐resolution crystal data for wild‐type creatinase and the first two mutants suggest isomorphism at least for these three proteins (R. Huber, pers. comm.). Physicochemical measurements confirm this prediction, showing that the mutations have no effect either on the quaternary structure and gross conformation or the catalytic properties as compared to wild‐type creatinase. The replacement of V182 (at the solvent‐exposed end of the first helix of the C‐terminal domain) does not cause significant differences in comparison with the wild‐type enzyme. The other point mutations stabilize the first step in the biphasic denaturation transition without affecting the second one. In sum, the enhanced stability seems to reflect slight improvements in the local packing without creating new well‐defined bonds. The increase in hydrophobicity generated by the introduction of additional methyl groups (A109V, V1821) must be compensated by minor readjustments of the global structure. Secondary or quaternary interactions are not affected. In going from single to double and triple mutants, to a first approximation, the increments of stabilization are additive.
Knowledge‐based model building of proteins: Concepts and examples
Authors: Bajorath Jürgen , Stenkamp Ronald , Aruffo Alejandro
Published: February 2009

We describe how to build protein models from structural templates. Methods to identify structural similarities between proteins in cases of significant, moderate to low, or virtually absent sequence similarity are discussed. The detection and evaluation of structural relationships is emphasized as a central aspect of protein modeling, distinct from the more technical aspects of model building. Computational techniques to generate and complement comparative protein models are also reviewed. Two examples, P‐selectin and gp39, are presented to illustrate the derivation of protein model structures and their use in experimental studies.
Toward computational determination of peptide‐receptor structure
Authors: Sezerman Ugur , Vajda Sandor , Cornette James , Delisi Charles
Published: February 2009

We introduce a method for docking small flexible ligands of the size of dipeptides and phosphocholine and test it against crystallographic complexes. We then show how the method can be used as the basis for a strategy for solving the much more difficult problem of docking fully flexible peptides in the 8–10‐residue size range. After developing the method we apply it to peptide–MHC class I systems and find that the predictions are in accord with biological and crystallographic data.
Resolution of the fluorescence equilibrium unfolding profile of trp aporepressor using single tryptophan mutants
Authors: Royer Catherine A. , Mann Craig J. , Matthews C. Robert
Published: February 2009

Single tryptophan mutants of the trp aporepressor, tryptophan 19 → phenylalanine (W19F) and tryptophan 99 → phenylalanine (W99F), were used in this study to resolve the individual steady‐state and time‐resolved fluorescence urea unfolding profiles of the two tryptophan residues in this highly intertwined, dimeric protein. The wild‐type protein exhibits a large increase in fluorescence intensity and lifetime, as well as a large red shift in the steady‐state fluorescence emission spectrum, upon unfolding by urea (Lane, A.N. & Jardetsky, O., 1987, Eur. J. Biochem. 164, 389–396; Gittelman, M.S. & Matthews, C.R., 1990, Biochemistry 29, 7011–7020; Fernando, T. & Royer, C.A., 1992, Biochemistry 31, 6683–6691). Unfolding of the W19F mutant demonstrated that Trp 99 undergoes a large increase in intensity and a red shift upon exposure to solvent. Lifetime studies revealed that the contribution of the dominant 0.5‐ns component of this tryptophan tends toward zero with increasing urea, whereas the longer lifetime components increase in importance. This lifting of the quenching of Trp 99 may be due to disruption of the interaction between the two subunits upon denaturation, which abolishes the interaction of Trp 99 on one subunit with the amide quenching group of Asn 32 on the other subunit (Royer, C.A., 1992, Biophys. J. 63, 741–750). On the other hand, Trp 19 is quenched in response to unfolding in the W99F mutant. Exposure to solvent of Trp 19, which is buried at the hydrophobic dimer interface in the native protein, results in a large red shift of the average steady‐state emission. Thus, changes in the fluorescence properties of the two intrinsic tryptophan residues in trp aporepressor upon unfolding are explained readily in terms of disruption of the dimer interface.
Kinetics of folding and association of differently glycosylated variants of invertase from saccharomyces cerevisiae
Authors: Kern Gunther , Kern Dorothee , Jaenicke Rainer , Seckler Robert
Published: February 2009

A core‐glycosylated form of the dimeric enzyme invertase has been isolated from secretion mutants of Saccharomyces cerevisiae blocked in transport to the Golgi apparatus. This glycosylation variant corresponds to the form that folds and associates during biosynthesis of the protein in vivo. In the present work, its largely homogeneous subunit size and well‐defined quaternary structure were utilized to characterize the folding and association pathway of this highly glycosylated protein in comparison with the nonglycosylated cytoplasmic and the high‐mannose‐glycosylated periplasmic forms of the same enzyme encoded by the suc2 gene.

Renaturation of core‐glycosylated invertase upon dilution from guanidinium‐chloride solutions follows a unibimolecular reaction scheme with consecutive first‐order subunit folding and second‐order association reactions. The rate constant of the rate‐limiting step of subunit folding, as detected by fluorescence increase, is k1 = 1.6 + 0.4 × 10−3 s−1 at 20 °C; it is characterized by an activation enthalpy of ΔH = 65 kJ/mol. The reaction is not catalyzed by peptidyl‐prolyl cis‐trans isomerase of the cyclophilin type. Reactivation of the enzyme depends on protein concentration and coincides with subunit association, as monitored by size‐exclusion high‐pressure liquid chromatography. The association rate constant, estimated by numerical simulation of reactivation kinetics, increases from 5 × 103 M−1 s−1 to 7 × 104 M−1 s−1 between 5 and 30 °C.

Although reactivation kinetics and yields of the core‐glycosylated and high‐mannose‐glycosylated invertase are essentially identical between 5 and 40 °C, the nonglycosylated cytoplasmic form displays strongly reduced reactivation yields at the high end and significantly reduced reactivation rates at the low end of this temperature range.
Mechanism of phage P22 tailspike protein folding mutations
Authors: Danner Martina , Seckler Robert
Published: February 2009

Temperature‐sensitive folding (tsf) and global‐tsf‐suppressor (su) point mutations affect the folding yields of the trimeric, thermostable phage P22 tailspike endorhamnosidase at elevated temperature, both in vivo and in vitro, but they have little effect on function and stability of the native folded protein. To delineate the mechanism by which these mutations modify the partitioning between productive folding and off‐pathway aggregation, the kinetics of refolding after dilution from acid‐urea solutions and the thermal stability of folding intermediates were analyzed. The study included five tsf mutations of varying severity, the two known su mutations, and four tsf/su double mutants.

At low temperature (10°C), subunit‐folding rates, measured as an increase in fluorescence, were similar for wild‐type and mutants. At 25°C, however, tsf mutations reduced the rate of subunit folding. The su mutations increased this rate, when present in the tsf‐mutant background, but had no effect in the wild‐type background. Conversely, tsf mutations accelerated, and su mutations retarded the irreversible off‐pathway reaction, as revealed by temperature down‐shifts after varied times during refolding at high temperature (40°C). The kinetic results are consistent with tsf mutations destabilizing and su mutations stabilizing an essential subunit folding intermediate.

In accordance with this interpretation, tsf mutations decreased, and su mutations increased the temperature resistance of folding intermediates, as disclosed by temperature up‐shifts during refolding at 25°C. The stabilizing and destabilizing effects were most pronounced early during refolding. However, they were not limited to subunit‐folding intermediates and were also observable during thermal unfolding of the native protein.
Macromolecular solvation energies derived from small molecule crystal morphology
Authors: Rees Douglas C. , Wolfe Gershon M.
Published: February 2009

The morphology of small molecule crystals provides a model for evaluating surface solvation energies in a system with similar packing density to that observed for amino acid residues in proteins. The solvation energies associated with the transfer of methylene and carboxyl groups between vacuum and aqueous phases are estimated to be approx. +40 and ‐260 cal/Å2, respectively, from an analysis of the morphology of succinic acid crystals. These solvation energies predict values for contact angles in reasonable agreement with measurements determined from macroscopic monolayer surfaces. Transfer free energies between vapor and water phases for a series of carboxylic acids are also predicted reasonably well by these solvation energies, provided the surface exposure of different groups is quantitated with the molecular surface area rather than the more traditional accessible surface area. In general, molecular surfaces and molecular surface areas are seen to have important advantages for characterizing the structure and energetics of macromolecular surfaces. Crystal faces of succinic acid with the lowest surface energies in aqueous solution are characteristically smooth. Increasing surface roughness and apolarity are associated with higher surface energies, which suggests an approach for modifying the surface properties of proteins and other macromolecules.
Aldehyde dehydrogenases: Widespread structural and functional diversity within a shared framework
Authors: Hempel John , Nicholas Hugh , Lindahl Ronald
Published: February 2009

Sequences of 16 NAD and/or NADP‐linked aldehyde oxidoreductases are aligned, including representative examples of all aldehyde dehydrogenase forms with wide substrate preferences as well as additional types with distinct specificities for certain metabolic aldehyde intermediates, particularly semialdehydes, yielding pairwise identities from 15 to 83%. Eleven of 23 invariant residues are glycine and three are proline, indicating evolutionary restraint against alteration of peptide chain‐bending points. Additionally, another 66 positions show high conservation of residue type, mostly hydrophobic residues. Ten of these occur in predicted β‐strands, suggesting important interior‐packing interactions.

A single invariant cysteine residue is found, further supporting its catalytic role. A previously identified essential glutamic acid residue is conserved in all but methyl malonyl semialdehyde dehydrogenase, which may relate to formation by that enzyme of a CoA ester as a product rather than a free carboxylate species. Earlier, similarity to a GXGXXG segment expected in the NAD‐binding site was noted from alignments with fewer sequences. The same region continues to be indicated, although now only the first glycine residue is strictly conserved and the second (usually threonine) is not present at all, suggesting greater variance in coenzyme‐binding interactions.
Import of cytochrome b2 to the mitochondrial intermembrane space: The tightly folded heme‐binding domain makes import dependent upon matrix ATP
Authors: Glick Benjamin S. , Wachter Clemens , Reid Graeme A. , Schatz Gottfried
Published: February 2009

Cytochrome b2 is synthesized as a precursor in the cytoplasm and imported to the intermembrane space of yeast mitochondria. We show here that the precursor contains a tightly folded heme‐binding domain and that translocation of this domain across the outer membrane requires ATP. Surprisingly, it is ATP in the mitochondrial matrix rather than external ATP that drives import of the heme‐binding domain. When the folded structure of the heme‐binding domain is disrupted by mutation or by urea denaturation, import and correct processing take place in ATP‐depleted mitochondria. These results indicate that (1) cytochrome b2 reaches the intermembrane space without completely crossing the inner membrane, and (2) some precursors fold outside the mitochondria but remain translocation‐competent, and import of these precursors in vitro does not require ATP‐dependent cytosolic chaperone proteins.
NMR and protein folding: Equilibrium and stopped‐flow studies
Authors: Frieden Carl , Hoeltzli Sydney D. , Ropson Ira J.
Published: February 2009

NMR studies are now unraveling the structure of intermediates of protein folding using hydrogen—deuterium exchange methodologies. These studies provide information about the time dependence of formation of secondary structure. They require the ability to assign specific resonances in the NMR spectra to specific amide protons of a protein followed by experiments involving competition between folding and exchange reactions. Another approach is to use 19F‐substituted amino acids to follow changes in side‐chain environment upon folding. Current techniques of molecular biology allow assignments of 19F resonances to specific amino acids by site‐directed mutagenesis. It is possible to follow changes and to analyze results from 19F spectra in real time using a stopped‐flow device incorporated into the NMR spectrometer.
Sequence‐specific
Authors: Fletcher C.M. , Harrison R.A. , Lachmann P.J. , Neuhaus D.
Published: February 2009

CD59 is a recently discovered cell‐surface glycoprotein that restricts lysis by homologous complement and has limited sequence similarity to snake venom neurotoxins. This paper describes the first results of a two‐dimensional NMR study of CD59 prepared from human urine. Nearly complete 1H‐NMR assignments were obtained for the 77 amino acid residues and partial assignments for the N‐glycan and the glycosylphosphatidylinositol (GPI) anchor. These results together confirm that the C‐terminal residue of the mature protein is Asn 77 and that the urine‐derived form retains the nonlipid part of the GPI anchor. The data further indicate that the GPI anchor and possibly the N‐glycan are structurally inhomogeneous and suggest that the phospholipid present in the intact GPI anchor was removed by phosphatidylinositol‐specific phospholipase‐D. The folding topology of the protein was determined from NOE enhancements and slowly exchanging backbone amide protons and consists primarily of five extended strands (denoted β1–β5 in sequence order), arranged into separate two‐stranded (β1 and β2) and three‐stranded (β3–β5) antiparallel β‐sheets. The same folding topology is found in all of the snake venom neurotoxins whose structures have been determined. The region between the β4 and β5 strands has helical character, a feature that is not present in the neurotoxins but that is seen in the topologically similar wheat germ agglutinin.
Characterization of the NR1, NR2A, and NR2C receptor proteins
Authors: Köpke Andreas K.E. , Bonk Ines , Sydow Sabine , Menke Hildegard , Spiess Joachim
Published: February 2009

N‐Methyl‐d‐aspartate (NMDA) receptor subunits were characterized with seven polyclonal antibodies. The antibodies were directed against NR1‐A, NR2A‐N1, and NR2C‐N1, representing N‐terminal sequences of the NR1, NR2A, and NR2C subunits, and against NR1‐E, NR2A‐C1, and NR2C‐C1, derived from C‐terminal sequences of these subunits. The anti‐NR1‐D antibody was raised against the putative internal loop of NR1.

A size of 118 kDa was found in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis for NR1 (from rat brain) detected by anti‐NR1‐D and ‐NR1‐E, but not anti‐NR1‐A. With the anti‐NR1‐A antibody, a 125‐kDa protein was discovered that may represent a glutamate receptor not yet characterized. NR2A and NR2C were identified as proteins with sizes of 175 and 140 kDa, respectively.

Enzymatic N‐deglycosylation generated a 97‐kDa protein from NR1, a 105‐kDa protein from the 125‐kDa protein, a 162‐kDa protein from NR2A, and a 127‐kDa protein from NR2C. In contrast to the deglycosylation product of the NR2A, the 97‐ and 127‐kDa proteins derived from NR1 and NR2C, respectively, were found significantly smaller than the molecular masses of 103 and 141 kDa, respectively, predicted on the basis of DNA data. These products may represent truncated proteins.

The tissue content of the NR1 and NR2A was high in bovine hippocampus and cortex but lower in the cerebellum. In contrast, NR2C was solely found in the cerebellum. The 125‐kDa protein was highest in the cerebellum and cortex.
Fluorescence characterization of Trp 21 in rat glutathione S‐transferase 1–1: Microconformational changes induced by S‐hexyl glutathione
Authors: Wang Regina W. , Bird Andrew W. , Newton Deborah J. , Lu Anthony Y. H. , Atkins William M.
Published: February 2009

The glutathione S‐transferase (GST) isoenzyme A1–1 from rat contains a single tryptophan, Trp 21, which is expected to lie within α‐helix 1 based on comparison with the X‐ray crystal structures of the pi‐ and mu‐class enzymes. Steady‐state and multifrequency phase/modulation fluorescence studies have been performed in order to characterize the fluorescence parameters of this tryptophan and to document ligand‐induced conformational changes in this region of the protein. Addition of S‐hexyl glutathione to GST isoenzyme A1–1 causes an increase in the steady‐state fluorescence intensity, whereas addition of the substrate glutathione has no effect. Frequency‐domain excited‐state lifetime measurements indicate that Trp 21 exhibits three exponential decays in substrate‐free GST. In the presence of S‐hexyl glutathione, the data are also best described by the sum of three exponential decays, but the recovered lifetime values change. For the substrate‐free protein, the short lifetime component contributes 9–16% of the total intensity at four wavelengths spanning the emission. The fractional intensity of this lifetime component is decreased to less than 3% in the presence of S‐hexyl glutathione. Steady‐state quenching experiments indicate that Trp 21 is insensitive to quenching by iodide, but it is readily quenched by acrylamide. Acrylamide‐quenching experiments at several emission wavelengths indicate that the long‐wavelength components become quenched more easily in the presence of S‐hexyl glutathione. Differential fluorescence polarization measurements also have been performed, and the data describe the sum of two anisotropy decay rates. The recovered rotational correlation times for this model are 26 ns and 0.81 ns, which can be attributed to global motion of the protein dimer, and fast local motion of the tryptophan side chain. These results demonstrate that regions of GST that are not in direct contact with bound substrates are mobile and undergo microconformational rearrangement when the “H‐site” is occupied.
MIF proteins are theta‐class glutathione S‐transferase homologs
Authors: Blocki Frank A. , Ellis LYNDA B.M. , Wackett Lawrence P.
Published: February 2009

MIF proteins are mammalian polypeptides of approximately 13, 000 molecular weight. This class includes human macrophage migration inhibitory factor (MIF), a rat liver protein that has glutathione S‐transferase (GST) activity (TRANSMIF), and the mouse delayed early response gene 6 (DER6) protein. MIF proteins were previously linked to GSTs by demonstrating transferase activity and observing N‐terminal sequence homology with a mu‐class GST (Blocki, F.A., Schlievert, P.M., & Wackett, L.P., 1992, Nature 360, 269–270). In this study, MIF proteins are shown to be structurally related to the theta class of GSTs. This is established in three ways. First, unique primary sequence patterns are developed for each of the GST gene classes. The patterns identify the three MIF proteins as theta‐like transferase homologs. Second, pattern analysis indicates that GST members of the theta class contain a serine residue in place of the N‐terminal tyrosine that is implicated in glutathione deprotonation and activation in GSTs of known structure (Liu, S., et al., 1992, J. Biol. Chem. 267, 4296–4299). The MIF proteins contain a threonine at this position. Third, polyclonal antibodies raised against recombinant human MIF cross‐react on Western blots with rat theta GST but not with alpha and mu GSTs. That MIF proteins have glutathione‐binding ability may provide a common structural key toward understanding the varied functions of this widely distributed emerging gene family. Because theta is thought to be the most ancient evolutionary GST class, MIF proteins may have diverged early in evolution but retained a glutathione‐binding domain.
Classification of doubly wound nucleotide binding topologies using automated loop searches
Authors: Swindells Mark B.
Published: February 2009

A classification is presented of doubly wound α/β nucleotide binding topologies, whose binding sites are located in the cleft formed by a topological switch point. In particular, the switch point loop nearest the N‐terminus is used to identify specific structural classes of binding protein. This yields seven structurally distinct loop conformations, which are subsequently used as motifs for scanning the Protein Data Bank. The searches, which are effective at identifying functional relationships within a large database of structures, reveal a remarkable and previously unnoticed similarity between the coenzyme binding sites of flavodoxin and tryptophan synthetase, even though there is no sequence or topological similarity between them.
Divalent metal ions influence catalysis and active‐site accessibility in the camp‐dependent protein kinase
Authors: Adams Joseph A. , Taylor Susan S.
Published: February 2009

Phosphorylation of the peptide LRRASLG by the catalytic subunit of cAMP‐dependent protein kinase was measured in the presence of various divalent metals to establish the role of electrophiles in the kinetic mechanism. Under conditions of low or high metal concentrations, the apparent second‐order rate constant, kcat/Kpeptide, and the maximal rate constant, kcat, followed the trend Mg2+ > Co2+ > Mn2+. Competitive inhibition studies indicate that the former effect is not due to destabilization of the substrate complex, E‐ATP‐S. The effects of solvent viscosity on the steady‐state kinetic parameters were interpreted according to a simple mechanism involving substrate binding, phosphotransfer, and product release steps and two metal chelation sites in the nucleotide pocket. Decreases in kcat and kcat/Kpeptide result mostly from attenuations in the dissociation rate constant for ADP and the association rate constant for the substrate, respectively. Decreases in the phosphoryl transfer rate constant have only negligible to moderate effects on these parameters. The low observed values for the association rate constant of the substrate indicate that the metals control the concentration of the productive binary form, Ea‐ATP, and indirectly the accessibility of the active site. By comparison, Mg2+ is the best divalent metal catalyst because it uniformly lowers the transition state energies for all steps in the kinetic mechanism, permitting maximum flux of substrate to product. The data suggest that cAMP‐dependent protein kinase uses metal ions to serve multiple roles in facilitating phosphotransfer and accelerating substrate association and product dissociation.
Probing weakly polar interactions in cytochrome c
Authors: Auld Douglas S. , Young Gregory B. , Saunders Aleister J. , Doyle Donald F. , Betz Stephen F. , Pielak Gary J.
Published: February 2009

Theoretical, statistical, and model studies suggest that proteins are stabilized by weakly polar attractions between sulfur atoms and properly oriented aromatic rings. The two sulfur‐containing amino acids, methionine and cysteine, occur frequently among functional alleles in random mutant libraries of Saccharomyces cerevisiae iso‐1‐cytochrome c genes at positions that form a weakly polar aromatic‐aromatic interaction in the wild‐type protein. To determine if a weakly polar sulfur‐aromatic interaction replaced the aromatic‐aromatic interaction, the structure and stability of two variants were examined. Phenylalanine 10, which interacts with tyrosine 97, was replaced by methionine and cysteine. The cysteine was modified to form the methionine and cystine analog, S‐methyl cysteine (CysSMe). Proton NMR studies indicate that changing Phe 10 to Met or CysSMe affects only local structure and that the structures of sulfur‐containing variants are nearly identical. Analysis of chemical shifts and nuclear Overhauser effect data indicates that both sulfur‐containing side chains are in position to form a weakly polar interaction with Tyr 97. The F10M and F10CSMe variants are 2–3 kcal mol−1 less stable than iso‐1‐cytochrome c at 300 K. Comparison of the stabilities of the F10M and F10CSMe variants allows evaluation of the potential weakly polar interaction between the additional sulfur atom of F10CSMe and the aromatic moiety of Tyr 97. The F10CSMe;C102T variant is 0.7 + 0.3 kcal mol−1 more stable than the F10M;C102T protein. The increased stability is explained by the difference in hydrophobicity of the sulfur‐containing side chains. We conclude that any weakly polar interaction between the additional sulfur and the aromatic ring is too weak to detect or is masked by destabilizing contributions to the free energy of denaturation.
Conservation of solvent‐binding sites in 10 crystal forms of T4 lysozyme
Authors: Zhang X.‐J. , Matthews B.W.
Published: February 2009

Solvent‐binding sites were compared in 10 different crystal forms of phage T4 lysozyme that were refined using data from 2.6 Å to 1.7 Å resolution. The sample included 18 crystallographically independent lysozyme molecules.

Despite different crystallization conditions, variable crystal contacts, changes due to mutation, and varying attention to solvent during crystallographic refinement, 62% of the 20 most frequently occupied sites were conserved. Allowing for potential steric interference from neighboring molecules in the crystal lattice, this fraction increased to 79% of the sites. There was, however, no solvent‐binding site that was occupied in all 18 lysozyme molecules. A buried double site was occupied in 17 instances and 2 other internal sites were occupied 15 times. Apart from these buried sites, the most frequently occupied sites were often at the amino‐termini of α‐helices. Solvent molecules at the most conserved sites tended to have crystallographic thermal factors lower than average, but atoms with low B‐factors were not restricted to these sites.

Although superficial inspection may suggest that only 50–60% (or less) of solvent‐binding sites are conserved in different crystal forms of a protein, it appears that many sites appear to be empty either because of steric interference or because the apparent occupancy of a given site can vary from crystal to crystal. The X‐ray method of identifying sites is somewhat subjective and tends to result in specification only of those solvent molecules that are well ordered and bound with high occupancy, even though there is clear evidence for solvent bound at many additional sites. Because similar sites are occupied under a variety of crystallization conditions, it seems likely that binding of solvent to such sites will be maintained in solution under physiological conditions.
A conformational change in the lactose permease of Escherichia coli is induced by ligand binding or membrane potential
Authors: Jung Heinrich , Jung Kirsten , Kaback H. Ronald
Published: February 2009

Lactose transport in membrane vesicles containing lactose permease with a single Cys residue in place of Val 315 is inactivated by N‐ethylmaleimide in a manner that is stimulated by substrate or by a H+ electrochemical gradient (δμ, Sahin‐Tóth M, Kaback HR, 1993, Protein Sci 2:1024–1033). The findings are confirmed and extended in this communication. Purified, reconstituted Val 315Ψ Cys permease reacts with N‐ethylmaleimide or hydrophobic fluorescent maleimides but not with a membrane impermeant thiol reagent, and β‐galactosides specifically stimulate the rate of labeling. Furthermore, the reactivity of purified Val 315 Ψ Cys permease is enhanced by imposition of a membrane potential (δΨ, interior negative). The results indicate that either ligand binding or ΔΨ induces a conformational change in the permease that brings the N‐terminus of helix X into an environment that is more accessible from the lipid phase.
Engineering of betabellin 14D: Disulfide‐induced folding of a β‐sheet protein
Authors: Yan Yibing , Erickson Bruce W.
Published: February 2009

The betabellin target structure consists of 2 32‐residue β sheets packed against each other by hydrophobic interactions. We have designed, chemically synthesized, and biophysically characterized betabellin 14S, a single chain, and betabellin 14D, the disulfide‐bridged double chain. The 32‐residue nongenetic betabellin‐14 chain (HSLTASIkaLTIHVQakTATCQVkaYTVHISE, a = D‐Ala, k = D‐Lys) has a palindromic pattern of polar (p), nonpolar (n), end (e), and β‐turn (t, r) residues (epnpnpnttnpnpnprrpnpnpnttnpnpnpe). Each half contains the same 14‐residue palindromic pattern (underlined). Pairs of D‐amino acid residues are used to favor formation of inverse‐common (type‐I') β turns. In water at pH 6.5, the single chain of betabellin 14S is not folded, but the disulfide‐linked betabellin 14D is folded into a stable β‐sheet structure. Thus, folding of the covalent dimer betabellin 14D is induced by formation of the single interchain disulfide bond. The binary pattern of alternating polar and nonpolar residues of its β sheets is not sufficient to induce folding. Betabellin 14D is a very water‐soluble (10 mg/mL), small (64 residues), nongenetic (12 D residues) β‐sheet protein with properties (well‐dispersed proton NMR resonances; Tm = 58 °C and δHm = 106 kcal/mol at pH 5.5) like those of a native protein structure.
Unexpected sequence similarity between nucleosidases and phosphoribosyltransferases of different specificity
Authors: Mushegian Arcady R. , Koonin Eugene V.
Published: February 2009

Amino acid sequences of enzymes that catalyze hydrolysis or phosphorolysis of the N‐glycosidic bond in nucleosides and nucleotides (nucleosidases and phosphoribosyltransferases) were explored using computer methods for database similarity search and multiple alignment. Two new families, each including bacterial and eukaryotic enzymes, were identified. Family I consists of Escherichia coli AMP hydrolase (Amn), uridine phosphorylase (Udp), purine phosphorylase (DeoD), uncharacterized proteins from E. coli and Bacteroides uniformis, and, unexpectedly, a group of plant stress‐inducible proteins. It is hypothesized that these plant proteins have evolved from nucleosidases and may possess nucleosidase activity. The proteins in this new family contain 3 conserved motifs, one of which was found also in eukaryotic purine nucleosidases, where it corresponds to the nucleoside‐binding site. Family II is comprised of bacterial and eukaryotic thymidine phosphorylases and anthranilate phosphoribosyltransferases, the relationship between which has not been suspected previously. Based on the known tertiary structure of E. coli thymidine phosphorylase, structural interpretation was given to the sequence conservation in this family. The highest conservation is observed in the N‐terminal α‐helical domain, whose exact function is not known. Parts of the conserved active site of thymidine phosphorylases and anthranilate phosphoribosyltransferases were delineated. A motif in the putative phosphate‐binding site is conserved in family II and in other phosphoribosyltransferases. Our analysis suggests that certain enzymes of very similar specificity, e.g., uridine and thymidine phosphorylases, could have evolved independently. In contrast, enzymes catalyzing such different reactions as AMP hydrolysis and uridine phosphorolysis or thymidine phosphorolysis and phosphoribosyl anthranilate synthesis are likely to have evolved from common ancestors.
Self‐splicing group I and group II introns encode homologous (putative) DNA endonucleases of a new family
Authors: Gorbalenya Alexander E.
Published: February 2009

A new family of protein domains consisting of 50–80 amino acid residues is described. It is composed of nearly 40 members, including domains encoded by plastid and phage group I introns; mitochondrial, plastid, and bacterial group II introns; eubacterial genomes and plasmids; and phages. The name “EX1H‐HX3H” was coined for both domain and family. It is based on 2 most prominent amino acid sequence motifs, each encompassing a pair of highly conserved histidine residues in a specific arrangement: EX1HH and HX3H. The “His” motifs often alternate with amino‐ and carboxy‐terminal motifs of a new type of Zn‐finger‐like structure CX2, 4CX29–54[CH]X2, 3[CH]. The EX1HH‐HX3H domain in eubacterial E2‐type bacteriocins and in phage RB3 (wild variant of phage T4) product of the nrdB group I intron was reported to be essential for DNA endonuclease activity of these proteins. In other proteins, the EX1HH‐EX3H domain is hypothesized to possess DNase activity as well. Presumably, this activity promotes movement (rearrangement) of group I and group II introns encoding the EX1HH‐HX3H domain and other gene targets. In the case of Escherichia coli restrictase McrA and possibly several related proteins, it appears to mediate the restriction of alien DNA molecules.
Multiple conformations of the sea anemone polypeptide anthopleurin‐A in solution
Authors: Scanlon Martin J. , Norton Raymond S.
Published: February 2009

Anthopleurin‐A (AP‐A) is a member of a family of sea anemone‐derived polypeptides that interact with sodium channels in a voltage‐dependent manner, producing a positive inotropic effect on the mammalian heart. There has been considerable interest in this molecule as a lead compound for the development of novel therapeutic agents. Earlier attempts to define the 3‐dimensional structure of AP‐A were complicated by the fact that it was found to exist in 2 conformations in solution. Using1H‐ and13C‐NMR spectroscopy, we have now shown that this conformational heterogeneity arises fromcis–transisomerization about the Gly 40–Pro 41 peptide bond and that in the major form of the protein this peptide bond adopts acisconformation. Furthermore, the increased sensitivity afforded by higher‐field NMR has allowed identification of additional minor conformations of AP‐A, the origin of which is presently unknown. We believe there will be many more examples of the detection by high‐field NMR of previously unobserved minor conformations of proteins in solution.
Crystallization and preliminary X‐ray crystallographic studies of UDP‐N‐acetylenolpyruvylglucosamine reductase
Authors: Benson Timothy E. , Walsh Christopher T. , Hogle James M.
Published: February 2009

The overexpression and purification of the second enzyme in Escherichia coli peptidoglycan biosynthesis, UDP‐N‐acetylenolpyruvylglucosamine reductase (MurB), provided sufficient protein to undertake crystallization and X‐ray crystallographic studies of the enzyme. MurB crystallizes in 14–20% PEG 8000, 100 mM sodium cacodylate, pH 8.0, and 200 mM calcium acetate in the presence of its substrate UDP‐N‐acetylglucosamine enolpyruvate. Crystals of MurB belong to the tetragonal space group P41212 with a = b = 49.6 Å, c = 263.2 Å, and α = β = γ = 90° at –160 °C and diffract to at least 2.5 Å. Screening for heavy atom derivatives has yielded a single site that is reactive with both methylmercury nitrate and Thimerosal.
Mouse liver NAD(P)H:quinone acceptor oxidoreductase: Protein sequence analysis by tandem mass spectrometry, cDNA cloning, expression inEscherichia coli, and enzyme activity analysis
Authors: Chen Shiuan , Clarke Patrick E. , Martino Paul A. , Deng Paulis S. K. , Yeh Che‐Hung , Lee Terry D. , Prochaska H. J. , Talalay Paul
Published: February 2009

The amino acid sequence of mouse liver NAD(P)H:quinone acceptor oxidoreductase (EC 1.6.99.2) has been determined by tandem mass spectrometry and deduced from the nucleotide sequence of the cDNA encoding for the enzyme. The electrospray mass spectral analyses revealed, as previously reported (Prochaska HJ, Talalay P, 1986,J Biol Chem 261:1372–1378), that the 2 forms – the hydrophilic and hydrophobic forms – of the mouse liver quinone reductase have the same molecular weight. No amino acid sequence differences were found by tandem mass spectral analyses of tryptic peptides of the 2 forms. Moreover, the amino‐termini of the mouse enzymes are acetylated as determined by tandem mass spectrometry. Further, only 1 cDNA species encoding for the quinone reductase was found. These results suggest that the 2 forms of the mouse quinone reductase have the same primary sequences, and that any difference between the 2 forms may be attributed to a labile posttranslational modification.

Analysis of the mouse quinone reductase cDNA revealed that the enzyme is 273 amino acids long and has a sequence homologous to those of rat and human quinone reductases. In this study, the mouse quinone reductase cDNA was also ligated into a prokaryotic expression plasmid pKK233.2, and the constructed plasmid was used to transformEscherichia colistrain JM109. TheE. coli‐expressed mouse quinone reductase was purified and characterized. Although mouse quinone reductase has an amino acid sequence similar to those of the rat and human enzymes, the mouse enzyme has a higher NAD(P)H‐menadione reductase activity and is less sensitive to flavones and dicoumarol, 2 known inhibitors of the enzyme. The results would indicate that the regions in mouse quinone reductase that contain amino acids different from the rat and human enzymes are critical for the binding of menadione, flavones, and dicoumarol.
Structure and functions of arrestins
Authors: Palczewski Krzysztof
Published: February 2009

Transmembrane signal transductions in a variety of cell types that mediate signals as diverse as those carried by neurotransmitters, hormones, and sensory signals share basic biochemical mechanisms that include: (1) an extracellular perturbation (neurotransmitter, hormone, odor, light); (2) specific receptors; (3) coupling proteins, such as G proteins; and (4) effector enzymes or ion channels. Parallel to these amplification reactions, receptors are precisely inactivated by mechanisms that involve protein kinases and regulatory proteins called arrestins. The structure and functions of arrestins are the focus of this review.
Subunit interface mutants of rabbit muscle aldolase form active dimers
Authors: Beernink Peter T. , Tolan Dean R.
Published: February 2009

We report the construction of subunit interface mutants of rabbit muscle aldolase A with altered quaternary structure. A mutation has been described that causes nonspherocytic hemolytic anemia and produces a thermolabile aldolase (Kishi H et al., 1987, Proc Natl Acad Sci USA 84:8623–8627). The disease arises from substitution of Gly for Asp‐128, a residue at the subunit interface of human aldolase A. To elucidate the role of this residue in the highly homologous rabbit aldolase A, site‐directed mutagenesis is used to replace Asp‐128 with Gly, Ala, Asn, Gln, or Val. Rabbit aldolase D128G purified from Escherichia coli is found to be similar to human D128G by kinetic analysis, CD, and thermal inactivation assays. All of the mutant rabbit aldolases are similar to the wild‐type rabbit enzyme in secondary structure and kinetic properties. In contrast, whereas the wild‐type enzyme is a tetramer, chemical crosslinking and gel filtration indicate that a new dimeric species exists for the mutants. In sedimentation velocity experiments, the mutant enzymes exist as mixtures of dimer and tetramer at 4 °C. Sedimentation at 20 °C shows that the mutant enzymes are >99.5% dimeric and, in the presence of substrate, that the dimeric species is active. Differential scanning calorimetry demonstrates that Tm values of the mutant enzymes are decreased by 12 °C compared to wild‐type enzyme. The results indicate that Asp‐128 is important for interface stability and suggest that 1 role of the quaternary structure of aldolase is to provide thermostability.
The NMR solution structure of the pheromone Er‐1 from the ciliated protozoan Euplotes raikovi—NMR structure of the pheromone Er‐I
Authors: Mronga S. , Luginbühl P. , Brown L. R. , Ortenzi C. , Luporini P. , Bradshaw R. A. , WÜthrich K.
Published: February 2009

The 3‐dimensional structure of the pheromone Er‐1 isolated from the ciliated protozoan Euplotes raikovi has been determined in aqueous solution by 1H NMR spectroscopy. The structure of this 40‐residue protein was calculated with the distance geometry program DIANA on the basis of 503 upper distance constraints derived from nuclear Overhauser effects and 77 dihedral angle constraints derived from spin—spin coupling constants, and refined by restrained energy minimization with the program OPAL. The Er‐1 solution structure is represented by a group of 20 conformers with an average RMS deviation relative to the mean structure of 0.55 Å for the backbone atoms N, Cα, and C', and 0.93 Å for all heavy atoms of the complete polypeptide chain, residues 1–40. The molecular architecture is dominated by an up‐down‐up bundle of 3 α‐helices formed by residues 2–9, 12–19, and 24–33. Although this core part coincides closely with the previously determined structure of the homologous pheromone Er‐10, the C‐terminal peptide segment adopts a novel conformation. This is of interest in view of previous suggestions, based on sequence comparisons, that this molecular region may be important for the different specificity of receptor recognition by different pheromones.