bluuebunny/crossref_metadata_2025 huggingface dataset:
Target recognition by calmodulin: Dissecting the kinetics and affinity of interaction using short peptide sequences
Authors: Bayley P.M. , Findlay W.A. , Martin S.R.
Published: February 2009

The interaction between calmodulin (CaM) and peptide M13, its target binding sequence from skeletal muscle myosin light chain kinase, involves predominantly two sets of interactions, between the N‐terminal target residues and the C‐domain of calmodulin, and between the C‐terminal target residues and the N‐domain of calmodulin (Ikura M et al., 1992, Science 256:632–638). Using short synthetic peptides based on the two halves of the target sequence, the interactions with calmodulin and its separate C‐domain have been studied by fluorescence and CD spectroscopy, calcium binding, and kinetic techniques. Peptide WF10 (residues 1–10 of M13) binds to CaM with Kd ≈︁ 1 μM; peptide FW10 (residues 9–18 of M13, with Phe‐17 → Trp substitution) binds to CaM with Kd ≈︁ 100 μM. The effect of peptide WF10 on calcium binding to calmodulin produces a biphasic saturation curve, with marked enhancement of affinity for the binding of two calcium ions to the C‐domain, forming a stable half‐saturated complex, Ca2‐CaM‐peptide, and confirming the functional importance of the interaction of this sequence with the C‐domain. Stopped‐flow studies show that the EGTA‐induced dissociation of WF10 from Ca4‐CaM proceeds by a reversible relaxation mechanism from a kinetic intermediate state, also involving half‐saturation of CaM, and the same mechanism is evident for the full target peptide. Interaction of the N‐terminal target residues with the C‐domain is energetically the most important component, but interaction of calmodulin with the whole target sequence is necessary to induce the full cooperative interaction of the two contiguous elements of the target sequence with both N‐ and C‐domains of calmodulin. Thus, the interaction of calmodulin with the M13 sequence can be dissected on both a structural and kinetic basis into partial reactions involving intermediates comprising distinct regions of the target sequence. We propose a general mechanism for the calcium regulation of calmodulin‐dependent enzyme activation, involving an intermediate complex formed by interaction of the calmodulin C‐domain and the corresponding part of the target sequence. This intermediate species can function to regulate the overall calcium sensitivity of activation and to determine the affinity of the calmodulin target interaction.
The structural alignment between two proteins: Is there a unique answer?
Authors: Godzik Adam
Published: February 2009

Structurally similar but sequentially unrelated proteins have been discovered and rediscovered by many researchers, using a variety of structure comparison tools. For several pairs of such proteins, existing structural alignments obtained from the literature, as well as alignments prepared using several different similarity criteria, are compared with each other. It is shown that, in general, they differ from each other, with differences increasing with diminishing sequence similarity. Differences are particularly strong between alignments optimizing global similarity measures, such as RMS deviation between Cα atoms, and alignments focusing on more local features, such as packing or interaction pattern similarity. Simply speaking, by putting emphasis on different aspects of structure, different structural alignments show the unquestionable similarity in a different way. With differences between various alignments extending to a point where they can differ at all positions, analysis of structural similarities leads to contradictory results reported by groups using different alignment techniques.

The problem of uniqueness and stability of structural alignments is further studied with the help of visualization of the suboptimal alignments. It is shown that alignments are often degenerate and whole families of alignments can be generated with almost the same score as the “optimal alignment.” However, for some similarity criteria, specially those based on side‐chain positions, rather than Cα positions, alignments in some areas of the protein are unique. This opens the question of how and if the structural alignments can be used as “standards of truth” for protein comparison.
Ala‐insertion scanning mutagenesis of the glycophorin a transmembrane helix: A rapid way to map helix‐helix interactions in integral membrane proteins
Authors: Mingarro Ismael , Whitley Paul , Heijne Gunnar Von , Lemmon Mark A.
Published: February 2009

Alanine insertions into the glycophorin A transmembrane helix are found to disrupt helix‐helix dimerization in a way that is fully consistent with earlier saturation mutagenesis data, suggesting that Ala‐insertion scanning can be used to rapidly map the approximate location of structurally and/or functionally important segments in transmembrane helices.
Novel isolation method and structural stability of a eukaryotic chaperonin: The TCP‐1 ring complex from rabbit reticulocytes
Authors: Norcum Mona Trempe
Published: February 2009

In the course of removing a contaminant from preparations of aminoacyl‐tRNA synthetase complexes, a novel purification method has been developed for the eukaryotic cytoplasmic chaperonin known as TRiC or CCT. This method uses only three steps: ammonium sulfate precipitation, pelleting into a sucrose cushion, and heparin‐agarose chromatography. As judged by electrophoresis, sedimentation, and electron microscopy, the preparations are homogeneous. The particle is identified as a chaperonin from electrophoretic polypeptide pattern, electron microscopic images, direct mass measurement by sedimentation velocity analysis, amino‐terminal sequencing, and ATP‐dependent refolding of rhodanese and actin.

Further investigation of the biochemical and physical properties of the particle demonstrates that its constituent polypeptides are not glycosylated. The particle as a whole binds strongly to polyanionic matrices. Of particular note is that negatively stained images of chaperonin adsorbed to a single carbon layer are distinctly different from those where it is sandwiched between two layers. In the former, the “characteristic” ring and four‐stripe barrel predominate. In the latter, most images are round with a highly reticulated surface, the average particle diameter increases from 15 to 18 nm, and additional side, end, and substrate‐containing views are observed. The particle structure is strikingly resistant to physical forces (long‐term storage, repeated cycles of freezing and thawing, sedimentation), detergents (Triton, deoxycholate), salts (molar levels of KC1 or LiCl), and pH changes (9‐6). Only a strongly chaotropic salt (NaSCN) and extremely acidic conditions (pH 4.5) cause aggregation and dissociation of TRiC, respectively. However, treatment with KC1 or deoxycholate reduces TRiC folding activity.
Conformational stability of apoflavodoxin
Authors: Genzor Carlos G. , Gómez‐Moreno Carlos , Sancho Javier , Beldarraín Alejandro , López‐Lacomba José Luis , Cortijo Manuel
Published: February 2009

Flavodoxins areα/βproteins that mediate electron transfer reactions. The conformational stability of apoflavodoxin fromAnabœnaPCC 7119 has been studied by calorimetry and urea denaturation as a function of pH and ionic strength. At pH > 12, the protein is unfolded. Between pH 11 and pH 6, the apoprotein is folded properly as judged from near‐ultraviolet (UV) circular dichroism (CD) and high‐field1H NMR spectra. In this pH interval, apoflavodoxin is a monomer and its unfolding by urea or temperature follows a simple two‐state mechanism. The specific heat capacity of unfolding for this native conformation is unusually low. Near its isoelectric point (3.9), the protein is highly insoluble. At lower pH values (pH 3.5‐2.0), apoflavodoxin adopts a conformation with the properties of a molten globule. Although apoflavodoxin at pH 2 unfolds cooperatively with urea in a reversible fashion and the fluorescence and far‐UV CD unfolding curves coincide, the transition midpoint depends on the concentration of protein, ruling out a simple two‐state process at acidic pH. Apoflavodoxin constitutes a promising system for the analysis of the stability and folding ofα/βproteins and for the study of the interaction between apoflavoproteins and their corresponding redox cofactors.
Membrane‐bound states of α‐lactalbumin: Implications for the protein stability and conformation
Authors: Cawthern Kevin M. , Permyakov Eugene , Berliner Lawrence J.
Published: February 2009

α‐Lactalbumin (α‐LA) associates with dimyristoylphosphatidylcholine (DMPC) or egg lecithin (EPC) liposomes. Thermal denaturation of isolated DMPC or EPC α‐LA complexes was dependent on the metal bound state of the protein. The intrinsic fluorescence of thermally denatured DMPC‐α‐LA was sensitive to two thermal transitions: the Tc of the lipid vesicles, and the denaturation of the protein. Quenching experiments suggested that tryptophan accessibility increased upon protein‐DMPC association, in contrast with earlier suggestions that the limited emission red shift upon association with the liposome was due to partial insertion of tryptophan into the apolar phase of the bilayer (Hanssens I et al., 1985, Biochim Biophys Acta 817:154‐166). On the other hand, above the protein transition (70 °C), the spectral blue shifts and reduced accessibility to quencher suggested that tryptophan interacts significantly with the apolar phase of either DMPC and EPC. At pH 2, where the protein inserts into the bilayer rapidly, the isolated DMPC‐α‐LA complex showed a distinct fluorescence thermal transition between 40 and 60 °C, consistent with a partially inserted form that possesses some degree of tertiary structure and unfolds cooperatively. This result is significant in light of earlier findings of increased helicity for the acid form, i.e., molten globule state of the protein (Hanssens I et al., 1985, Biochim Biophys Acta 817:154‐166). These results suggest a model where a limited expansion of conformation occurs upon association with the membrane at neutral pH and physiological temperatures, with a concomitant increase in the exposure of tryptophan to external quenchers; i.e., the current data do not support a model where an apolar, tryptophan‐containing surface is covered by the lipid phase of the bilayer.
Experimentally observed conformation‐dependent geometry and hidden strain in proteins
Authors: Karplus P. Andrew
Published: February 2009

A database has been compiled documenting the peptide conformations and geometries from 70 diverse proteins refined at 1.75 Å or better. Analysis of the well‐ordered residues within the database shows ϕ, Ψ‐distributions that have more fine structure than is generally observed. Also, clear evidence is presented that the peptide covalent geometry depends on conformation, with the interpeptide N‐Cα‐C bond angle varying by nearly ±5 degrees from its standard value. The observed deviations from standard peptide geometry are greatest near the edges of well‐populated regions, consistent with strain occurring in these conformations. Minimization of such hidden strain could be an important factor in thermostability of proteins. These empirical data describing how equilibrium peptide geometry varies as a function of conformation confirm and extend quantum mechanics calculations, and have predictive value that will aid both theoretical and experimental analyses of protein structure.
Crystallization, X‐ray studies, and site‐directed cysteine mutagenesis of the DNA‐binding domain of OmpR
Authors: Martinez‐Hackert Erik , Berman Helen M. , Harlocker Susan , Inouye Masayori , Stock Ann M.
Published: February 2009

A C‐terminal fragment of the transcription factor OmpR has been crystallized using the sitting drop vapor‐diffusion method. Crystals belong to the trigonal spacegroup P3n12 with cell dimensions a = b = 54.4 Å, c = 135.5 Å, and γ = 120.00°. A second crystal form has been obtained by soaking this crystal form in a cryo‐buffer and flash‐cooling to 108 K in a cold nitrogen stream. Crystals belong to the trigonal space‐group P3n12 with cell dimensions a = b = 108.07 Å, c = 131.81Å, and γ = 120.00°. Both crystal forms diffract to at least 2.3 Å at a synchrotron light source. Single‐site cysteine mutations have been introduced to provide mercury‐binding sites for multiple isomorphous replacement.
Protein folding in the endoplasmic reticulum: Lessons from the human chorionic gonadotropin β subunit
Authors: Ruddon Raymond W. , Sherman Simon A. , Bedows Elliott
Published: February 2009

There have been few studies of protein folding in the endoplasmic reticulum of intact mammalian cells. In the one case where the in vivo and in vitro folding pathways of a mammalian secretory protein have been compared, the folding of the human chorionic gonadotropin β subunit (hCG‐β), the order of formation of the detected folding intermediates is the same. The rate and efficiency with which multidomain proteins such as hCG‐β fold to native structure in intact cells is higher than in vitro, although intracellular rates of folding of the β subunit can be approached in vitro in the presence of an optimal redox potential and protein disulfide isomerase. Understanding how proteins fold in vivo may provide a new way to diagnose and treat human illnesses that occur due to folding defects.
Structure of equine infectious anemia virus proteinase complexed with an inhibitor
Authors: Gustchina Alla , Kervinen Jukka , Powell David J. , Zdanov Alexander , Kay John , Wlodawer Alexander
Published: February 2009

Equine infectious anemia virus (EIAV), the causative agent of infectious anemia in horses, is a member of the lentiviral family. The virus‐encoded proteinase (PR) processes viral polyproteins into functional molecules during replication and it also cleaves viral nucleocapsid protein during infection. The X‐ray structure of a complex of the I54G mutant of EIAV PR with the inhibitor HBY‐793 was solved at 1.8 Å resolution and refined to a crystallographic R‐factor of 0.136. The molecule is a dimer in which the monomers are related by a crystallographic twofold axis. Although both the enzyme and the inhibitor are symmetric, the interactions between the central part of the inhibitor and the active site aspartates are asymmetric, and the inhibitor and the two flaps are partially disordered. The overall fold of EIAV PR is very similar to that of other retroviral proteinases. However, a novel feature of the EIAV PR structure is the appearance of the second α‐helix in the monomer in a position predicted by the structural template for the family of aspartic proteinases. The parts of the EIAV PR with the highest resemblance to human immunodeficiency virus type 1 PR include the substrate‐binding sites; thus, the differences in the specificity of both enzymes have to be explained by enzyme‐ligand interactions at the periphery of the active site as well.
A catalytic function for the structurally conserved residue Phe 100 of ribonuclease T1
Authors: Doumen Jan , Gonciarz Malgorzata , Zegers Ingrid , Loris Remy , Wyns Lode , Steyaert Jan
Published: February 2009

The function of the conserved Phe 100 residue of RNase T1 (EC 3.1.27.3) has been investigated by site‐directed mutagenesis and X‐ray crystallography. Replacement of Phe 100 by alanine results in a mutant enzyme with kcat reduced 75‐fold and a small increase in Km for the dinucleoside phosphate substrate GpC. The Phe 100 Ala substitution has similar effects on the turnover rates of GpC and its minimal analogue GpOMe, in which the leaving cytidine is replaced by methanol. The contribution to catalysis is independent of the nature of the leaving group, indicating that Phe 100 belongs to the primary site. The contribution of Phe 100 to catalysis may result from a direct van der Waals contact between its aromatic ring and the phosphate moiety of the substrate. Phe 100 may also contribute to the positioning of the pentacovalent phosphorus of the transition state, relative to other catalytic residues. If compared to the corresponding wild‐type data, the structural implications of the mutation in the present crystal structure of Phe 100 Ala RNase T1 complexed with the specific inhibitor 2′‐GMP are restricted to the active site. Repositioning of 2′‐GMP, caused by the Phe 100 Ala mutation, generates new or improved contacts of the phosphate moiety with Arg 77 and His 92. In contrast, interactions with the Glu 58 carboxylate appear to be weakened. The effects of the His 92 Gln and Phe 100 Ala mutations on GpC turnover are additive in the corresponding double mutant, indicating that the contribution of Phe 100 to catalysis is independent of the catalytic acid His 92. The present results lead to the conclusion that apolar residues may contribute considerably to catalyze conversions of charged molecules to charged products, involving even more polar transition states.
RhNGF slow unfolding is not due to proline isomerization: Possibility of a cystine knot loop‐threading mechanism
Authors: de Young Linda R. , Burton Louis E. , Liu Jun , Powell Michael F. , Schmelzer Charles H. , Skelton Nicholas J.
Published: February 2009

The unfolding of recombinant human β‐NGF (NGF) in guanidine hydrochloride (GdnHCl) was found to be time dependent with the denaturation midpoint moving to lower GdnHCl concentration over time. Dissociation and extensive unfolding of the NGF dimer occurred rapidly in 5 M GdnHCl, but further unfolding of the molecule occurred over many days at 25 °C. Fluorescence spectroscopy, size‐exclusion and reversed‐phase HPLC, ultra‐centrifugation, and proton NMR spectroscopy were used to ascertain that the slow unfolding step was between two denatured monomeric states of NGF (M1 and M2). Proton NMR showed the monomer formed at early times in GdnHCl (M1) had little β‐sheet structure, but retained residual structure in the tryptophan indole and high‐field methyl regions of the spectrum. This residual structure was lost after prolonged incubation in GdnHCl giving a more fully unfolded monomer, M2. From kinetic unfolding experiments in 5 M GdnHCl it was determined that the conversion of M1 to M2 had an activation energy of 26.5 kcal/mol, a half‐life of 23 h at 25 °C, and the rate of formation of M2 was dependent on the GdnHCl concentration between 5 and 7.1 M GdnHCl. These properties of the slow unfolding step are inconsistent with a proline isomerization mechanism. The rate of formation of the slow folding monomer M2 increases with truncation of five and nine amino acids from the NGF N‐terminus. A model for the slow unfolding reaction is proposed where the N‐terminus threads through the cystine knot to form M2, a loop‐threading reaction, increasing the conformational freedom of the denatured state.
Solid‐state NMR studies of the prion protein H1 fragment
Authors: Heller Jonathan , Kolbert Andrew C. , Larsen Russell , Ernst Matthias , Bekker Tatiana , Baldwin Michael , Prusiner Stanley B. , Pines Alexander , Wemmer David E.
Published: February 2009

Conformational changes in the prion protein (PrP) seem to be responsible for prion diseases. We have used conformation‐dependent chemical‐shift measurements and rotational‐resonance distance measurements to analyze the conformation of solid‐state peptides lacking long‐range order, corresponding to a region of PrP designated H1. This region is predicted to undergo a transformation of secondary structure in generating the infectious form of the protein. Solid‐state NMR spectra of specifically 13C‐enriched samples of H1, residues 109–122 (MKHMAGAAAAGAVV) of Syrian hamster PrP, have been acquired under cross‐polarization and magic‐angle spinning conditions. Samples lyophilized from 50% acetonitrile/50% water show chemical shifts characteristic of a β‐sheet conformation in the region corresponding to residues 112–121, whereas samples lyophilized from hexa‐fluoroisopropanol display shifts indicative of α‐helical secondary structure in the region corresponding to residues 113–117. Complete conversion to the helical conformation was not observed and conversion from α‐helix back to β‐sheet, as inferred from the solid‐state NMR spectra, occurred when samples were exposed to water. Rotational‐resonance experiments were performed on seven doubly 13C‐labeled H1 samples dried from water. Measured distances suggest that the peptide is in an extended, possibly β‐strand, conformation. These results are consistent with the experimental observation that PrP can exist in different conformational states and with structural predictions based on biological data and theoretical modeling that suggest that H1 may play a key role in the conformational transition involved in the development of prion diseases.
Models for the 310‐helix/coil, π‐helix/coil, and α‐helix/310‐helix/coil transitions in isolated peptides
Authors: Rohl Carol A. , Doig Andrew J.
Published: February 2009

Models for the 310‐helix/coil and π‐helix/coil equilibria have been derived. The theory is based on classifying residues into helical or nonhelical (coil) conformations. Statistical weights are assigned to residues in a helical conformation with an associated helical hydrogen bond, a helical conformation with no hydrogen bond, an N‐cap position, a C‐cap position, or the reference coil conformation. The models for α‐helix formation and 310‐helix formation have also been combined to describe a three‐state equilibrium in which α‐helical, 310‐helical, and coil conformations are populated. The results are compared with the modified Lifson‐Roig theory for the α‐helix/coil equilibrium. The comparison accounts for the experimental observations that 310‐helices tend to be short and π‐helices are not favored for any length. This work may provide a framework for quantitatively rationalizing experimental work on isolated 310‐helices and mixed 310‐/α‐helices.
Topology prediction for helical transmembrane proteins at 86% accuracy–Topology prediction at 86% accuracy
Authors: Rost Burkhard , Fariselli Piero , Casadio Rita
Published: February 2009

Previously, we introduced a neural network system predicting locations of transmembrane helices (HTMs) based on evolutionary profiles (PHDhtm, Rost B, Casadio R, Fariselli P, Sander C, 1995, Protein Sci 4:521–533). Here, we describe an improvement and an extension of that system. The improvement is achieved by a dynamic programming‐like algorithm that optimizes helices compatible with the neural network output. The extension is the prediction of topology (orientation of first loop region with respect to membrane) by applying to the refined prediction the observation that positively charged residues are more abundant in extra‐cytoplasmic regions. Furthermore, we introduce a method to reduce the number of false positives, i.e., proteins falsely predicted with membrane helices. The evaluation of prediction accuracy is based on a cross‐validation and a double‐blind test set (in total 131 proteins). The final method appears to be more accurate than other methods published: (1) For almost 89% (π3%) of the test proteins, all HTMs are predicted correctly. (2) For more than 86% (π3%) of the proteins, topology is predicted correctly. (3) We define reliability indices that correlate with prediction accuracy: for one half of the proteins, segment accuracy raises to 98%; and for two‐thirds, accuracy of topology prediction is 95%. (4) The rate of proteins for which HTMs are predicted falsely is below 2% (π1%). Finally, the method is applied to 1,616 sequences of Haemophilus influenzae. We predict 19% of the genome sequences to contain one or more HTMs. This appears to be lower than what we predicted previously for the yeast VIII chromosome (about 25%).
Covalent structure of the flavoprotein subunit of the flavocytochrome c: Sulfide dehydrogenase from the purple phototrophic bacterium chromatium vinosum
Authors: Driessche Gonzalez Van , Koh Monjoo , Van Beeumen Jozef J. , Chen Zhi‐Wei , Mathews F. Scott , Meyer Terry E. , Bartsch Robert G. , Cusanovich Michael A.
Published: February 2009

The amino acid sequence of the flavoprotein subunit of Chromatium vinosum flavocytochrome c—sulftde dehydrogenase (FCSD) was determined by automated Edman degradation and mass spectrometry in conjunction with the three‐dimensional structure determination (Chen Z et al., 1994, Science 266:430–432). The sequence of the diheme cytochrome c subunit was determined previously. The flavoprotein contains 401 residues and has a calculated protein mass, including FAD, of 43,568 Da, compared with a mass of 43,652 ± 44 Da measured by LDMS. There are six cysteine residues, among which Cys 42 provides the site of covalent attachment of the FAD. Cys 161 and Cys 337 form a disulfide bond adjacent to the FAD. The flavoprotein subunit of FCSD is most closely related to glutathione reductase (GR) in three‐dimensional structure and, like that protein, contains three domains. However, approximately 20 insertions and deletions are necessary for alignment and the overall identity in sequence is not significantly greater than for random comparisons. The first domain binds FAD in both proteins. Domain 2 of GR is the site of NADP binding, but has an unknown role in FCSD. We postulate that it is the binding site for a cofactor involved in oxidation of reduced sulfur compounds. Domains 1 and 2 of FCSD, as of GR, are homologous to one another and represent an ancient gene doubling. The third domain provides the dimerization interface for GR, but is the site of binding of the cytochrome subunit in FCSD. The four functional entities, predicted to be near the FAD from earlier studies of the kinetics of sulfite adduct formation and decay, have now been identified from the three‐dimensional structure and the sequence as Cys 161/Cys 337 disulfide, Tip 391, Glu 167, and the positive end of a helix dipole.
Crystal structure of the 2[4Fe‐4S] ferredoxin from Chromatium vinosum: Evolutionary and mechanistic inferences for [3/4Fe‐4S] ferredoxins
Authors: Moulis Jean‐Marc , Sieker Larry C. , Wilson Keith S. , Dauter Zbigniew
Published: February 2009

The crystal structure of the 2[4Fe‐4S] ferredoxin from Chromatium vinosum has been solved by molecular replacement using data recorded with synchrotron radiation. The crystals were hexagonal prisms that showed a strong tendency to develop into long tubes. The hexagonal prisms diffracted to 2.1 Å resolution at best, and a structural model for C. vinosum ferredoxin has been built with a final R of 19.2%. The N‐terminal domain coordinates the two [4Fe‐4S] clusters in a fold that is almost identical to that of other known ferredoxins. However, the structure has two unique features. One is a six‐residue insertion between two ligands of one cluster forming a two‐turn external loop; this short loop changes the conformation of the Cys 40 ligand compared to other ferredoxins and hampers the building of one NH—S H‐bond to one of the inorganic sulfurs. The other remarkable structural element is a 3.5‐turn α‐helix at the C‐terminus that covers one side of the same cluster and is linked to the cluster‐binding domain by a six‐residue external chain segment. The charge distribution is highly asymmetric over the molecule. The structure of C. vinosum ferredoxin strongly suggests divergent evolution for bacterial [3/4Fe‐4S] ferredoxins from a common ancestral cluster‐binding core. The unexpected slow intramolecular electron transfer rate between the clusters in C. vinosum ferredoxin, compared to other similar proteins, may be attributed to the unusual electronic properties of one of the clusters arising from localized changes in its vicinity rather than to a global structural rearrangement.
Peptide rescue of an N‐terminal truncation of the stoffel fragment of Taq DNA polymerase
Authors: Vainshtein Inna , Malcolm Bruce A. , Atrazhev Alexey , Elliott John F. , Eom Soo Hyun , Wishart David S.
Published: February 2009

Deletion of the first 289 amino acids of the DNA polymerase from Thermus aquaticus (Taq polymerase) removes the 5′ to 3′ exonuclease domain to yield the thermostable Stoffel polymerase fragment (Lawyer et al., 1989). Preliminary N‐terminal truncation studies of the Stoffel fragment suggested that removal of an additional 12 amino acids (the StofΔ12 mutant) had no significant effect on activity or stability, but that the further truncation of the protein (the StofΔ47, in which 47 amino acids were deleted), resulted in a significant loss of both activity and thermostability. A 33‐amino acid synthetic peptide, based on this critical region (i.e., residues 303–335 inclusive), was able to restore 85% of the StofΔ12 activity when added back to the truncated StofΔ47 protein as well as return the temperature optimum to that of the StofΔ12 and Stoffel proteins. Examination of the crystal structure of Taq polymerase (Kim et al., 1995) shows that residues 302–336 of the enzyme form a three‐stranded β‐sheet structure that interacts with the remainder of the protein. CD analysis of the 33‐amino acid peptide indicates that the free peptide also adopts an ordered structure in solution with more than 50% β‐sheet content. These data suggest that this 33‐amino acid peptide constitutes a stable β‐sheet structure capable of rescuing the truncated polymerase in a fashion analogous to the well‐documented complementation of Ribonuclease S protein by the 15‐residue, α‐helical, S peptide.
Circularly permuted dihydrofolate reductase possesses all the properties of the molten globule state, but can resume functional tertiary structure by interaction with its ligands
Authors: Uversky Vladimir N. , Protasova Natalya YU. , Rogov Vladimir V. , Vassilenko Konstantin S. , Gudkov Anatoly T. , Kutyshenko Viktor P.
Published: February 2009

It is obvious that functional activity of a protein molecule is closely related to its structure. On the other hand, the understanding of structure‐function relationship still remains one of the intriguing problems of molecular biology. There is widespread belief that mutagenesis presents a real way to solve this problem. Following this assumption, we have investigated the effect of circular permutation in dihydrofolate reductase from E. coli on protein structure and functioning. It has been shown that in the absence of ligands two circularly permuted variants of dihydrofolate reductase possess all the properties of the molten globule state. However, after addition of ligands they gain the native‐like structural properties and specific activity. This means that the in vitro folding of permuted dihydrofolate reductase is terminated at the stage of the molten globule formation. Interaction of permuted protein with ligands leads to the structural adjustment and formation of active protein molecules.
Two partially unfolded states oftorpedo californicaacetylcholinesterase
Authors: Kreimer David I. , Shin Irina , Silman Israel , Shnyrov Valery L. , Villar Enrique , Weiner Lev
Published: February 2009

Chemical modification with sulfhydryl reagents of the single, nonconserved cysteine residue Cys231in each subunit of a disulfide‐linked dimer ofTorpedo californicaacetylcholinesterase produces a partially unfolded inactive state. Another partially unfolded state can be obtained by exposure of the enzyme to 1–2 M guanidine hydrochloride. Both these states display several important features of a molten globule, but differ in their spectroscopic (CD, intrinsic fluorescence) and hydrodynamic (Stokes radii) characteristics. With reversal of chemical modification of the former state or removal of denaturant from the latter, both states retain their physicochemical characteristics. Thus, acetylcholinesterase can exist in two molten globule states, both of which are long‐lived under physiologic conditions without aggregating, and without either intraconverting or reverting to the native state. Both states undergo spontaneous intramolecular thiol‐disulfide exchange, implying that they are flexible. As revealed by differential scanning calorimetry, the state produced by chemical modification lacks any heat capacity peak, presumably due to aggregation during scanning, whereas the state produced by guanidine hydrochloride unfolds as a single cooperative unit, thermal transition being completely reversible. Sucrose gradient centrifugation reveals that reduction of the interchain disulfide of the native acetylcholinesterase dimer converts it to monomers, whereas, after such reduction, the two subunits remain completely associated in the partially unfolded state generated by guanidine hydrochloride, and partially associated in that produced by chemical modification. It is suggested that a novel hydrophobic core, generated across the subunit interfaces, is responsible for this noncovalent association. Transition from the unfolded state generated by chemical modification to that produced by guanidine hydrochloride is observed only in the presence of the denaturant, yielding, on extrapolation to zero guanidine hydrochloride, a high free energy barrier (ca. 23.8 kcal/mol) separating these two flexible, partially unfolded states.
Homology modeling of human methylmalonyl‐CoA mutase: A structural basis for point mutations causing methylmalonic aciduria
Authors: Thomäu Nicolas H. , Leadlay Peter F.
Published: February 2009

Point mutations in the human gene encoding coenzyme B12 (adenosylcobalamin)‐dependent methylmalonyl‐CoA mutase give rise to an inherited disorder of propionic acid metabolism termed mut methylmalonic aciduria. Almost all such mutations alter amino acids in the homodimeric human enzyme that are identical to residues in the catalytic α‐subunit of the heterodimeric methylmalonyl‐CoA mutase from the bacterium Propionibacte‐rium shermanii, to which the mature human enzyme shows an overall 65% sequence identity. To explore how specific mutations might cause the observed clinical phenotype, 12 known mutations were mapped onto a three‐dimensional homology model of the subunit of the human enzyme, generated using the program MODELLER on the basis of the recently published 2.0 A X‐ray crystal structure of the P. shermanii methylmalonyl‐CoA mutase. Eight mutations are found in the C‐terminal B12‐binding domain, of which 4 (G623R, G626C, G630E, G703R) are in direct contact with the corrin and are clustered around the histidine ligand (H627) provided by the protein to coordinate the cobalt atom of the B12 cofactor. Introduction of a side chain, particularly one that is charged, at any of these positions is expected to disrupt the flavodoxin‐like fold and severely impair its binding of B12. Mutation at either of two other highly conserved glycine residues in this domain (G648D, G717V) also disrupts critical elements in the fold as would the introduction of an additional positive charge in the mutation H678R. Mutation of an arginine in a solvent‐exposed loop to a hydrophobic residue (R694W) is also pathogenic. The remaining mutations have been mapped to the N‐terminal region of the mutase, two of which introduce a buried, uncompensated charge, either near the subunit interface (A377E), or near the narrow channel through which acyl‐CoA esters gain access to the active site (W105R). The extreme N‐terminus of methylmalonyl‐CoA mutase is predicted to make extensive contacts with the other subunit, and a mutant in this region (R93H) may prevent the correct assembly of the dimer.
Crystallization and preliminary X‐ray diffraction studies of the Lb proteinase from foot‐and‐mouth disease virus
Authors: Guarnéa Alba , Verdaguer Núaria , Fita Ignasi , Kirchweger Regina , Liebig Hans‐Dieter , Blaas Dieter , Skern Tim
Published: February 2009

Different crystal forms of the C23A mutant from the leader proteinase of foot‐and‐mouth disease virus were obtained by the hanging drop vapor diffusion technique, using MgCl2and PEG 6000 as precipitants. Well‐developed crystals, with cubic morphology growing to approximately 1.0 mm3in size, presented a large unit cell parameter of 274.5 Å and diffracted to, at most, 5 Å resolution. A second type of crystal had a tetragonal appearance and these were obtained in droplets soaked in a silica gel matrix. These crystals, with an approximate size of 0.3 × 0.3 × 0.7 mm3, diffracted to approximately 4.0 Å resolution, but presented a strong anisotropic mosaicity around the longest crystal axis. Crystals with a needlelike morphology and reaching sizes of about 0.2 × 0.3 × 1.2 mm3diffracted beyond 3.5 Å resolution and were stable to X‐ray radiation for approximately one day when using a conventional source at room temperature. These crystals are orthorhombic with space group 1222 (or I212121) and unit cell dimensionsa= 65.9 Å,b= 104.3 Å, andc= 124.0 Å, and appear well suited for high‐resolution studies. Density packing considerations are consistent with the presence of two molecules in the asymmetric unit and a solvent content of approximately 54%.
Conformational flexibility and crystallization of tandemly linked type III modules of human fibronectin
Authors: Lombardo A. , Wang Y. , Ni C.‐Z. , Dai X. , Kodandapani R. , Chiang S. , White C.A. , Pio F. , Ruoslahti E. , Ely K.R. , Xuong N‐H. , Hamlin R.C. , Dickinson C.D.
Published: February 2009

Fibronectin is a large cell adhesion molecule that is composed of several functional domains. The cell‐binding domain that binds to cell surface integrins consists of repeated homologous type III modules. In this study, recombinant fragments from the cell‐binding domain of human fibronectin that participate in a newly characterized fibronectin‐fibronectin interaction with FNIII, were crystallized. In each case, the crystals had more than one fibronectin fragment in the asymmetric unit. Crystals of FNIII10–11 grew in the space group C2 with a = 117.1 Å, b = 38.6 Å, c = 80.6 Å, β = 97.2°, and two molecules in the asymmetric unit. These crystals diffracted to 2.5 Å resolution. Fragment FNIII8–11 and a shorter fragment, FNIII8–10, crystallized in hexagonal space groups with large unit cells and two to four molecules per asymmetric unit. Even very large crystals of these fragments did not diffract beyond 4 Å. The crystal packing for this collection of fibronectin fragments suggests conformational flexibility between linked type III modules. The functional relevance of this flexibility for elongated versus compact models of the cell‐binding domain of fibronectin is discussed.
A fragment of staphylococcal nuclease with an OB‐fold structure shows hydrogen‐exchange protection factors in the range reported for “molten globules”
Authors: Alexandrescu Andrei T. , Dames Sonja A. , Wiltscheck Ronald
Published: February 2009

Hydrogen‐exchange rates for an OB‐fold subdomain fragment of staphylococcal nuclease have been measured at pH 4.7 and 4 °C, conditions close to the minimum of acid/base catalyzed exchange. The strongest protection from solvent exchange is observed for residues from a five‐stranded β‐barrel in the NMR structure of the protein. Protection factors, calculated from the experimental hydrogen‐exchange rates, range between 1 and 190. Similarly small protection factors have in many cases been attributed to “molten globule” conformations that are supposed to lack a specific tertiary structure. The present results suggest that marginal protection from solvent exchange does not exclude well‐defined structure.
High‐resolution structures of the bifunctional enzyme and transcriptional coactivator DCoH and its complex with a product analogue
Authors: Cronk Jeff D. , Endrizzi James A. , Alber Tom
Published: February 2009

DCoH, the dimerization cofactor of hepatocyte nuclear factor 1 (HNF‐1), functions as both a transcriptional coactivator and a pterin dehydratase. To probe the relationship between these two functions, the X‐ray crystal structures of the free enzyme and its complex with the product analogue 7,8‐dihydrobiopterin were refined at 2.3 Å resolution. The ligand binds at four sites per tetrameric enzyme, with little apparent conformational change in the protein. Each active‐site cleft is located in a subunit interface, adjacent to a prominent saddle motif that has structural similarities to the TATA binding protein. The pterin binds within an arch of aromatic residues that extends across one dimer interface. The bound ligand makes contacts to three conserved histidines, and this arrangement restricts proposals for the enzymatic mechanism of dehydration. The dihedral symmetry of DCoH suggests that binding to the dimerization domain of HNF‐1 likely involves the superposition of two‐fold rotation axes of the two proteins.
Surface point mutations that significantly alter the structure and stability of a protein's denatured state
Authors: Smith Catherine K. , Bu Zimei , Engelman Donald M. , Regan Lynne , Anderson Karen S. , Sturtevant Julian M.
Published: February 2009

Significantly different m values (1.9–2.7 kcal mol−1 M−1) were observed for point mutations at a single, solvent‐exposed site (T53) in a variant of the B1 domain of streptococcal Protein G using guanidine hydrochloride (GuHCl) as a denaturant. This report focuses on elucidating the energetic and structural implications of these m‐value differences in two Protein G mutants, containing Ala and Thr at position 53. These two proteins are representative of the high (m+) and low (m−) m‐value mutants studied. Differential scanning calorimetry revealed no evidence of equilibrium intermediates. A comparison of GuHCl denaturation monitored by fluorescence and circular dichroism showed that secondary and tertiary structure denatured concomitantly. The rates of folding (286 s−1 for the m+ mutant and 952 s−1 for the m− mutant) and the rates of unfolding (11 s−1 for m+ mutant and 3 s−1 for the m− mutant) were significantly different, as determined by stopped‐flow fluorescence. The relative solvation free energies of the transition states were identical for the two proteins (α++ = 0.3). Small‐angle X‐ray scattering showed that the radius of gyration of the denatured state (Rgd) of the m+ mutant did not change with increasing denaturant concentrations (Rgd≈︁23 Å); whereas, the Rgd of the m− mutant increased from approximately 17 Å to 23 Å with increasing denaturant concentration. The results indicate that the mutations exert significant effects in both the native and GuHCl‐induced denatured state of these two proteins.
Bending of the calmodulin central helix: A theoretical study
Authors: Spoel David Van Der , Groot Bert L. De , Hayward Steven , Berendsen Herman J. C. , Vogel Hans J.
Published: February 2009

The crystal structure of calcium‐calmodulin (CaM) reveals a protein with a typical dumbbell structure. Various spectroscopic studies have suggested that the central linker region of CaM, which is α‐helical in the crystal structure, is flexible in solution. In particular, NMR studies have indicated the presence of a flexible backbone between residues Lys 77 and Asp 80. This flexibility is related directly to the function of the protein because it enables the N‐ and C‐terminal domains of the protein to move toward each other and bind to the CaM‐binding domain of a target protein. We have investigated the flexibility of the CaM central helix by a variety of computational techniques: molecular dynamics (MD) simulations, normal mode analysis (NMA), and essential dynamics (ED) analysis. Our MD results reproduce the experimentally determined location of the bend in a simulation of only the CaM central helix, indicating that the bending point is an intrinsic property of the α‐helix, for which the remainder of the protein is not important. Interestingly, the modes found by the ED analysis of the MD trajectory are very similar to the lowest frequency modes from the NM analysis and to modes found by an ED analysis of different structures in a set of NMR structures. Electrostatic interactions involving residues Arg 74 and Asp 80 seem to be important for these bending motions and unfolding, which is in line with pH‐dependent NMR and CD studies.
In vitro renaturation of bovine β‐lactoglobulin A leads to a biologically active but incompletely refolded state
Authors: Subramaniam Vinod , Steel Duncan G. , Gafni Ari
Published: February 2009

When bovine β‐lactoglobulin (β‐LG) was refolded after extensive denaturation in 4.8 M guanidine hydrochloride (GuHCl), the functional activity of the protein, retinol binding, as measured by the enhancement of this ligand's fluorescence, was completely recovered. In contrast, the room‐temperature tryptophan phosphorescence lifetime of the refolded protein, a local measure of the residue environment, was ∽10 ms, significantly shorter than the phosphorescence lifetime of the untreated native protein (∽20 ms). The lability of the freshly refolded protein, as monitored by following the time course of its unfolding when incubated in 2.5 M GuHCl through the change in fluorescence intensity at 385 nm, was also determined and found to be increased significantly relative to untreated native protein. In contrast to the long term postactivation conformational changes detected previously in Escherichia coli alkaline phosphatase (Subramaniam V, Bergenhem NCH, Gafni A, Steel DG, 1995, Biochemistry 34:1133–1136), we found no changes in either the lability or phosphorescence decays of β‐LG during a period of 24 h. Our results are in agreement with the report by Hattori et al. (1993, J Biol Chem 268:22414–22419), using conformation‐specific monoclonal antibodies to recognize native‐like structure, that long‐term changes occur in the protein conformation, compared with the native structure, on refolding.
Crystallization and preliminary X‐ray diffraction studies of α‐amylase from the antarctic psychrophile Alteromonas haloplanctis A23
Authors: Aghajari Nushin , Haser Richard , Feller Georges , Gerday Charles
Published: February 2009

A cold‐active α‐amylase was purified from culture supernatants of the antarctic psychrophile Alteromonas haloplanctis A23 grown at 4 °C. In order to contribute to the understanding of the molecular basis of cold adaptations, crystallographic studies of this cold‐adapted enzyme have been initiated because a three‐dimensional structure of a mesophilic counterpart, pig pancreatic α‐amylase, already exists. α‐Amylase from A. haloplanctis, which shares 53% sequence identity with pig pancreatic α‐amylase, has been crystallized and data to 1.85 Å have been collected. The space group is found to be C2221 with a = 71.40 Å, b = 138.88 Å, and c = 115.66 Å. Until now, a three‐dimensional structure of a psychrophilic enzyme is lacking.
Cleavage site analysis in picornaviral polyproteins: Discovering cellular targets by neural networks
Authors: Blom Nikolaj , Hansen Jan , Brunak Søren , Blaas Dieter
Published: February 2009

Picornaviral proteinases are responsible for maturation cleavages of the viral polyprotein, but also catalyze the degradation of cellular targets. Using graphical visualization techniques and neural network algorithms, we have investigated the sequence specificity of the two proteinases 2Apro and 3Cpro. The cleavage of VP0 (giving rise to VP2 and VP4), which is carried out by a so‐far unknown proteinase, was also examined. In combination with a novel surface exposure prediction algorithm, our neural network approach successfully distinguishes known cleavage sites from noncleavage sites and yields a more consistent definition of features common to these sites. The method is able to predict experimentally determined cleavage sites in cellular proteins. We present a list of mammalian and other proteins that are predicted to be possible targets for the viral proteinases. Whether these proteins are indeed cleaved awaits experimental verification. Additionally, we report several errors detected in the protein databases.

A computer server for prediction of cleavage sites by picornaviral proteinases is publicly available at the e‐mail address NetPicoRNA@cbs.dtu.dk or via WWW at http://www.cbs.dtu.dk/services/NetPicoRNA/.