bluuebunny/crossref_metadata_2025_split huggingface dataset:
Probing the conformation of a human apolipoprotein C‐1 by amino acid substitutions and trimethylamine‐N‐oxide
Authors: Gursky Olga 
Published: February 2009

AbstractTo test, at the level of individual amino acids, the conformation of an exchangeable apolipoprotein in aqueous solution and in the presence of an osmolyte trimethylamine‐N‐oxide (TMAO), six synthetic peptide analogues of human apolipoprotein C‐1 (apoC‐1, 57 residues) containing point mutations in the predicted α‐helical regions were analyzed by circular dichroism (CD). The CD spectra and the melting curves of the monomeric wild‐type and plasma apoC‐1 in neutral low‐salt solutions superimpose, indicating 31 ± 4% α‐helical structure at 22 °C that melts reversibly with Tm,WT = 50 ± 2°C and van't Hoff enthalpy ΔHv,WT(Tm) = 18 ± 2 kcal/mol. G15A substitution leads to an increased α‐helical content of 42 ± 4% and an increased Tm,G15A = 57 ± 2°C, which corresponds to stabilization by ΔGapp = +0.4 ± 1.5 kcal/mol. G15P mutant has ∼∼20% α‐helical content at 22 °C and unfolds with low cooperativity upon heating to 90 °C. R23P and T45P mutants are fully unfolded at 0–90 °C. In contrast, Q31P mutation leads to no destabilization or unfolding. Consequently, the R23 and T45 locations are essential for the stability of the cooperative α‐helical unit in apoC‐1 monomer, G15 is peripheral to it, and Q31 is located in a nonhelical linker region. Our results suggest that Pro mutagenesis coupled with CD provides a tool for assigning the secondary structure to protein groups, which should be useful for other self‐associating proteins that are not amenable to NMR structural analysis in aqueous solution. TMAO induces a reversible cooperative coil‐to‐helix transition in apoC‐1, with the maximal α‐helical content reaching 74%. Comparison with the maximal α‐helical content of 73% observed in lipid‐bound apoC‐1 suggests that the TMAO‐stabilized secondary structure resembles the functional lipid‐bound apolipoprotein conformation.
New evidence for the denaturant binding model
Authors: Wu Jia‐Wei , Wang Zhi‐Xin 
Published: February 2009

AbstractDenaturation with guanidine hydrochloride (GdnHCl) or urea is one of the primary ways of measuring the conformational stability of proteins and comparing the stability of mutant proteins. Despite the widespread use of these two denaturants to provide quantitative data for the free energies of unfolding, the mode of action of these agents is not well understood. We are not even certain whether the action of these agents on proteins is direct and can be regarded as ligand binding, or indirect and involves a change in the properties of solvent (water) in the presence of GdnHCl and urea. In this paper, an extensive kinetic study of the inhibition of ribonuclease A and papain by urea has been performed. The results suggest that the effect of urea on activities of these enzymes can be well described by the denaturant binding model. The binding constants of urea determined by the present method are nearly identical to that determined from a variety of different studies on model compounds and proteins.
Global and local dynamics of the human U1A protein determined by tryptophan fluorescence
Authors: Jean John M. , Clerte Caroline , Hall Kathleen B. 
Published: February 2009

AbstractTryptophan residues have been introduced into two domains of the human U1A protein to probe solution dynamics. The full length protein contains 282 residues, separated into three distinct domains: the N‐terminal RBD1 (RNA Binding Domain I), consisting of amino acids 1‐101; the C‐terminal RBD2, residues 202‐282; and the intervening linker region. Tryptophan residues have been substituted for specific phenylalanine residues on the surface of the β‐sheet of either RBD1 or RBD2, thus introducing a single solvent exposed tryptophan as a fluorescence reporter. Both steady‐state and time‐resolved fluorescence measurements of the isolated RBD domains show that each tryptophan experiences a unique environment on the β‐sheet surface. The spectral properties of each tryptophan in RBD1 and RBD2 are preserved in the context of the U1A protein, indicating these domains do not interact with each other or with the linker region. The rotational correlation times of the isolated RBDs and the whole U1A, determined by dynamic polarization measurements, show that the linker region is highly flexible such that each RBD exhibits uncorrelated motion.
Position dependence of amino acid intrinsic helical propensities II: Non‐charged polar residues: Ser, Thr, Asn, and Gln
Authors: Petukhov Michael , Uegaki Koichi , Yumoto Noboru , Yoshikawa Susumu , Serrano Luis 
Published: February 2009

AbstractThe assumption that the intrinsic α‐helical propensities of the amino acids are position independent was critical in several helix/coil transition theories. In the first paper of these series, we reported that this is not the case for Gly and nonpolar aliphatic amino acids (Val, Leu, Met, and Ile). Here we have analyzed the helical intrinsic propensities of noncharged polar residues (Ser, Thr, Asn, and Gln) at different positions of a model polyalanine‐based peptide. We found that Thr is more favorable (by ∼0.3 kcal/mol) at positions N1 and N2 than in the helix center, although for Ser, Asn, and Gln the differences are smaller (±0.2 kcal/mol), and in many cases within the experimental error. There is a reasonable agreement (±0.2 kcal/mol) between the calculated free energies, using the ECEPP/2 force field equipped with a hydration potential, and the experimental data, except at position N1.
Conformational changes in the activation loop of the insulin receptor's kinase domain
Authors: Frankel Mark , Bishop Steven M. , Ablooglu Ararat J. , Han Yuan‐Ping , Kohanski Ronald A. 
Published: February 2009

AbstractLow catalytic efficiency of basal‐state protein kinases often depends on activation loop residues blocking substrate access to the catalytic cleft. Using the recombinant soluble form of the insulin receptor's kinase domain (IRKD) in its unphosphorylated state, activation loop conformation was analyzed by limited proteolysis. The rate of activation loop cleavage by trypsin is slow in the apo‐IRKD. Bound Mg‐adenine nucleoside di‐ and triphosphates increased the cleavage rate with half‐maximal effects observed at 0.4‐0.9 mM nucleotide. Adenosine monophosphate at concentrations up to 10 mM was not bound appreciably by the IRKD and had virtually no impact on activation loop cleavage. Amino‐terminal and carboxy‐terminal core‐flanking regions of the IRKD had no statistically significant impact on the ligand‐dependent or ‐independent activation loop cleavages. Furthermore, the core‐flanking regions did not change the inherent conformational stability of the active site or the global stability of the IRKD, as determined by guanidinium chloride‐induced denaturation. These measurements indicate that the intrasterically inhibitory conformation encompasses ≥90% of the ligand‐free basal state kinase. However, normal intracellular concentrations of Mg‐adenine nucleotides, which are in the millimolar range, would favor a basal‐state conformation of the activation loop that is more accessible.
Design of highly stable functional GroEL minichaperones
Authors: Wang Qinghua , Buckle Ashley M. , Foster Nicholas W. , Johnson Christopher M. , Fersht Alan R. 
Published: February 2009

AbstractGroEL minichaperones have potential in the biotechnology industry for the refolding of recombinant proteins. With the aim of enhancing and widening their use, we have created two highly stable functional variants of minichaperone GroEL(193‐345). A sequence alignment of 130 members of the chaperonin 60 (Cpn60) family was used to design 37 single mutations. Two small‐to‐large mutations, A223T, A223V and one similar‐size mutation, M233L, all located in the hydrophobic core were found to stabilize the protein by more than 1 kcal mol−1 each. Six stabilizing mutations were combined, yielding two multiple mutants that were 6.99 and 6.15 kcal mol−1 more stable than wild‐type protein. Even though some of the substituted residue pairs are close to each other in the protein structure, the energetic effects of mutation are approximately additive. In particular, the stabilizing substitution A223T is unexpected and would have been missed by purely structural analysis. In the light of previously reported successes employing similar methods with several other proteins, our results show that a homology based approach is a simple and efficient method of increasing the stability of a protein.
Crystal structure of the EF‐hand parvalbumin at atomic resolution (0.91 Å) and at low temperature (100 K). Evidence for conformational multistates within the hydrophobic core
Authors: Declercq Jean‐Paul , Evrard Christine , Lamzin Victor , Parello Joseph 
Published: February 2009

AbstractSeveral crystal structures of parvalbumin (Parv), a typical EF‐hand protein, have been reported so far for different species with the best resolution achieving 1.5 Å. Using a crystal grown under microgravity conditions, cryotechniques (100 K), and synchrotron radiation, it has now been possible to determine the crystal structure of the fully Ca2+‐loaded form of pike (component pI 4.10) Parv.Ca2 at atomic resolution (0.91 Å). The availability of such a high quality structure offers the opportunity to contribute to the definition of the validation tools useful for the refinement of protein crystal structures determined to lower resolution. Besides a better definition of most of the elements in the protein three‐dimensional structure than in previous studies, the high accuracy thus achieved allows the detection of well‐defined alternate conformations, which are observed for 16 residues out of 107 in total. Among them, six occupy an internal position within the hydrophobic core and converge toward two small buried cavities with a total volume of about 60 Å. There is no indication of any water molecule present in these cavities. It is probable that at temperatures of physiological conditions there is a dynamic interconversion between these alternate conformations in an energy‐barrier dependent manner. Such motions for which the amplitudes are provided by the present study will be associated with a time‐dependent remodeling of the void internal space as part of a slow dynamics regime (millisecond timescales) of the parvalbumin molecule. The relevance of such internal dynamics to function is discussed.
The interaction of neurotrophins with the p75NTR common neurotrophin receptor: A comprehensive molecular modeling study
Authors: Shamovsky Igor L. , Weaver Donald F. , Ross Gregory M. , Riopelle Richard J. 
Published: February 2009

AbstractNeurotrophins are a family of proteins with pleiotropic effects mediated by two distinct receptor types, namely the Trk family, and the common neurotrophin receptor p75NTR. Binding of four mammalian neurotrophins, nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3), and neurotrophin‐4/5 (NT‐4/5), to p75NTR is studied by molecular modeling based on X‐ray structures of the neurotrophins and the extracellular domain of p55TNFR, a homologue of p75NTR. The model of neurotrophin/receptor interactions suggests that the receptor binding domains of neurotrophins (loops I and IV) are geometrically and electrostatically complementary to a putative binding site of p75NTR, formed by the second and part of the third cysteine‐rich domains. Geometric match of neurotrophin/receptor binding domains in the complexes, as characterized by shape complementarity statistic Sc, is comparable to known protein/protein complexes. All charged residues within the loops I and IV of the neurotrophins, previously determined as being critical for p75NTR binding, directly participate in receptor binding in the framework of the model. Principal residues of the binding site of p75NTR include Asp47, Lys56, Asp75, Asp76, Asp88, and Glu89. The additional involvement of Arg80 and Glu53 is specific for NGF and BDNF, respectively, and Glu73 participates in binding with NT‐3 and NT‐4/5. Neurotrophins are likely to induce similar, but not identical, conformational changes within the p75NTR binding site.
The turn sequence directs β‐ strand alignment in designed β‐hairpins
Authors: Alba Eva De , Rico Manuel , Jiménez M. Angeles 
Published: February 2009

AbstractA previous NMR investigation of model decapeptides with identical β‐strand sequences and different turn sequences demonstrated that, in these peptide systems, the turn residues played a more predominant role in defining the type of β‐hairpin adopted than cross‐strand side‐chain interactions. This result needed to be tested in longer β‐hairpin forming peptides, containing more potentially stabilizing cross‐strand hydrogen bonds and side‐chain interactions that might counterbalance the influence of the turn sequence. In that direction, we report here on the design and 1H NMR conformational study of three β‐hairpin forming pentadecapeptides. The design consists of adding two and three residues at the N‐ and C‐termini, respectively, of the previously studied decapeptides. One of the designed pentadecapeptides includes a potentially stabilizing R‐E salt bridge to investigate the influence of this interaction on β‐hairpin stability. We suggest that this peptide self‐associates by forming intermolecular salt bridges. The other two pentadecapeptides behave as monomers. A conformational analysis of their 1H NMR spectra reveals that they adopt different types of β‐hairpin structure despite having identical strand sequences. Hence, the β‐turn sequence drives β‐hairpin formation in the investigated pentadecapeptides that adopt β‐hairpins that are longer than the average protein β‐hairpins. These results reinforce our previous suggestion concerning the key role played by the turn sequence in directing the kind of β‐hairpin formed by designed peptides.
Intrabody construction and expression III: Engineering hyperstable VH domains
Authors: Wirtz Peter , Steipe Boris 
Published: February 2009

AbstractThe folding of immunoglobulin domains requires the formation of a conserved structural disulfide. Therefore, as a general rule, they cannot be functionally expressed in the reducing environment of the cellular cytoplasm. We have previously reported that stability engineering can lead to the cytoplasmic expression of functional immunoglobulin VL domains. Here we apply rational stability engineering by consensus sequence analysis to VH domains. Isolated VH domains tend to aggregate more easily than VL domains; they do not refold quantitatively and are generally more difficult to handle in vitro. To overcome these problems, we successfully predicted and experimentally verified several stabilizing point mutations in the VH domain of a designed, catalytic Fv fragment. The effect of single mutations was additive, and they could be combined in a prototype domain with significantly improved stability against chemical denaturation and a 20‐fold increased half time of irreversible thermal denaturation, at physiological temperature. This stabilized, isolated VH domain could be expressed solubly in the reducing cellular cytoplasm of Escherichia coli, at a yield of approximately 1.2 mg/L of shake flask culture. It remains fully functional, as evidenced by the successful reconstitution of an esterolytic Fv fragment with the VL domain. This success provides further evidence that consensus sequence engineering is a rational, plannable route to the construction of intrabodies.
Folding of an isolated ribonuclease H core fragment
Authors: Chamberlain Aaron K. , Fischer Kael F. , Reardon Deirdre , Handel Tracy M. , Marqusee Susan 
Published: February 2009

AbstractBased on results from both equilibrium and kinetic hydrogen exchange studies of Escherichia coli ribonuclease HI (RNase H), a fragment of RNase H (eABCD) was designed. The sequence of eABCD contains less than half of the protein's primary sequence and includes the regions that were shown to be the most protected from hydrogen exchange in all previous studies of RNase H. This core fragment of RNase H encodes a well‐ordered protein with native‐like properties. When isolated from the full‐length monomeric protein, the eABCD fragment forms a stable dimer. However, we show indirectly that the monomeric form of eABCD is folded and has an overall secondary structure similar to the dimeric form.
Crystal structure of brain‐type creatine kinase at 1.41 Å resolution
Authors: Eder Michael , Schlattner Uwe , Wallimann Theo , Becker Andreas , Kabsch Wolfgang , Fritz‐Wolf Karin 
Published: February 2009

AbstractExcitable cells and tissues like muscle or brain show a highly fluctuating consumption of ATP, which is efficiently regenerated from a large pool of phosphocreatine by the enzyme creatine kinase (CK). The enzyme exists in tissue–as well as compartment‐specific isoforms. Numerous pathologies are related to the CK system: CK is found to be overexpressed in a wide range of solid tumors, whereas functional impairment of CK leads to a deterioration in energy metabolism, which is phenotypic for many neurodegenerative and age‐related diseases. The crystal structure of chicken cytosolic brain‐type creatine kinase (BB‐CK) has been solved to 1.41 Å resolution by molecular replacement. It represents the most accurately determined structure in the family of guanidino kinases. Except for the N‐terminal region (2‐12), the structures of both monomers in the biological dimer are very similar and closely resemble those of the other known structures in the family. Specific Ca2+‐mediated interactions, found between two dimers in the asymmetric unit, result in structurally independent heterodimers differing in their N‐terminal conformation and secondary structure.The high‐resolution structure of BB‐CK presented in this work will assist in designing new experiments to reveal the molecular basis of the multiple isoform‐specific properties of CK, especially regarding different subcellular locations and functional interactions with other proteins. The rather similar fold shared by all known guanidino kinase structures suggests a model for the transition state complex of BB‐CK analogous to the one of arginine kinase (AK). Accordingly, we have modeled a putative conformation of CK in the transition state that requires a rigid body movement of the entire N‐terminal domain by rms 4 Å from the structure without substrates.
Crystal structure analysis of a pentameric fungal and an icosahedral plant lumazine synthase reveals the structural basis for differences in assembly
Authors: Schneider Gunter , Sandalova Tatyana , Persson Karina , Jordan Douglas B. , Viitanen Paul V. 
Published: February 2009

AbstractLumazine synthase catalyzes the penultimate step in the synthesis of riboflavin in plants, fungi, and microorganisms. The enzyme displays two quaternary structures, the pentameric forms in yeast and fungi and the 60‐meric icosahedral capsids in plants and bacteria. To elucidate the structural features that might be responsible for differences in assembly, we have determined the crystal structures of lumazine synthase, complexed with the inhibitor 5‐nitroso‐6‐ribitylamino‐2,4‐pyrimidinedione, from spinach and the fungus Magnaporthe grisea to 3.3 and 3.1 Å resolution, respectively. The overall structure of the subunit and the mode of inhibitor binding are very similar in these enzyme species. The core of the subunit consists of a four‐stranded parallel β‐sheet sandwiched between two helices on one side and three helices on the other. The packing of the five subunits in the pentameric M. grisea lumazine synthase is very similar to the packing in the pentameric substructures in the icosahedral capsid of the plant enzyme. Two structural features can be correlated to the differences in assembly. In the plant enzyme, the N‐terminal β‐strand interacts with the β‐sheet of the adjacent subunit, thus extending the sheet from four to five strands. In fungal lumazine synthase, an insertion of two residues after strand β1 results in a completely different orientation of this part of the polypeptide chain and this conformational difference prevents proper packing of the subunits at the trimer interface in the icosahedron. In the spinach enzyme, the b‐hairpin connecting helices α4 and α5 participates in the packing at the trimer interface of the icosahedron. Another insertion of two residues at this position of the polypeptide chain in the fungal enzyme disrupts the hydrogen bonding in the hairpin, and the resulting change in conformation of this loop also interferes with proper intrasubunit contacts at the trimer interface.
Crystal structure of reduced thioredoxin reductase from Escherichia coli: Structural flexibility in the isoalloxazine ring of the flavin adenine dinucleotide cofactor
Authors: Lennon Brett W. , Williams Charles H. , Ludwig Martha L. 
Published: February 2009

AbstractCatalysis by thioredoxin reductase (TrxR) from Escherichia coli requires alternation between two domain arrangements. One of these conformations has been observed by X‐ray crystallography (Waksman G, Krishna TSR, Williams CH Jr, Kuriyan J, 1994, J Mol Biol 236:800‐816). This form of TrxR, denoted FO, permits the reaction of enzyme‐bound reduced FAD with a redox‐active disulfide on TrxR. As part of an investigation of conformational changes and intermediates in catalysis by TrxR, an X‐ray structure of the FO form of TrxR with both the FAD and active site disulfide reduced has been determined. Reduction after crystallization resulted in significant local conformation changes. The isoalloxazine ring of the FAD cofactor, which is essentially planar in the oxidized enzyme, assumes a 34° “butterfly” bend about the N(5)‐N(10) axis in reduced TrxR. Theoretical calculations reported by others predict ring bending of 15‐28° for reduced isoalloxazines protonated at N(1). The large bending in reduced TrxR is attributed in part to steric interactions between the isoalloxazine ring and the sulfur of Cys138, formed by reduction of the active site disulfide, and is accompanied by changes in the positions and interactions of several of the ribityl side‐chain atoms of FAD. The bending angle in reduced TrxR is larger than that for any flavoprotein in the Protein Data Bank. Distributions of bending angles in published oxidized and reduced flavoenzyme structures are different from those found in studies of free flavins, indicating that the protein environment has a significant effect on bending.