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PubMed1999

Anomeric specificity and protein-substrate interactions support the 3D model for the hemagglutinin-neuraminidase from sendai virus.

The 3D structure of paramyxovirus hemagglutinin-neuraminidase has not yet been resolved; however, a theoretical model has been built by using influenza virus and bacterial neuraminidases as template [V. C. Epa (1997) Proteins Struct. Funct. Gen. 29, 264-281]. Two common features of the catalytic mechanism of the neuraminidases of known 3D structure are the anomeric specificity and the involvement of a tyrosine residue in the stabilization of the transition state. These key features have been investigated on the water-soluble ectodomain of the hemagglutinin-neuraminidase from Sendai virus (cHN). The anomeric specificity of the hydrolysis of the substrate by cHN has been investigated by NMR spectroscopy. The immediate product of the reaction was the alpha-anomer, meaning that cHN belongs between glycohydrolases retaining anomeric configuration like influenza virus neuraminidase. Measurements of the UV difference spectrum upon binding of the substrate analogue 2,3-dehydro 2-deossi N-acetyl neuraminic acid indicate the ionization of a tyrosine residue and decreased polarity in the environment of a tryptophan residue. Functional significance of the spectral data was derived from the known structure of influenza neuraminidase, where a tyrosinate ion is involved in the stabilization of the transition-state carbonium ion, and a tryptophan residue is involved in the binding of the acetyl moiety of the substrate. The data give experimental support to the 3D model of paramyxovirus neuraminidase.

Catalytic Domain↗

PubMed2005

The relative stabilities of 11-vertex nido- and 12-vertex closo-heteroboranes and -borates: facile estimation by structural or connection increments.

The relative thermodynamic stabilities of ortho-, meta- and para-isomers of 12-vertex closo-heteroboranes and -borates with different p-block heteroatoms were determined using density functional theory. More electronegative (smaller) heteroatoms tend to occupy non-adjacent, whereas less electronegative (larger) heteroatoms tend to occupy adjacent vertices in the thermodynamically most stable closo-diheterododecaborane isomers. The computed relative stabilities agree perfectly with experimental observations. The energy differences of para- and meta- relative to ortho-isomers of 12-vertex closo-heteroboranes generally depend on the extent of electron localization by a given heteroatom and show highly periodic trends, i.e. increase along the period and decrease down the group. The energy penalties for the HetHet structural feature (two heteroatoms adjacent to each other) for the 12-vertex closo-cluster are apparently significantly different from those for the 11-vertex nido-cluster. Reformulating two 11-vertex nido-structural features, i.e. Het(5k)(2) and HetHet, in terms of connection increments along with the additional structural feature HetHet(m) give the relative stabilities of various isomeric 11-vertex nido- as well as 12-vertex closo-heteroboranes and -borates, using one unique set of increments.

Journal Article↗

PubMed2003

Posttranscriptional control of renin synthesis: identification of proteins interacting with renin mRNA 3'-untranslated region.

Stabilization and correct localization of mRNA are important features of renin synthesis. To elucidate the molecular basis of cAMP-mediated posttranscriptional control via mRNA stabilization, we analyzed the interaction of human preprorenin (hREN) mRNA 3'-untranslated region (3'-UTR) with proteins of renin synthesizing Calu-6 cells and investigated their functional impact on messenger integrity. To identify hREN mRNA binding proteins, electrophoretic mobility shift assays, UV cross-linking and RNA-affinity chromatography with subsequent matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were performed. The following six proteins were unambiguously identified as hREN mRNA 3'-UTR binding proteins: hnRNP E1 (synonyms alpha-CP or PCBP), hnRNP K, dynamin, nucleolin, YB-1, and MINT-homologous protein. All proteins contain various RNA binding motifs, and most have been described in the context of mRNA binding and mRNA stabilization. Four proteins for which antibodies were available were verified by immunological techniques (dynamin, nucleolin, hnRNP E1, and YB-1). Forskolin, an activator of cAMP synthesis, considerably stimulates renin synthesis via inhibition of REN mRNA decay. Functionally, this cAMP-based mRNA stabilization is accompanied by a 3- to 6-fold upregulation of REN mRNA binding proteins. RNase degradation assays confirm that 3'-UTR binding proteins are able to protect and stabilize REN mRNA in vitro.

3' Untranslated Regions↗

PubMed1989

Denaturation of poliovirus procapsids.

The denaturation of poliovirus procapsids at pH 6.5 was studied, both in the frozen and liquid condition. Denaturation involved alteration of antigenic and physical features (isoelectric pH, alkali dissociability, sedimentation coefficient). Magnesium was a stabilizing factor, and the sedimentation coefficient was the most denaturation-sensitive feature.

Capsid↗

PubMed2001

Activities of monomeric insulin analogs at position A8 are uncorrelated with their thermodynamic stabilities.

Previous studies have demonstrated that the potency and thermodynamic stability of human insulin are enhanced in concert by substitution of Thr(A8) by arginine or histidine. These surface substitutions stabilize the N-terminal alpha-helix of the A chain, a key element of hormone-receptor recognition. Does enhanced stability necessarily imply enhanced activity? Here, we test by structure-based mutagenesis the relationship between the stability and activity of the hormone. To circumvent confounding effects of insulin self-association, A chain analogs were combined with a variant B chain (Asp(B10), Lys(B28), and Pro(B29) (DKP)) to create a monomeric template. Five analogs were obtained by chain combination; disulfide pairing proceeded in each case with native yield. CD and (1)H NMR spectra of the DKP analogs are essentially identical to those of DKP-insulin, indicating a correspondence of structures. Receptor binding affinities were determined by competitive displacement of (125)I-insulin from human placental membranes. Thermodynamic stabilities were measured by CD titration; unfolding was monitored as a function of guanidine concentration. In this broader collection of analogs receptor binding affinities are uncorrelated with stability. We suggest that receptor binding affinities of A8 analogs reflect local features of the hormone-receptor interface rather than the stability of the free hormone or the intrinsic C-capping propensity of the A8 side chain.

Amino Acid Sequence↗

PubMed2007

Occurrence of protein structure elements in conserved sequence regions.

BACKGROUND: Conserved protein sequence regions are extremely useful for identifying and studying functionally and structurally important regions. By means of an integrated analysis of large-scale protein structure and sequence data, structural features of conserved protein sequence regions were identified. RESULTS: Helices and turns were found to be underrepresented in conserved regions, while strands were found to be overrepresented. Similar numbers of loops were found in conserved and random regions. CONCLUSION: These results can be understood in light of the structural constraints on different secondary structure elements, and their role in protein structural stabilization and topology. Strands can tolerate fewer sequence changes and nonetheless keep their specific shape and function. They thus tend to be more conserved than helices, which can keep their shape and function with more changes. Loop behavior can be explained by the presence of both constrained and freely changing loops in proteins. Our detailed statistical analysis of diverse proteins links protein evolution to the biophysics of protein thermodynamic stability and folding. The basic structural features of conserved sequence regions are also important determinants of protein structure motifs and their function.

Amino Acid Sequence↗

PubMed1998

Morphology of protein-protein interfaces.

BACKGROUND: Most soluble proteins are active as low-number oligomers. Statistical surveys of oligomeric proteins have defined the roles of hydrophobicity and complementarity in the stability of protein interfaces, but tend to average structural features over a diverse set of protein-protein interfaces, blurring information on how individual interfaces are stabilized. RESULTS: We report a visual survey of 136 homodimeric proteins from the Brookhaven Protein Data Bank, with images that highlight the major structural features of each protein-protein interaction surface. Nearly all of these proteins have interfaces formed between two globular subunits. Surprisingly, the pattern of hydrophilicity over the surface of these interfaces is quite variable. Approximately one-third of the interfaces show a recognizable hydrophobic core, with a single large, contiguous, hydrophobic patch surrounded by a ring of intersubunit polar interactions. The remaining two-thirds of the proteins show a varied mixture of small hydrophobic patches, polar interactions and water molecules scattered over the entire interfacial area. Ten proteins in the survey have intertwined interfaces formed by extensive interdigitation of the two subunit chains. These interfaces are very hydrophobic and are associated with proteins that require both stability and internal symmetry. CONCLUSIONS: The archetypal protein interface, with a defined hydrophobic core, is present in only a minority of the surveyed homodimeric proteins. Most homodimeric proteins are stabilized by a combination of small hydrophobic patches, polar interactions and a considerable number of bridging water molecules. The presence or absence of a hydrophobic core within these interfaces does not correlate with specific protein functions.

Adenylosuccinate Synthase↗

PubMed1996

Crystal structure at 2.3 A resolution and revised nucleotide sequence of the thermostable cyclodextrin glycosyltransferase from Thermonanaerobacterium thermosulfurigenes EM1.

The crystal structure of the cyclodextrin glycosyltransferase (CGTase) from the thermophilic microorganism Thermoanaerobacterium thermosulfurigenes EM1 has been elucidated at 2.3 A resolution. The final model consists of all 683 amino acid residues, two calcium ions and 343 water molecules, and has a crystallographic R-factor of 17.9% (Rfree 24.9%) with excellent stereochemistry. The overall fold of the enzyme is highly similar to that reported for mesophilic CGTases and differences are observed only at surface loop regions. Closer inspection of these loop regions and comparison with other CGTase structures reveals that especially loops 88-95, 335-339 and 534-539 possibly contribute with novel hydrogen bonds and apolar contacts to the stabilization of the enzyme. Other structural features that might confer thermostability to the T. thermosulfurigenes EM1 CGTase are the introduction of five new salt-bridges and three Gly to Ala/Pro substitutions. The abundance of Ser, Thr and Tyr residues near the active site and oligosaccharide binding sites might explain the increased thermostability of CGTase in the presence of starch, by allowing amylose chains to bind non-specifically to the protein. Additional stabilization of the A/E domain interface through apolar contacts involves residues Phe273 and Tyr187. No additional or improved calcium binding is observed in the structure, suggesting that the observed stabilization in the presence of calcium ions is caused by the reduced exchange of calcium from the protein to the solvent, rendering it less susceptible to unfolding. The 50% decrease in cyclization activity of the T. thermosulfurigenes EM1 CGTase compared with that of B. circulans strain 251 appears to be caused by the changes in the conformation and amino acid composition of the 88-95 loop. In the T. thermosulfurigenes EM1 CGTase there is no residue homologous to Tyr89, which was observed to take part in stacking interactions with bound substrate in the case of the B. circulans strain 251 CGTase. The lack of this interaction in the enzyme-substrate complex is expected to destabilize bound substrates prior to cyclization. Apparently, some catalytic functionality of CGTase has been sacrificed for the sake of structural stability by modifying loop regions near the active site.

Amino Acid Sequence↗

PubMed2003

[Acromelanosis].

Acromelanosis is an independent disease entity, characterized by increased skin pigmentation, usually located on the acral areas of the fingers and toes. It is mostly seen in newborns or during the first years of life. Only a few cases of this entity have previously been described in the medical literature. In some of these cases, the cutaneous lesions spread to affect large parts of the skin surface. A possible association with other benign and malignant diseases has been proposed. Differential diagnosis must be made with a wide variety of systemic and dermatologic conditions, especially dermatoses with acral distribution of macular hyperpigmentation, including acropigmentation. In this article, we report a new case of acromelanosis in a 5-week-old girl showing two peculiar clinical features: associated melanosis of the genital mucosa and stabilization of the lesions after an initial phase of progression and proximal spread. In addition, the most important features of this rare cutaneous disease are discussed.

Female↗

PubMed2002

Coiled-coils: stability, specificity, and drug delivery potential.

The coiled-coil is a ubiquitous protein folding and assembly motif made of alpha-helices wrapping around each other forming a supercoil. The sequences of coiled-coils are made of seven-residue repeats, called heptads, and thus are polymer-like. Due to its simplicity and regularity, the coiled-coil is the most extensively studied protein motif. In this review, results on coiled-coil stability and specificity from structural and biophysical studies are summarized. It is pointed out that the primary sequences of coiled-coils over specify the secondary structure but under specify the tertiary/quaternary structure. This leads to two unique features of coiled-coil structure: linkage between stability and specificity and decoupling of secondary and tertiary/quaternary structural specificity. This is followed by a discussion of the potential of coiled-coils as drug delivery vehicles, particularly the prospect in two-staged pretargeted delivery. Such potentials are intimately related to the unique structural features of coiled-coils. The aim of this review is to illustrate how knowledge on protein stability and specificity can be used in the de novo design of peptide-based drug delivery vehicles with well-defined structure and interaction features.

Autophagy-Related Proteins↗

PubMed2002

Identification, cloning, and expression of uracil-DNA glycosylase from Atlantic cod (Gadus morhua): characterization and homology modeling of the cold-active catalytic domain.

Two distinct forms of the highly conserved uracil-DNA glycosylase (UNG) have been isolated from Atlantic cod (Gadus morhua) liver cDNA by rapid amplification of cDNA ends (RACE). From the cDNA sequences, both forms were deduced to encode an open reading frame of 301 amino acids, with an identical 267-amino-acid C-terminal region and different N-terminal regions of 34 amino acids. By comparison with the human UNG sequences, the two forms were identified as possible mitochondrial (cUNG1) and nuclear (cUNG2) forms. Several constructs of recombinant cUNG (rcUNG) were expressed in Escherichia coli in order to optimize the yield. The recombinant enzyme was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Activity and stability experiments showed that rcUNG was similar to cUNG previously purified from Atlantic cod liver, and was more pH- and temperature labile than a recombinant human UNG (rhUNG). Under optimal assay conditions for both rcUNG and rhUNG, the turnover number (k(cat)) was three times higher for rcUNG compared with rhUNG, with an identical K(M), resulting in a threefold higher catalytic efficiency (k(cat)/K(M)) for rcUNG. These activity and stability experiments reveal cold-adapted features in rcUNG. Homology models of the catalytic domains of Atlantic cod (cUNG) and mouse uracil-DNA glycosylase (mUNG) were built using the human UNG (hUNG) crystal structure as a template. The unique amino acid substitutions observed in cod UNG were mainly located in the N- and C-terminal parts of the sequence. The analysis indicated a more stable N-terminal, a more flexible C-terminal, and a less stabilized core in cUNG as compared with the mammalian UNGs. Substitution of several amino acids in or near the DNA-binding site in cUNG could give rise to a more positively charged surface and a higher electrostatic potential near the active site compared with the mammalian UNGs. The higher potential may increase the electrostatic interactions between the enzyme and DNA, and may explain the increased substrate affinity and, in combination with the higher flexibility, the higher catalytic efficiency observed for rcUNG.

Amino Acid Sequence↗

PubMed1995

Increased stability of nucleic acids containing 7-deaza-guanosine and 7-deaza-adenosine may enable rapid DNA sequencing by matrix-assisted laser desorption mass spectrometry.

The use of matrix-assisted laser desorption mass spectrometry (MALDI-MS) has been suggested as an ultrafast readout of Sanger DNA sequencing ladders in a manner analogous to that used with sequencing gels. Currently, a serious limitation of MALDI-MS for the analysis of DNA results from the tendency for oligonucleotides to undergo facile fragmentation in the gas phase. The present study was undertaken to gain an understanding of the influence of various chemical structural features of purine bases on the stability of oligodeoxynucleotide ions produced by MALDI. The study focused on the stability of model compounds of the type d(TTTTTTTTTTXTTTTTTTTT TTTT), where T designates deoxythymidine and X a purine-containing 2'-deoxynucleotide. A variety of different purine derivatives were chosen as the base in the nucleotide X. The mass spectra of the model compounds containing 7-deaza analogues of guanine and adenine reveal a significantly increased stability compared to the 7-aza analogues under the conditions of MALDI-MS. The previously reported incorporation of the 7-deaza-2'-deoxy-adenosine triphosphate and the 7-deaza-2'-deoxy-guanosine triphosphate into DNA by polymerases suggests their use in a Sanger dideoxy sequencing experiment. The dideoxy termination products with the 7-deaza-purines instead of the 7-aza-purines might be sufficiently stable to allow separation and detection of the sequencing ladder by MALDI-MS. Thus, an ultrafast (seconds) read-out of DNA sequence may become feasible.

Base Sequence↗

PubMed1998

Novel analogues of arachidonylethanolamide (anandamide): affinities for the CB1 and CB2 cannabinoid receptors and metabolic stability.

Several analogues of the endogenous cannabinoid receptor ligand arachidonylethanolamide (anandamide) were synthesized and evaluated in order to study (a) the structural requirements for high-affinity binding to the CB1 and CB2 cannabinoid receptors and (b) their hydrolytic stability toward anandamide amidase. The series reported here was aimed at exploring structure-activity relationships (SAR) primarily with regard to stereoelectronic requirements of ethanolamido headgroup for interaction with the cannabinoid receptor active site. Receptor affinities, reported as Ki values, were obtained by a standard receptor binding assay using [3H]CP-55,940 as the radioligand, while stability toward the amidase was evaluated by comparing the Ki of each analogue in the presence and absence of phenylmethanesulfonyl fluoride (PMSF), a serine protease blocker and inhibitor of anandamide amidase. Introduction of a methyl group in the 1'- and 2'-positions or substitution of the ethanolamido headgroup with a butylamido group gave analogues with vastly improved biochemical stability. This is accomplished in some cases with increased receptor affinity. Conversely, oxazolyl and methyloxazolyl headgroups led to low-affinity analogues. Substitution of the hydroxyl group with electronegative substituents such as fluoro, chloro, allyl, and propargyl groups significantly increased receptor affinity but did not influence the biochemical stability. The 2'-chloro analogue of anandamide was found to have the highest affinity for CB1. Additionally, reversing the positions of the carbonyl and NH in the amido group produces retro-anandamides possessing considerably higher metabolic stability. Replacement of the arachidonyl tail with oleyl or linoleyl results in analogues with low affinities for both receptors. All of the analogues in this study showed high selectivity for the CB1 receptor over the peripheral CB2 receptor. The most potent analogues were tested for their ability to stimulate the binding of [35S]GTPgammaS to G-proteins and were shown to be potent cannabimimetic agonists. The results are discussed in terms of pharmacophoric features affecting receptor affinity and enzymatic stability.

Amidohydrolases↗

PubMed2006

Subcellular pH and predicted pH-dependent features of proteins.

A characteristic of two-dimensional proteomics gels is a general bimodal distribution of isoelectric (pI) values. Discussion of this feature has focussed on the balance of acidic and basic ionisable residues, and potential relationships between pI distributions and organism classification or protein subcellular location. Electrostatics calculations on a set of protein structures with known subcellular location show that predicted folded state pI are similar to those calculated from sequence alone, but adjusted according to a general stabilising effect from interactions between ionisable groups. Bimodal distributions dominate both pI and the predicted pH of maximal stability. However, there are significant differences between these features. The average pH of maximal stability generally follows organelle pH. Average pI values are well removed from organelle pH in most subcellular environments, consistent with the view that proteins have evolved to carry (on average) net charge in a given subcellular location, and relevant to discussion of solubility in crowded environments. Correlation of the predicted pH of maximum stability with subcellular pH suggests an evolutionary pressure to adjust folded state interactions according to environment. Finally, our analysis of ionisable group contributions to stability suggests that Golgi proteins have the largest such term, although this dataset is small.

Cell Nucleus↗

PubMed2003

Stimulated interaction between and subunits of tryptophan synthase from hyperthermophile enhances its thermal stability.

Tryptophan synthase from hyperthermophile, Pyrococcus furiosus, was found to be a tetrameric form (22) composed of and 2 subunits. To elucidate the relationship between the features of the subunit association and the thermal stability of the tryptophan synthase, the subunit association and thermal stability were examined by isothermal titration calorimetry and differential scanning calorimetry, respectively, in comparison with those of the counterpart from Escherichia coli. The association constants between the and subunits in the hyperthermophile protein were of the order of 108 M1, which were higher by two orders of magnitude than those in the mesophile one. The negative values of the heat capacity change and enthalpy change upon the subunit association were much lower in the hyperthermophile protein than in the mesophile one, indicating that the conformational change of the hyperthermophile protein coupled to the subunit association is slight. The denaturation temperature of the subunit from the hyperthermophile was enhanced by 17 degrees C due to the formation of the 22 complex. This increment in denaturation temperature due to complex formation could be quantitatively estimated by the increase in the association constant compared with that of the counterpart from E. coli.

Amino Acid Sequence↗

PubMed1993

Posterior stabilization of the cervical spine.

Operative management of the patient with cervical spinal instability depends on the specific anatomy and location of the injury. The unique features of the atlanto-axial complex require stabilizing procedures that are quite different from those used in the subaxial spine. A clear idea of the etiology of the instability, a definition of the pathological anatomy, and knowledge of the presence and extent of neural compression are essential for optimizing a successful outcome. In some instances the clinical circumstances mandate the use of an anterior approach, and in others a posterior operation is more appropriate and likely to be successful. In the majority of patients instability can be successfully managed utilizing either an anterior or a posterior approach. Even when a posterior approach is used, there is usually more than one surgical option. It cannot be emphasized too strongly, however, that the surgeon should choose the procedure with which he or she is most comfortable and which he or she is technically able to perform. It makes little sense to attempt a procedure such as C1-C2 transarticular screw fixation or posterior cervical plating without having had "hands on" experience with cadavers or anatomical models or having previously assisted in the procedure. Wiring and other means of fixation, although "less modern," have been used successfully for many years and are usually quite satisfactory if the surgeon is experienced and comfortable in their use.

Atlanto-Axial Joint↗

PubMed1996

Risperidone in the treatment of mania.

BACKGROUND: Risperidone, a 5-HT2 and D2 antagonist, has been shown to be an effective antipsychotic in the treatment of schizophrenia but has unclear efficacy in the treatment of psychotic affective disorders. The purpose of the study was to assess the efficacy of risperidone in the treatment of acute mania with psychotic features. METHOD: We conducted an open-label pilot study of risperidone and concurrent mood-stabilizing drugs in the treatment of acute mania with psychotic features. Patients were diagnosed with the Structured Clinical Interview for DSM-III-R (SCID). Efficacy was measured weekly with the use of the Young Mania Rating Scale (YMRS) and the Brief Psychiatric Rating Scale (BPRS). RESULTS: Ten women and 5 men (mean age = 38 years) were included in the study. Of the 13 patients who completed 2 weeks of treatment, 8 of these 13 had a 50% improvement of the BPRS, and all 13 had at least a 25% improvement (p = .002, 95% confidence interval [CI] = 46.0 to 57.8). Of the 8 patients who completed 6 weeks of treatment, 7 of the 8 had a 50% improvement, and all 8 had a 25% improvement (p = .012, 95% CI = 52.4 to 69.3). Similar results were obtained with the YMRS. By the second week of treatment, 10 of the 13 patients remaining in treatment had at least a 50% improvement, and 12 of these 13 had a 25% improvement (p = .002, 95% CI = 55.1 to 89.9). By the sixth week, all of the 8 patients remaining in treatment had a 75% improvement (p = .012, 95% CI = 90.5 to 102.8). The medication was well tolerated, and no case worsened. CONCLUSION: When used with concomitant mood-stabilizing drugs, risperidone may be effective and well tolerated in patients with acute mania with psychotic features. Considering the open design, small sample size, and limited period of observation, further studies need to be conducted.

Acute Disease↗

PubMed1988

Helix stability in prokaryotic promoter regions.

Prokaryotic promoters have been extensively studied to relate sequence features to promoter function. Here we examine the relationship between double-helix stability and promoter activity. The double-helix stability is evaluated from sequence data by free energy computation, based on reported values of dinucleotide free energies for strand separation. For a collection of 168 promoters, we find that within a 500-nucleotide span around the transcription initiation site the -10 region is the least stable. There is no correlation between the free energies and the rates of RNA polymerase-promoter open complex formation measured for 25 promoters. We also compare the free energies of 121 promoter mutations across the -35 and -10 consensus regions with the free energies of the corresponding wild-type sequences. These pairwise mutant-wild-type comparisons provide a particularly good test since the examined sequences differ only in one nucleotide so that all other sequence-dependent effects remain the same. About 80% of the mutations in the -10 region that show increased/reduced promoter activity are less/more stable than the wild types. The observed high free energy peak and the mutation data strongly support the conjecture that the instability, or melting properties, of the -10 region plays a significant role in promoter function.

Cells↗