Biomedical subjects

J Santoro

Publications and source records attributed to J Santoro.

At least 37 records · Page 2Linked to original sources

PubMed1998

Characterization of pKa values and titration shifts in the cytotoxic ribonuclease alpha-sarcin by NMR. Relationship between electrostatic interactions, structure, and catalytic function.

The electrostatic behavior of titrating groups in alpha-sarcin was investigated using 1H NMR spectroscopy. A total of 209 chemical shift titration curves corresponding to different protons in the molecule were determined over the pH range of 3.0-8.5. Nonlinear least-squares fits of the data to simple relationships derived from the Henderson-Hasselbalch equation led to the unambiguous determination of pKa values for all glutamic acid and histidine residues, as well as for the C-terminal carboxylate and most of the aspartic acids in the free enzyme. The ionization constants of catalytically relevant histidines, His50 and His137, and glutamic acid, Glu96, in the alpha-sarcin-2'-GMP complex were also determined. The pKa values of 15 ionizable groups (C-carboxylate, six aspartic acids, four glutamic acids, and four histidines) were found to be close to their normal values. On the other hand, a number of side chain groups, including those in the active center, showed pKa values far from their intrinsic values. Thus, the pKa values for active site residues His50, Glu96, and His137 were 7.7, 5.2, and 5.8 in the free enzyme and 7.6, approximately 4.8, and 6.8 in the alpha-sarcin-2'-GMP complex, respectively. The pKa values and the activity profile against ApA, as a function of pH, are in agreement with the proposed enzymatic mechanism (in common with RNase T1 and the family of the microbial ribonucleases), in which Glu96 and His137 act as a general base and general acid, respectively. In almost all microbial ribonucleases, a Phe-His interaction is present, which affects the pKa of one of the His residues at the active site (His137). The absence of this interaction in alpha-sarcin would explain the lower pKa value of this His residue, and provides an explanation for the decreased RNase activity of this protein as compared to those of other microbial ribonucleases.

Aspergillus↗

PubMed1998

Solution structure of acidic fibroblast growth factor bound to 1,3, 6-naphthalenetrisulfonate: a minimal model for the anti-tumoral action of suramins and suradistas.

Recent data show that anti-angiogenesis may provide a promising route to treat cancer. Fibroblast growth factors (FGFs) are powerful angiogenic polypeptides, whose mitogenic activity requires the presence of heparin-like compounds. It has been shown that angiogenesis promoted by FGFs on inhibition by monoclonal antibodies and antisense targeting can also inhibit tumour growth. Derivatives of suramin, a polysulfonated binaphthyl urea and binaphthylsulfonated derivatives of distamycin, suradistas, constitute an important group of potential anti-cancer agents. These compounds compete with heparin in forming tight complexes with FGFs. This inhibits the recognition of these growth factors by their tyrosine kinase membrane receptors thereby suppressing their angiogenic activity. Here we show that 1,3,6-naphthalenetrisulfonate, a common chemical function of the suramins and suradistas with the highest anti-angiogenic activity inhibits the mitogenic activity of acidic fibroblast growth factor, and that this inhibition is relieved by increasing concentrations of heparin in the assay. We have also solved the three-dimensional structure in solution of the protein complexed to this compound. The structural data provide clues that may help in understanding the inhibitory effect of suramins and suradistas, and could contribute to the development of new anti-tumoral drugs.

Amino Acid Sequence↗

PubMed1996

Structural basis for the catalytic mechanism and substrate specificity of the ribonuclease alpha-sarcin.

alpha-Sarcin is a ribosome-inactivating protein which selectively cleaves a single phosphodiester bond in a universally conserved sequence of the major rRNA. The solution structure of a-sarcin has been determined on the basis of 1898 distance and angular experimental constraints from NMR spectroscopy. It reveals a catalytic mechanism analogous to that of the T1 family of ribonucleases while its exquisite specificity resides in the contacts provided by its distinctive loops.

Binding Sites↗

PubMed1996

Three-dimensional structure of acidic fibroblast growth factor in solution: effects of binding to a heparin functional analog.

Acidic and basic fibroblast growth factors (aFGF and bFGF; FGFs) are paradigms of a group of nine closely related proteins known as the fibroblast growth factor family. FGFs induce mitosis in most mesoderm- and neuroectoderm-derived cells, and appear to be involved in diseases caused by anomalous cell proliferation. In vitro assays show that binding to heparin-like glycosaminoglycans is required to elicit the mitogenic activity of these proteins. It has been shown that myo-inositol hexasulfate (MIHS) emulates heparin in the mitogenesis assays of aFGF, and a low-resolution three-dimensional structure in solution of this protein bound to MIHS has been reported. Here we describe the 1H-NMR three-dimensional structure in solution of the free aFGF. Comparison of this structure with that of the protein bound to MIHS, upgraded to a level of refinement equivalent to that of the free protein, shows that MIHS binding causes some slight conformational changes with an increase in the definition of the structure. In addition, amide exchange H/2H rates of the most protected protons, and exchange data of the intermediate and fast-exchanging ones show that the free protein is less stable (< or = 2 kcal/mol) and more flexible in terms of local unfolding equilibria, respectively, than the MIHS-bound one. Thus, MIHS binding to aFGF causes a decrease of its flexibility, which translates into an enhancement of the definition of its three-dimensional structure. The increase of aFGF rigidity affects regions that include those involved in recognizing the cell membrane receptor. Thus, our data suggest that enhancement of structural definition may play a key role in the modulation of the affinity of aFGF by its receptor, and, consequently, of its specific mitogenic activity.

Amides↗

PubMed1996

1H and 15N nuclear magnetic resonance assignment and secondary structure of the cytotoxic ribonuclease alpha-Sarcin.

The ribosome-inactivating protein alpha-Sarcin (alpha S) is a 150-residue fungal ribonuclease that, after entering sensitive cells, selectively cleaves a single phosphodiester bond in an universally conserved sequence of the major rRNA to inactivate the ribosome and thus exert its cytotoxic action. As a first step toward establishing the structure-dynamics-function relationships in this system, we have carried out the assignment of the 1H and 15N NMR spectrum of alpha S on the basis of homonuclear (1H-1H) and heteronuclear (1H-15N) two-dimensional correlation spectra of a uniformly 15N-labeled sample, and two selectively 15N-labeled (Tyr and Phe) samples, as well as a single three-dimensional experiment. The secondary structure of alpha S, as derived from the characteristic patterns of dipolar connectivities between backbone protons, conformational chemical shifts, and the protection of backbone amide protons against exchange, consists of a long N-terminal beta-hairpin, a short alpha-helical segment, and a C-terminal beta-sheet of five short strands arranged in a + 1, + 1, + 1, + 1 topology, connected by long loops in which the 13 Pro residues are located.

Amino Acid Sequence↗

PubMed1995

Three-dimensional structure of chemotactic Che Y protein in aqueous solution by nuclear magnetic resonance methods.

The three-dimensional structure of chemotactic Che Y protein from Escherichia coli in aqueous solution has been determined by nuclear magnetic resonance (NMR) spectroscopy combined with restrained molecular dynamics calculations. A total of 20 converged structures were computed from 1545 conformationally relevant distance restraints derived from 1858 unambiguously assigned NOE cross-correlations. The resulting average pairwise root-mean-square deviation is 1.03 A for the backbone atoms and 1.69 A for all heavy atoms. If residues in the regions structurally least defined (1 to 5, 47 to 50, 76 to 79, 88 to 91 and 124 to 129) are excluded from the analysis, the root-mean-square deviations are reduced to 0.53 A and 1.23 A, respectively. The solution structure is closely similar to the refined X-ray crystal structure, except in the regions found to be less defined by NMR spectroscopy. The root-mean-square deviation between the average solution structure and the X-ray crystal structure is 0.92 A for the backbone residues (2 to 129). The highly refined solution structure determined herewith provides an essential background to delineate functionally important conformational changes brought about by different effectors.

Bacterial Proteins↗

PubMed1995

NMR solution structure of the antifungal protein from Aspergillus giganteus: evidence for cysteine pairing isomerism.

The solution structure of the antifungal protein (AFP, 51 residues, 4 disulfide bridges) from Aspergillus giganteus has been determined by using experimentally derived interproton distance constraints from nuclear magnetic resonance (NMR) spectroscopy. Complete sequence-specific proton assignments were obtained at pH 5.0 and 35 degrees C. A set of 834 upper limit distance constraints from nuclear Overhauser effect measurements was used as input for the calculation of structures with the program DIANA. An initial family of 40 structures calculated with no disulfide constraints was used to obtain information about the disulfide connectivities, which could not be determined by standard biochemical methods. Three possible disulfide patterns were selected and the corresponding disulfide constraints applied to generate a family of 20 DIANA conformers for each pattern. Following energy minimization, the average pairwise RMSD of the 20 conformers of each family is 1.01, 0.89, and 1.01 A for backbone atoms and 1.82, 1.74, and 1.81 A for all heavy atoms. One of these three families contains the disulfide bridge arrangement actually present in the solution structure of AFP. Although the three families fulfill the NMR constraints, one of the disulfide patterns considered (cysteine pairs 7-33, 14-40, 26-49, 28-51) is favored among the others on the basis of previous chemical studies. It thus probably corresponds to the actual pattern of disulfide bridges present in the protein, and the corresponding family represents the solution structure of AFP. The folding of AFP consists of five antiparallel beta strands connected in a -1, -1, +3, +1 topology and highly twisted, defining a small and compact beta barrel stabilized by four internal disulfide bridges. A cationic site formed by up to three lysine side chains adjacent to a hydrophobic stretch, both at the protein surface, may constitute a potential binding site for phospholipids which would be the basis of its biological function. On the other hand, a second, minor form of AFP has been detected. NMR data, together with results from mass spectrometry, chemical analysis, and sedimentation equilibrium, suggest that this species differs from the major form in the pairs of cysteines involved in the four disulfide bridges.

Amino Acid Sequence↗

PubMed1995

1H-nmr studies on the structure of a new thionin from barley endosperm.

A new thionin from barley, omega-hordothionin, has been shown to exist in aqueous solution as a mixture of two different isoforms in a 3:2 ratio, as revealed by a complete analysis of its two-dimensional 1H-nmr spectra. The conformational heterogeneity arises from cis-trans isomerism about the Phe 12-Pro 13 peptide bond, where the major form corresponds to the cis conformation. The complete assignment of chemical shifts and nuclear Overhauser effects (NOEs) of the two isoforms allows a detailed comparative analysis of their conformational properties, even though a complete calculation of their solution structures is not possible because of a somewhat limited number of NOE constraints. Structures for the two isomers could be modeled, however, on the basis of the high structural homology between omega-hordothionin and related gamma-thionins, and under the conditions of satisfying all observed experimental data. The two isoforms adopt practically identical global folds and the structural changes imposed by cis-trans isomerization are confined to the region proximal to Pro 13. The cis-trans isomerism occurs in a conserved loop connecting the first beta-strand of the triple-stranded antiparallel beta-sheet and the alpha-helix. A comparative analysis of the sequences of this loop in the different thionins suggests that the cis-trans equilibrium about the X-Pro peptide bond depends on the size of the side chain of X (X = Gly in gamma-thionins and Phe in omega-thionin). The structural homology of this new thionin with gamma-thionins as well as with some scorpion toxins and insect defensins suggests that these proteins may share a common mode of functional activity.

Amino Acid Sequence↗

PubMed1995

Antibacterial agents in renal failure.

This article provides background information on the pharmacokinetics of antimicrobial agents in patients with normal and impaired renal function. Tables are provided to allow quick determination of appropriate dosages for varying degrees of renal failure. Specific mention is made of aminoglycoside dosing, dosage adjustment in dialysis, and antibiotic toxicity in renal failure.

Anti-Infective Agents↗

PubMed1994

1H-NMR assignment and solution structure of human acidic fibroblast growth factor activated by inositol hexasulfate.

A major fragment of human acidic fibroblast growth factor of 132 amino acid residues is shown to be as active and stable as the 139 residue molecule initially described, and commonly used in physiological studies. It is shown that inositol hexasulfate is a good substitute for heparin in both activating and protecting acidic fibroblast growth factor. The complex between the shortened form of the protein and inositol hexasulfate was used to determine the structure of activated acidic fibroblast growth factor in solution. The 1H-NMR spectrum of the complex was totally assigned, and a low-resolution, three-dimensional structure of the protein computed. The global fold of the activated acidic fibroblast growth factor is similar to that proposed for a crystallized variant of the protein obtained by genetic engineering whose activity is not dependent on heparin. The inositol hexasulfate binds to the protein through the positively charged groups of Lys126, Lys127, Arg133 and Lys142 side-chains. The computed three-dimensional structure suggests that inositol hexasulfate may stabilize and activate the protein by conferring rigidity to the hairpin involving beta-strands 10 and 11.

Amino Acid Sequence↗

PubMed1994

NMR solution structure of the isolated N-terminal fragment of protein-G B1 domain. Evidence of trifluoroethanol induced native-like beta-hairpin formation.

The solution structure of the isolated N-terminal fragment of streptococcal protein-G B1 domain has been investigated in H2O and TFE/H2O solution by CD and NMR to gain insight into the possible role that native beta-hairpin secondary structure elements may have in early protein folding steps. The fragment also has been studied under denaturing conditions (6 M urea), and the resulting NMR chemical shifts were used as a reference for the disordered state. On the basis of CD and NMR data, it is concluded that in aqueous solution the fragment is basically flexible, with two local low populated chain bends involving residues 8-9 and 14-15, respectively, in close agreement with secondary structure predictions, a structure that is different from the final folded state of that segment of the protein. The changes in the CD spectrum, the presence of several medium-range NOEs plus two long-range NOEs, and the sign of the H alpha conformational shifts reveal that the addition of TFE facilitates the formation of a set of transient beta-hairpins involving essentially the same residues that form the native beta-hairpin found in the final three-dimensional structure of the B1 domain. The stabilization of native-like structures by TFE is known to occur for helices, but, to our knowledge, this is the first time the stabilization of a native-like beta-hairpin structure by TFE is reported. Since long-range tertiary interactions are absent in the isolated fragment, our results support the idea that, in addition to helices, beta-hairpins may play an active role in directing the protein folding process.

Amino Acid Sequence↗

PubMed1994

Social cognitive factors in brain injury-associated personality change.

Often social difficulties are the most enduring difficulties after a head injury. The social deficits most frequently reported by relatives 6-12 months post-injury include egocentric styles of social interaction characterized by family members as a change in the head-injured patient's personality style. The described characteristics of head-injured patients in terms of social perspective-taking abilities are similar to those of children reported in the cognitive developmental literature. To demonstrate the nature of the social deficits in head-injured participants, social perspective-taking paradigms in the development literature were used to construct a task to be administered to both head-injured and non-head-injured adults. Results of the comparison between the two groups suggest deficits in social perspective-taking abilities in head-injured adults similar to the level demonstrated by pre-adolescent children.

Adolescent↗

PubMed1994

Use of bone scintigraphy to select patients with multiple myeloma for treatment with strontium-89.

Strontium-89 is an effective agent for palliation of pain due to bony metastases from breast and prostate carcinoma. As a functional analog of calcium, 89Sr is taken up by bone in areas of osteoblastic activity. Since patients with multiple myeloma frequently have osteolytic metastases, 89Sr might not be considered to be a therapeutic option. However, metastases which appear osteolytic by radiographs may demonstrate osteoblastic activity on bone scans. Consequently, the bone scan may be used to identify a subset of patients with osteolytic metastases who may benefit from 89Sr treatment. This report describes a patient with severe rib pain due to multiple myeloma whose chest radiograph showed multiple lucent lesions throughout the bones of the chest wall but whose bone scan showed marked osteoblastic activity. The patient was treated with 89Sr and received substantial pain relief. Bone scans may be useful in selecting myeloma patients or other cancer patients with osteolytic radiographic lesions who may benefit from 89Sr therapy.

Bone Neoplasms↗

PubMed1993

1H- and 15N-NMR assignment and solution structure of the chemotactic Escherichia coli Che Y protein.

Che Y is a 129-residue parallel alpha/beta protein involved in bacterial chemotaxis. We have used this protein as a model to study the folding reaction of parallel alpha/beta proteins. As a first step we carried out the complete assignment of the 1H and 15N spectra from Escherichia coli Che Y protein on the basis of two-dimensional 1H homonuclear and 1H-15N heteronuclear experiments by using sequence-specific methods. Our assignments differ from the preliminary assignments made by Kar et al. [Kar, L., Matsumura, P. & Johnson, M.E. (1992) Biochem. J. 287, 521-531] of aromatic residues obtained by comparison of NOEs with short proton-proton distances in the crystal structure of Che Y. The analysis of the extension of the secondary elements, as well as a preliminary calculation of the three-dimensional structure, indicate that the solution structure is closely coincident with the single crystal structure determined by X-ray diffraction.

Amino Acid Sequence↗

PubMed1993

Three-dimensional structure of omega-conotoxin GVIA determined by 1H NMR.

omega-Conotoxin GVIA, a peptide of 27 amino acid residues and three disulfide bridges, has been studied by NMR techniques. The complete assignment of the corresponding proton NMR spectra was performed by two-dimensional sequence specific methods at 288 K and pH 3.5. On the basis of 169 distance restraints derived from this analysis, the three-dimensional structure was obtained. A total of 30 initial structures were generated by distance geometry methods and further refined by restrained energy minimization techniques yielding a final set of 8 structures. The mean root-mean-square deviation between each of the 8 structures and the mean atomic coordinates for all residues is 0.82 +/- 0.06 A for the backbone atoms and 1.45 +/- 0.18 A for all non-H atoms. The structure shows a globular folding pattern that is stabilized by the three disulfide linkages and a number of intramolecular hydrogen bonds. A total of 14 hydroxyl groups are found at the periphery fully exposed to the solvent. These groups, together with the charged side chains of Lys and Arg residues emerging radially from the peptide core, provide specific recognition elements for the interaction of this toxin with neuronal calcium channels.

Amino Acid Sequence↗

PubMed1993

High-resolution three-dimensional structure of ribonuclease A in solution by nuclear magnetic resonance spectroscopy.

High-resolution three-dimensional structures of bovine pancreatic ribonuclease A in aqueous solution have been determined by nuclear magnetic resonance (NMR) spectroscopy combined with restrained molecular dynamics calculations. The structures are based on: (1) 464 interproton distance constraints with accurate upper and lower limits, determined from build-up rates of nuclear Overhauser effects (NOE) by using the complete relaxation matrix; (2) 999 more approximate upper limits for interproton distances; and (3) 42 dihedral angle constraints (37 for phi and 5 for chi 1). A total of 16 structures were calculated, which show a root-mean-square (r.m.s.) deviation of 0.66 A for the backbone atoms and 1.68 A for all heavy-atoms. The converged structures are highly similar to those found in the crystal state. r.m.s. deviation of backbone atom positions in the crystal as compared to those in the average solution structure is 0.92 A. Observed differences are concentrated in loop regions and in the neighborhood of His119 and His48 side-chains. Dynamic aspects, such as H/D amide proton exchange and side-chain mobility have been examined.

Amides↗

PubMed1993

Solution structure of gamma 1-H and gamma 1-P thionins from barley and wheat endosperm determined by 1H-NMR: a structural motif common to toxic arthropod proteins.

The complete assignment of the proton NMR spectra of the homologous gamma 1-hordothionin and gamma 1-purothionin (47 amino acids, 4 disulfide bridges) from barley and wheat, respectively, has been performed by two-dimensional sequence-specific methods. A total of 299 proton-proton distance constraints for gamma 1-H and 285 for gamma 1-P derived from NOESY spectra have been used to calculate the three-dimensional solution structures. Initial structures have been generated by distance geometry methods and further refined by dynamical simulated annealing calculations. Both proteins show identical secondary and tertiary structure with a well-defined triple-stranded antiparallel beta-sheet (residues 1-6, 31-34, and 39-47), an alpha-helix (residues 16-28), and the corresponding connecting loops. Three disulfide bridges are located in the hydrophobic core holding together the alpha-helix and the beta-sheet and forming a cysteine-stabilized alpha-helical (CSH) motif. Moreover, a clustering of positive charges is observed on the face of the beta-sheet opposite to the helix. The three-dimensional structures of the gamma-thionins differ remarkably from plant alpha- and beta-thionins and crambin. However, they show a higher structural analogy with scorpion toxins and insect defensins which also present the CSH motif.

Amino Acid Sequence↗

PubMed1992

Periodic properties of proton conformational shifts in isolated protein helices. An experimental study.

In this work, the helix-forming residues in fragments of several proteins (ribonuclease, thermolysin, tendamistat and angiogenin) were identified by NOE and the helix proton shifts were measured as delta changes associated with helix-population increments driven by trifluoroethanol addition. When estimated in this way, a regular pattern of helix conformational shifts was clearly seen in the delta delta versus sequence profiles of all the peptides studied. The helix periodicity of the H alpha and H beta resonances was especially clear, an observation that earlier statistical studies of protein delta values failed to predict. Amide protons showed the largest helix shifts, but with a less-sharply defined periodic character. Aromatic residues considerably distorted the periodicity of the helix amide shifts in some peptides, as evidenced by the delta shifts of a RNase A fragment 1-15 analog in which the two aromatic residues were replaced by Ala. The relationship between helix periodicity and peptide amphiphatic character is discussed.

Amino Acid Sequence↗