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C R Middaugh

Publications and source records attributed to C R Middaugh.

At least 55 records · Page 3Linked to original sources
PubMed1994

Delta-sleep-inducing peptide: solution conformational studies of a membrane-permeable peptide.

Peptides and peptide-like molecules as a class have very poor permeability through biological membranes, which severely compromises their potential effectiveness as therapeutic agents. In order to gain insight into the problem of delivering peptide and protein drugs and to establish a model in which the effects of systematic structural variations on transport can be explored, an investigation of the solution conformation of a membrane-permeable peptide was undertaken. Delta-sleep-inducing peptide (DSIP, MW 849) was used in this investigation. DSIP is a charged, hydrophilic peptide that possesses the unusual ability to diffuse passively across the blood-brain barrier (BBB) in vivo [Kastin, A. J., Banks, W. A., Castellanos, P. F., Nissen, C., & Coy, D. H. (1982) Pharmacol. Biochem. Behav. 17, 1187-1191] and across monolayers of brain microvessel endothelial cells in vitro, a model of the BBB [Raeissi, S., & Audus, K. L. (1989) J. Pharm. Pharmacol. 41, 848-852]. This nonapeptide was studied in solution using one- and two-dimensional nuclear magnetic resonance (NMR), circular dichroism (CD), Fourier transform infrared (FT-IR), and fluorescence spectroscopies in conjunction with molecular modeling. Our spectroscopic findings suggest that DSIP exists in a dynamic equilibrium between unordered and folded structures. Residues 2-5 and 6-9 tend to form type I beta-turns in aqueous solution and a similar, but more ordered, helix-like structure inducible in 40% trifluoroethanol (TFE). NMR, FT-IR, and CD studies in aqueous solution support the dynamic equilibrium hypothesis with the IR data, suggesting that the beta-turn population is approximately 40%.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

The analysis of protein pharmaceuticals: near future advances.

The analysis of protein pharmaceuticals currently involves a complex series of chromatographic, electrophoretic, spectroscopic, immunological and biological measurements to unequivocally establish their identity, purity and integrity. In this review, I briefly consider the possibility that at least the functional identity and integrity of a protein drug might be established by either a single analysis involving X-ray diffraction, NMR or mass spectrometry, or by a chromatographically based multi-detector system in which a number of critical parameters are essentially simultaneously determined. The use of a protein standard to obtain comparative measurements and new advances in the technology of each of these methods is emphasized. A current major obstacle to the implementation of these approaches is the frequent microheterogeneity of protein preparations. The evolution of biological assays into measurements examining more defined intracellular signal transduction events or based on novel biosensors as well as the analysis of vaccines is also briefly discussed.

Biotechnology↗
PubMed1993

Sucralfate and soluble sucrose octasulfate bind and stabilize acidic fibroblast growth factor.

The actions of the anti-ulcer drug sucralfate have been proposed to be mediated through interaction with fibroblast growth factors (Folkman, J., Szabo, S., Strovroff, M., McNeil, P., Li, W. and Shing, Y. (1991) Ann. Surg. 214, 414-427). We show here that acidic fibroblast growth factor (aFGF; FGF-1) binds in vitro to both the soluble potassium salt and the insoluble aluminum salt of sucrose octasulfate, as demonstrated by a variety of biophysical techniques. Similar to the well-described interaction and stabilization of aFGF by heparin, soluble sucrose octasulfate (SOS) stabilizes aFGF against thermal, urea and acidic pH-induced unfolding as determined by a combination of circular dichroism, fluorescence spectroscopy and differential scanning calorimetry. In addition, SOS also enhances the mitogenic activity of aFGF and partially protects the protein's three cysteine residues from copper-catalyzed oxidation. SOS competes with heparin and suramin for the aFGF polyanion binding site as measured by both fluorescence and light scattering based competitive binding assays. Front-face fluorescence measurements show that the native, folded form of aFGF binds to the insoluble aluminum salt of sucrose octasulfate (sucralfate). Moreover, sucralfate stabilizes aFGF against thermal and acidic pH-induced unfolding to the same extent as observed with SOS. Thus, due to their high charge density, SOS and sucralfate bind and stabilize aFGF via interaction with the aFGF polyanion binding site.

Binding, Competitive↗
PubMed1993

Partially structured self-associating states of acidic fibroblast growth factor.

A combination of near- and far-UV circular dichroism, Fourier-transform infrared spectroscopy, tryptophan fluorescence, size-exclusion chromatography, and a fluorescent extrinsic hydrophobic probe has been employed to characterize partially structured states of human recombinant acidic fibroblast growth factor (aFGF). At low pH, the addition of specific polyanionic ligands or moderate amounts of salts induces states with high secondary but low tertiary structure content. At neutral pH, intermediate amounts of chaotropic agents impose similar partially structured conformational states which also display noncooperative unfolding transitions. Kinetic evidence indicates that similar forms of the protein exist in the first few hundred milliseconds in the refolding pathway of aFGF. The kinetics of their formation appear to be temperature-independent, implying lack of an energy barrier, which is characteristic for further slow folding into the native state. Unlike the native and fully unfolded states, these partially structured conformations exhibit very low solubility, resulting in irreversible aggregation. Potential physiological implications of the existence of such "molten globule" states with regard to the growth factor's transport and biological activity are considered.

Chromatography, Gel↗
PubMed1993

Effect of polyanions on the unfolding of acidic fibroblast growth factor.

The urea-induced unfolding of acidic fibroblast growth factor (aFGF) in the presence and absence of various polyanions has been quantitatively examined by fluorescence spectroscopy. In the absence of a stabilizing polyanion, the apparent free energy of unfolding of aFGF is 6.5 kcal mol-1. The presence of equimolar or greater amounts of heparin stabilizes aFGF from unfolding by more than 2.5 kcal mol-1 and slows the rate of unfolding by greater than 2000-fold. The ability of heparin to stabilize aFGF is critically dependent upon many factors including the number of aFGF molecules bound to the heparin chain, ionic strength, temperature, and the extent of sulfation of the polysaccharide. The presence of similar amounts of other polyanions such as sulfated beta-cyclodextrin or heparan sulfate also stabilizes aFGF to a similar extent as heparin. Additional experiments demonstrate that increasing charge density enhances the ability of polyanions such as sulfated beta-cyclodextrins, phosphorylated inositols, and modified heparins to protect aFGF from urea-induced unfolding.

Calorimetry↗
PubMed1993

Nature of the interaction of heparin with acidic fibroblast growth factor.

The binding of human acidic fibroblast growth factor (aFGF) to heparin has been analyzed by a variety of different approaches to better elucidate the nature of this protein/sulfated polysaccharide interaction. Static and dynamic light scattering as well as analytical ultracentrifugation analyses indicates that 14-15 molecules of a FGF can bind to a 16-kDa heparin chain, with approximately 10 of these bound relatively uniformly to high-affinity sites. The dissociation constants of these latter sites are estimated to be approximately 50-140 nM on the basis of surface plasmon resonance experiments in which the association and dissociation rates of aFGF interaction with immobilized heparin were measured. The size of the binding site of a FGF on heparin was also determined by heparin lyase digestion of a FGF/heparin complexes followed by isolation and characterization of protected oligosaccharides. The smallest aFGF-protected oligosaccharide comigrated with delta UA2S(1-->4)-alpha-D-GlcNp2S6S(1-->4)-alpha-L-IdoAp-2S( 1-->4)-alpha-D-GlcNp2S6S (where delta UA represents 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid and S is sulfate). Thus, aFGF appears to bind at high density (one molecule every 4-5 polysaccharide units) and with high affinity to heparin. This potentially provides a concentrated, stabilized storage form of the growth factor that can be released for receptor-mediated cellular activation in response to the proper stimuli. It is also possible that close proximity of aFGF molecules on the highly sulfated regions of heparan chains may be involved in the induction of receptor aggregation as suggested by Ornitz et al. [Ornitz, D. M., Yayon, A., Flanagan, J. G., Svahn, C. M., Levi, E., & Leder, P. (1992) Mol. Cell. Biol. 12, 240-247].

Binding Sites↗
PubMed1993

A transforming growth factor-alpha-Pseudomonas exotoxin hybrid protein undergoes pH-dependent conformational changes conducive to membrane interaction.

TP40 is a chimeric protein containing transforming growth factor alpha (TGF-alpha) at the N-terminus and a derivative of a 40,000-Da segment (PE40 delta cys) of Pseudomonas exotoxin (PE). PE40 delta cys contains domains Ib, II, and III of PE in which the cysteines are mutated to alanines. The rationale for inclusion of TGF-alpha is to provide TP40 with selective targeting toward cells expressing the epidermal growth factor receptor (EGFr) on their surface [Pastan, I., & FitzGerald, D. (1989) J. Biol. Chem. 264, 15157-15160]. Translocation across endosomal membranes is thought to be a required step for cytotoxic activity of PE. This step is presumably facilitated by the low pH in endosomes which induces exposure of a hydrophobic surface of the protein, which in turn becomes available to interact with and translocate across the membrane. We have employed the hydrophobic fluorescence probe 2-p-toludinylnaphthalene-6-sulfonate (TNS) and the intrinsic tryptophan fluorophores of TP40 to investigate pH-induced changes in the tertiary structure of this protein. The pH dependence of TP40 interaction with liposomes also provided a model for studying protein-membrane interactions. TNS fluorescence was markedly enhanced in the presence of TP40 below pH 4 and to a lesser degree between pH 7 and 5. A progressive red shift of tryptophan fluorescence with decreasing pH was also seen with the approximate midpoint for this transition occurring around pH 3. Both observations suggest that acidic pH induces exposure of hydrophobic regions of TP40, making them accessible to solvent and TNS. No major alteration of the secondary structure was manifested in the far-UV CD spectrum of TP40 upon a reduction in pH from 7 to 2. Thus, the low-pH-induced structural change of TP40 appears to involve a subtle exposure of one or more hydrophobic surfaces without an extensive unfolding of the protein's secondary structure. In the presence of anionic liposomes, a low-pH-induced blue shift of the TP40 tryptophan fluorescence was observed, suggesting that interaction with liposomes also required the low-pH conformation of the protein. However, the midpoint of this fluorescence blue shift occurred at approximately pH 5, which is presumably closer to the physiological pH within endosomes. Neutral liposomes failed to induce these spectral changes in TP40, implying a lack of interaction with these lipids. At acidic pH values between 2 and 4, self-association of TP40 in solution was detected by equilibrium sedimentation and quasielastic light scattering measurements. This probably results from intermolecular interaction between exposed hydrophobic surfaces.(ABSTRACT TRUNCATED AT 400 WORDS)

ADP Ribose Transferases↗
PubMed1993

Changes in structure of alpha 2-macroglobulin upon reaction with trypsin as assessed by light scattering and differential scanning calorimetry.

Employing a combination of static and dynamic light scattering, as well as differential scanning calorimetry (DSC), the structural changes which appear in alpha 2-macroglobulin (alpha 2M) upon trypsin binding have been further characterized. Light-scattering measurements suggest that a 15% reduction in both the hydrodynamic radius and radius of gyration occurs when two molecules of trypsin complex to alpha 2M. Approx. 85% of this trypsin-induced compaction results from the binding of the first proteinase. A complementary result was obtained from DSC measurements in which the major fraction of the trypsin-induced conversion of alpha 2M to a single more thermally stable form results from interaction with the first proteinase molecule. These observations support a functionally asymmetric model of trypsin binding to alpha 2M in which the significant reduction in size of the complex is primarily due to the initial interaction of alpha 2M with a single proteinase molecule.

Calorimetry, Differential Scanning↗
PubMed1993

Structure of synthetic peptide analogues of an eggshell protein of Schistosoma mansoni.

The peptide (Gly-L-Tyr-L-Asp-L-Lys-L-Tyr)6, referred to as F4-6, was synthesized as a model for a schistosome eggshell protein in which the Gly-Tyr-Asp-Lys-Tyr consensus sequence is repeated over 40 times. Analysis by CD, Fourier transform infrared spectroscopy, potentiometric and spectrophotomertric titrations, NMR, and molecular modeling suggests that F4-6 forms some type of left-handed structure. A likely possibility appears to be a left-handed alpha-helix stabilized by Lysi-Aspi +4 salt bridges and possibly Aspi-Tyri +4 hydrogen bonding and Tyr-Tyr interactions. Spectroscopic studies of a number of F4-6 analogues support this conclusion. For example, substitution of D-Ala for Gly produces a peptide with enhanced left-handed helical spectral characteristics, whereas an L-Ala substitution results in a peptide with minimal structure. These studies suggest that the F4 protein from Schistosoma mansoni may be the first example of a naturally occurring protein devoid of proline and carbohydrate that forms a left-handed helix composed of L-amino acids, although alternative forms of other left-handed structures have yet to be rigorously excluded.

Amino Acid Sequence↗
PubMed1993

Physical stabilization of acidic fibroblast growth factor by polyanions.

Acidic fibroblast growth factor (aFGF) is markedly stabilized by heparin. Partially due to the heterogeneity of heparin preparations, the nature of the aFGF polyanion binding site is still ill-defined. We have, therefore, investigated a wide variety of well-defined polyanions in terms of their ability to stabilize human recombinant aFGF (15-154) against thermal denaturation. The specificity of the interaction between aFGF and polyanions is shown to be remarkably weak with a surprising number of polyanions (including small phosphorylated and sulfated compounds as well as highly charged biopolymers) able to induce physical stability. Temperature-dependent fluorescence and circular dichroism measurements show that many of these polyanionic compounds stabilize aFGF to the same extent as heparin. The ability of these agents to protect the three free thiol groups of aFGF from copper-catalyzed oxidation was also explored and significant protection was observed. The extent and electrostatic requirements of the protein's polyanion binding site were probed by the use of a series of well-defined heparin fragments and differentially phosphorylated inositol compounds. A tetrasaccharide fragment of heparin is the smallest unit of heparin capable of stabilizing aFGF against thermal denaturation. Increasing phosphorylation of inositol compounds (up to six phosphate groups per molecule) enhances the thermal stability of aFGF. These results are discussed in the context of a model of human aFGF based on the X-ray crystal structure of the bovine protein and previous studies by others of the heparin binding site of both acidic and basic FGF.

Amino Acid Sequence↗
PubMed1993

Origin of the isoelectric heterogeneity of monoclonal immunoglobulin h1B4.

The origin of the microheterogeneity of a highly purified antiinflammatory humanized monoclonal antibody prepared in mammalian cell culture has been investigated. This antibody is an IgG directed toward human CD18 (a subunit of leukocyte integrins). When the IgG preparation is subjected to isoelectric focusing, it is found to contain four major species with pI values ranging from 6 to 7. Although the relative amounts of each form differ and some species are present only in small quantities, each has been isolated by a combination of high-resolution anion-exchange chromatography and isoelectric focusing. Comparative studies reveal no detectable differences in overall secondary (far UV circular dichroism) or tertiary (intrinsic fluorescence) structure, molecular weight (laser-desorption mass spectroscopy), or antigen binding activity. When each of the isolated species is incubated under conditions which favor deamidation, it is converted to forms of lower pI which appear to correspond to naturally observed species. While the isolated light chain is relatively homogeneous, the heavy chain exhibits a pattern of isoelectric focusing bands similar to that of the intact immunoglobulin. These results suggest that in this case, charge microheterogeneity is due to the sequential deamidation of the immunoglobulin heavy chain.

Antibodies, Monoclonal↗
PubMed1993

Formulation design of acidic fibroblast growth factor.

The design of an aqueous formulation for acidic fibroblast growth factor (aFGF) requires an understanding of the type of compounds that can either directly or indirectly stabilize the protein. To this end, spectrophotometric turbidity measurements were initially employed to screen the ability of polyanionic ligands, less specific compounds, and variations in solution conditions (temperature and pH) to stabilize aFGF against heat-induced aggregation. It was found that in addition to the well-known protection of aFGF by heparin, a surprisingly wide variety of polyanions (including small sulfated and phosphorylated compounds) also stabilizes aFGF. These polyanionic ligands are capable of raising the temperature at which the protein unfolds by 15-30 degrees C. Many commonly used excipients were also observed to stabilize aFGF in both the presence and the absence of heparin. High concentrations of some of these less specific agents are also able to increase the temperature of aFGF thermal unfolding by as much as 6-12 degrees C as shown by circular dichroism and differential scanning calorimetry. Other compounds were found which protect the chemically labile cysteine residues of aFGF from oxidation. Aqueous formulations of aFGF were thus designed to contain both a polyanionic ligand that enhances structural integrity by binding to the protein and chelating agents (e.g., EDTA) to prevent metal ion-catalyzed oxidation of cysteine residues. While room-temperature storage (30 degrees C) leads to rapid inactivation of aFGF in physiological buffer alone, several of these aFGF formulations are stable in vitro for at least 3 months at 30 degrees C. Three aFGF topical formulations were examined in an impaired diabetic mouse model and were found to be equally capable of accelerating wound healing.

3T3 Cells↗
PubMed1992

Nature of the interaction of growth factors with suramin.

Suramin inhibits the binding of a variety of growth factors to their cell surface receptors. The direct interaction of suramin with acidic fibroblast growth factor has been detected by the enhancement of the drug's fluorescence in the presence of the protein with the maximum effect occurring at a molar ratio of suramin to aFGF of 2:1. This interaction stabilizes aFGF to thermal denaturation and partially protects a free thiol in its polyanion binding site from oxidation. The binding of suramin to aFGF also induces aggregation of the growth factor to at least a hexameric state as detected by static and dynamic light scattering as well as by gel filtration studies. Both CD and amide I' FTIR spectra of aFGF in the presence and absence of suramin suggest that the drug may also be causing a small conformational change in the growth factor. Suramin produces an even greater aggregation of bFGF and PDGF but not of EGF or IGF-1. Evidence for a suramin-induced conformational change in IGF-1 but not EGF is found by CD, however. It is concluded that suramin binds to many growth factors and that this induces microaggregation and, in some cases, conformational changes. In the case of aFGF, suramin interacts at or near its heparin binding site. The relationship between these phenomena and the anti-growth factor activity of suramin remains to be clearly elucidated.

3T3 Cells↗
PubMed1992

Structural implications of spectroscopic characterization of a putative zinc finger peptide from HIV-1 integrase.

The N-terminal domain of human immunodeficiency virus (HIV-1) integrase (IN) contains the sequence motif His-Xaa3-His-Xaa23-Cys-Xaa2-Cys, which is strongly conserved in all retroviral and retrotransposon IN proteins. This structural motif constitutes a putative zinc finger in which a metal ion may be coordinately bound by the His and Cys residues. A recombinant peptide, IN(1-55), composed of the N-terminal 55 amino acids of HIV-1 IN was expressed in Escherichia coli and purified. Utilizing a combination of techniques including UV-visible absorption, circular dichroism, Fourier transform infrared, and fluorescence spectroscopies, we have demonstrated that metal ions (Zn2+, Co2+, and Cd2+) are bound with equimolar stoichiometry by IN(1-55). The liganded peptide assumes a highly ordered structure with increased alpha-helical content and exhibits remarkable thermal stability. UV-visible difference spectra of the peptide-Co2+ complexes directly implicate thiols in metal coordination, and Co2+ d-d transitions in the visible range indicate that Co2+ is tetrahedrally coordinated. Mutant peptides containing conservative substitutions of one of the conserved His or either of the Cys residues displayed no significant Zn(2+)-induced conformational changes as monitored by CD and fluorescence spectra. We conclude that the N terminus of HIV-1 IN contains a metal-binding domain whose structure is stabilized by tetrahedral coordination of metal by histidines 12 and 16 and cysteines 40 and 43. A preliminary structural model for this zinc finger is presented.

Amino Acid Sequence↗
PubMed1992

Tight folding of acidic fibroblast growth factor prevents its translocation to the cytosol with diphtheria toxin as vector.

A fusion protein of acidic fibroblast growth factor and diphtheria toxin A-fragment was disulfide-linked to the toxin B-fragment. The complex bound specifically to diphtheria toxin receptors, and subsequent exposure to low pH induced the fusion protein to translocate to the cytosol. Heparin, inositol hexaphosphate and inorganic sulfate strongly increased the trypsin resistance of the growth factor part of the fusion protein, indicating tight folding, and they prevented translocation of the fusion protein to the cytosol. The data indicate that only a more disordered form of the growth factor is translocation competent.

Animals↗
PubMed1992

Detection of proteins and phenol in DNA samples with second-derivative absorption spectroscopy.

We have employed near-uv second-derivative spectra of DNA, N-acetyl-L-tryptophanamide, N-acetyl-L-tyrosinamide, N-acetyl-L-phenylalanine ethyl ester, and phenol in a matrix least-squares multicomponent analysis algorithm to detect the presence of tryptophan, tyrosine, phenylalanine, and/or phenol in DNA preparations. With this method, each of these compounds can be detected in a DNA sample (absorbance, 0.1) at absorbance levels of less than 0.002. In practice, the presence of proteins can be detected at absorbance levels of less than 0.003. Using second-derivative spectra of proteins, contents of mixtures of proteins and DNA can be determined with less than 1% error. Mixtures of DNA and RNA can also be quantitatively analyzed with an error of approximately 2%. This technique can be easily implemented with computer-controlled spectrophotometers equipped with standard spectral analysis software. With prerecorded standard spectra, the time of analysis does not exceed a few seconds.

Animals↗
PubMed1992

Statistical determination of the average values of the extinction coefficients of tryptophan and tyrosine in native proteins.

Spectroscopic measurement of protein concentration requires knowledge of the value of the relevant extinction coefficient. If the amino acid composition of a protein is known, however, extinction coefficients can be calculated approximately, provided that the values of the molar absorptivities for tryptophan and tyrosine residues in the protein are known. We have applied a matrix linear regression procedure and a mapping of average absolute deviations between experimental and calculated values to find molar extinction coefficients (epsilon M, 1 cm, 280 nm) of 5540 M-1 cm-1 for tryptophan and 1480 M-1 cm-1 for tyrosine residues in an "average" protein, as defined by a set of experimentally determined extinction coefficients for more than 30 proteins. Use of these values provides a significant improvement in extinction coefficient estimation over that obtained with the commonly used values obtained from solutions of model compounds in guanidine-HCl. The consistency of these results when compared to the large deviations often observed between experimentally determined extinction coefficients suggest that this method may offer acceptable accuracy in the initial estimation of molar absorptivities of globular proteins.

Animals↗