Biomedical subjects

B Brodsky

Publications and source records attributed to B Brodsky.

At least 55 records · Page 3Linked to original sources

PubMed1996

Disrupted collagen architecture in the crystal structure of a triple-helical peptide with a Gly-->Ala substitution.

The crystal structure of the collagen-based peptide (Pro-Hyp-Gly)4-Pro-Hyp-Ala-(Pro-Hyp-Gly)5 has provided for the first time a highly detailed picture of the architectural elements that come into play in the collagen triple helix. The center of the molecule, which harbors a Gly-->Ala substitution, shows subtle conformational changes that result in a local untwisting of the triple helix. The characteristic hydrogen bonding pattern of collagen triple helices is replaced by interstitial water bridges. These effects may be relevant to the diseased states derived from Gly-->X mutations in collagens. The possible implications of this disrupted architecture for collagen assemblies are discussed.

Alanine↗

PubMed1995

Hydration structure of a collagen peptide.

BACKGROUND: The collagen triple helix is a unique protein motif defined by the supercoiling of three polypeptide chains in a polyproline II conformation. It is a major domain of all collagen proteins and is also reported to exist in proteins with host defense function and in several membrane proteins. The triple-helical domain has distinctive properties. Collagen requires a high proportion of the post-translationally modified imino acid 4-hydroxyproline and water to stabilize its conformation and assembly. The crystal structure of a collagen-like peptide determined to 1.85 Angstrum showed that these two features may be related. RESULTS: A detailed analysis of the hydration structure of the collagen-like peptide is presented. The water molecules around the carbonyl and hydroxyprolyl groups show distinctive geometries. There are repetitive patterns of water bridges that link oxygen atoms within a single peptide chain, between different chains and between different triple helices. Overall, the water molecules are organized in a semi-clathrate-like structure that surrounds and interconnects triple helices in the crystal lattice. Hydroxyprolyl groups play a crucial role in the assembly. CONCLUSIONS: The roles of hydroxyproline and hydration are strongly interrelated in the structure of the collagen triple helix. The specific, repetitive water bridges observed in this structure buttress the triple-helical conformation. The extensively ordered hydration structure offers a good model for the interpretation of the experimental results on collagen stability and assembly.

Alanine↗

PubMed1995

Acid destabilization of a triple-helical peptide model of the macrophage scavenger receptor.

Electrostatic interactions were studied in a triple-helical peptide, (POG)3PKGQKGEKG(POG)4, which contains a lysine-rich 9 residue sequence from the collagen-like domain of the macrophage scavenger receptor (MSR). This peptide adopts a stable triple-helical conformation only when the pH is higher than 4.5, corresponding to ionization of the Glu side chain. Modeling shows Glu forms ion pairs with one of the Lys residues, stabilizing the structure. Previously studied collagen-like peptides show relatively small contributions of electrostatic interactions to stability. The large magnitude of the pH mediated structural changes seen for this peptide suggests that specific placement of charged residues in the triple-helix conformation can generate strong electrostatic interactions.

Amino Acid Sequence↗

PubMed1995

Atypical Gly-X-Y sequences surround interruptions in the repeating tripeptide pattern of basement membrane collagen.

The triple-helical domains of type IV collagen chains have more than 20 sites at which the repeating (Gly-X-Y)n pattern is interrupted. Analysis of alpha 1 (IV) and alpha 2 (IV) chains indicates the residues in the three Gly-X-Y triplets preceding or following interruptions differ statistically from the rest of the chain. Unusually high frequencies of charged residues are seen at a number of X and Y sites, with the charge density being particularly high C-terminal to the interruption site. Analyses were carried out on individual categories of interruptions, classified as insertions or deletions in the Y position. All of the residues in the X and Y positions of the triplets flanking insertion sites are atypical, with a high concentration of charged residues. Triplets flanking sites where there has been a deletion in the Y position show unusually high frequencies of charged residues at some sites, hydrophobic residues at other sites, and an invariant imino acid N-terminal to the interruption. The presence of atypical sequences surrounding interruptions could be important at a molecular level, related to triple-helix stability, or at a supramolecular level, related to the association of molecules to form networks in basement membranes.

Animals↗

PubMed1995

Protein motifs. 8. The triple-helix motif in proteins.

The triple helix is an important motif found in the family of collagens as well as a set of host-defense proteins. This conformation may be identified by its strict sequence constraints, including glycine as every third residue and a high content of imino acids. The first high-resolution structure available for a triple helix has confirmed the model of three supercoiled polyproline II-like helices and has defined a highly ordered water network whose regularity depends on the presence of 4-hydroxyproline. The role of the rod-like triple helix lies in its capacity to self-associate in a variety of forms as well as its ability to bind a wide range of ligands. The extensive hydrogen-bonded water network, together with the high content of sterically restricted imino acids, are the major contributors to the stabilization of triple helices, whereas electrostatic and hydrophobic interactions define intermolecular association and ligand binding. Mutations in the repeating Gly-X-Y sequences of triple helices have been shown to cause a variety of human diseases.

Amino Acid Sequence↗

PubMed1994

Collagen fibril structure in lamprey.

X-ray diffraction and electron microscopy are used to compare the molecular and higher order structure of collagen fibrils in three tissues of the lamprey: the dermis, perinotochord and notochord sheath. These lamprey tissues are known to contain five distinct genetic types of fibrillar collagen. The modes of axial and lateral packing of collagen molecules in fibrils of the lamprey tissues demonstrate the three major motifs seen in higher vertebrate D-periodic collagen fibrils. In particular, lamprey dermis was found to have a decreased D period resulting from a molecular tilt that is seen in skins of higher vertebrates. Our results suggest the molecular-packing motifs for collagen fibrils were in place at the dawn of vertebrate evolution and have been conserved since. In contrast, the diameters of fibrils and their spatial orientation in lamprey tissues do not in general, correspond to features found in mammalian tissues. Only for lamprey notochord is there a strong similarity, with fibril diameters and organization closely resembling those seen in type II tissues of higher vertebrates. This suggests that for all tissues except those with type II collagen, higher level organization of fibrils evolved along with the diversification of vertebrates.

Animals↗

PubMed1994

Crystal and molecular structure of a collagen-like peptide at 1.9 A resolution.

The structure of a protein triple helix has been determined at 1.9 angstrom resolution by x-ray crystallographic studies of a collagen-like peptide containing a single substitution of the consensus sequence. This peptide adopts a triple-helical structure that confirms the basic features determined from fiber diffraction studies on collagen: supercoiling of polyproline II helices and interchain hydrogen bonding that follows the model II of Rich and Crick. In addition, the structure provides new information concerning the nature of this protein fold. Each triple helix is surrounded by a cylinder of hydration, with an extensive hydrogen bonding network between water molecules and peptide acceptor groups. Hydroxyproline residues have a critical role in this water network. The interaxial spacing of triple helices in the crystal is similar to that in collagen fibrils, and the water networks linking adjacent triple helices in the crystal structure are likely to be present in connective tissues. The breaking of the repeating (X-Y-Gly)n pattern by a Gly-->Ala substitution results in a subtle alteration of the conformation, with a local untwisting of the triple helix. At the substitution site, direct interchain hydrogen bonds are replaced with interstitial water bridges between the peptide groups. Similar conformational changes may occur in Gly-->X mutated collagens responsible for the diseases osteogenesis imperfecta, chondrodysplasias, and Ehlers-Danlos syndrome IV.

Alanine↗

PubMed1994

Electrostatic interactions in collagen-like triple-helical peptides.

Collagen-like peptides with potential for ion pair formation were studied to investigate the role of electrostatic interactions in the triple-helix conformation. Three peptides--(POG)10, the EK-containing peptide (POG)4EKG(POG)5, and T3-487, a peptide with 18 residues of type III collagen and a C-terminal (GPO)4 tail--all form stable triple helices in aqueous solution, with melting temperatures of 58, 46, and 26 degrees C, respectively, at neutral pH. The thermal stabilities of these peptides correlate with their imino acid content, which is 66%, 60%, and 41%, respectively. Variation of pH over the range of 1-13 led to 8-9 degrees C changes in the Tm of the EK-containing peptide and peptide T3-487, with the greatest stability seen at pH values where both acidic and basic residues are ionized. Equilibrium ultracentrifugation shows these peptides are largely trimeric at low temperature, with no hexamers or larger aggregates, indicating that the pH-dependent stability arises from intramolecular interaction. Computer modeling indicates both intrachain ion pairs and interchain ion pairs can form and stabilize the triple helix. Studies of the pH dependence of the thermal stability of (POG)10 and the N-terminal acetylated form of T3-487 indicate that repulsion of the three charged N-terminal or C-terminal ends has a destabilizing effect. Taking into account these end effects, the energy contribution of two oppositely charged residues in a triple helix which are sterically capable of participating in ion pairs and backbone hydrogen bonding is 0.5-1 kcal/mol ion pair. It is possible that the stabilizing influence of ion pairs arises indirectly, through elimination of like charge repulsion, formation of ion pairs in the single chain form, or solvent effects.

Amino Acid Sequence↗

PubMed1994

Collagen organization in an oriented fibrous capsule.

X-ray diffraction and polarized light microscopy studies were carried out on a fibrous capsule to examine the collagen organization produced in a granuloma response. The capsule had been formed around a silicone tube covered with polyester mesh following implantation in a sheep. The fibrous capsule has previously been shown to include a substantial amount of type III collagen, in addition to type I collagen. Whereas granulomas generally show little order, the sample in the present study has a high degree of orientation of the collagen fibrils along the tube direction. X-ray diffraction patterns of the capsule showed an axial D period near 65 nm, a value similar to that of other type III collagen-containing tissues. This axial period is less than the 67 nm period seen in tendon and bone, which contain only type I collagen. The decreased D period is consistent with an increase in the tilting of the collagen molecules with respect to the fibril axis.

Animals↗

PubMed1993

Backbone dynamics of (Pro-Hyp-Gly)10 and a designed collagen-like triple-helical peptide by 15N NMR relaxation and hydrogen-exchange measurements.

The backbone dynamics of specific residues in two collagen-like triple-helical peptides with (X-Y-Gly)n sequences have been investigated using two-dimensional inverse-detected 15N NMR relaxation measurements and hydrogen-exchange experiments. One peptide, (POG)10, has the highest possible imino acid content and is considered to be a very stable prototype of a triple helix. The second peptide, (POG)3ITGARGLAGPOG(POG)3 (denoted T3-785), models an imino acid poor region of type III collagen and contains 12 residues from near the unique collagenase cleavage site. 15N relaxation parameters and hydrogen-exchange data were obtained for a glycine residue in the center of (POG)10 and for the tripeptide unit Gly-Leu-Ala in the middle of T3-785. Analysis of the relaxation data of the rodlike triple-helical peptides required the assumption of anisotropic overall motion, and the model-free approach of Lipari and Szabo (1982) was used to derive overall motional parameters and the order parameter, S2, that describes the amplitudes of the internal motion. First the mobilities of the Gly, Leu, and Ala residues in peptide T3-785 were compared. Both hydrogen-exchange methods and relaxation measurements indicated that the residue in the Y position (Ala) is more mobile than residues in the Gly and X positions (Leu). The slower exchange rates of Gly and Leu compared to that of Ala are consistent with the two-hydrogen-bonded model for the triple helix. Then the backbone mobilities of the central Gly residue were compared for the two peptides (POG)10 and T3-785. In this case, 15N relaxation measurements give different results from hydrogen exchange. The glycine residues in the trimer form of both T3-785 and (POG)10 have high values for the order parameter (near 0.85), suggesting similar small-amplitude internal motions and rigid backbones in both peptides. In contrast to the similar values of the order parameters, hydrogen-exchange data indicate that the central Gly exchanges at a faster rate in the trimer form of T3-785 than in (POG)10. These results suggest that a Gly in the imino acid rich environment of (POG)10 is dynamically different from a Gly in the imino acid poor environment of T3-785 and that the difference lies in the slower motion related to stability, rather than the faster motion on the picosecond time scale. This sequence-dependent difference in dynamical properties may have important consequences for recognition processes in collagen.

Amino Acid Sequence↗

PubMed1993

Characterization of collagen-like peptides containing interruptions in the repeating Gly-X-Y sequence.

Glycine is found as every third residue along the entire length of triple helices in fibrillar collagens, but the triple-helix regions of nonfibrillar collagens and other proteins usually contain one or more interruptions in this repeating pattern. A set of four peptides was designed to model the effect of interruptions in the (Gly-X-Y)n repeating pattern on triple-helix formation, stability, and folding. Into the middle of the stable triple-helical peptide (Pro-Hyp-Gly)10, an interruption was introduced representing one of the four possible categories: a glycine deletion, a deletion of a hydroxyproline (Y position), an alanine insertion, or a glycine to alanine substitution. As shown by sedimentation equilibrium, NMR, and CD studies, the introduction of an interruption still allowed formation of trimers in solution, but with marked decrease in stability. The degree of destabilization and the thermodynamic basis for the loss of stability depended on the type of interruption. The glycine substitution and alanine insertion were the least disruptive, followed by the hydroxyproline deletion, with the glycine deletion being the most destabilizing. Our results suggest that the breaks in these peptides affect both the triple-helical conformation and the monomer conformation. These studies provide a basis for considering the structural and functional consequences of different kinds of interruptions in collagen.

Amino Acid Sequence↗

PubMed1993

Two-dimensional NMR assignments and conformation of (Pro-Hyp-Gly)10 and a designed collagen triple-helical peptide.

Homonuclear and heteronuclear 2D NMR methods are used to study two triple-helical peptides. One peptide, (POG)10, is considered to be the most stable prototype of a triple helix. The second peptide, (POG)3ITGARGLAGPOG(POG)3 (denoted T3-785), was designed to model an imino acid poor region of collagen and contains 12 residues from near the unique collagenase cleavage site in type III collagen. Both peptides associated as trimers, with melting temperatures of 60 degrees C for (POG)10 and 25 degrees C for the T3-785 peptide. Sequence-specific assignments were made for a tripeptide unit POG in (POG)10, and 80% of the POG triplets are found to be in an equivalent environment. In T3-785, with nonrepeating X-Y-Gly units incorporated in the sequence, the three chains of the homotrimer can be distinguished from one another by NMR. The solution conformation of (POG)10 is very similar to the model derived from X-ray fiber diffraction data, although the peptide contains less ordered regions at the peptide ends. In the trimer from of T3-785, the central residues of the three chains are closely packed, and the data are consistent with a triple-helical model with a one-residue stagger of three parallel chains. For T3-785, in contrast to (POG)10, there are also resonances from a less ordered form, which are probably due to the presence of a small amount of monomer. The similarity of the backbone conformations of T3-785 and (POG)10 suggests that an alternative conformation is not present in the imino acid poor region.

Amino Acid Sequence↗

PubMed1992

X-ray diffraction studies on human tendon show age-related changes in collagen packing.

In this report, X-ray diffraction on native hydrated tendon is established as a monitor of human aging. X-ray diffraction patterns were recorded on toe extensor tendons of persons ranging from 1.6 years to 87 years old. All patterns show a set of 67 nm meridional reflections derived from the collagen fibril axial repeat, and the ratio of the intensities of the 16:17 orders showed a linear increase with age. The spacing of the equatorial maximum, which relates to the lateral packing of molecules in collagen fibrils, was also greater in older tendons. The observed changes in X-ray parameters follow those seen for rat-tail tendons subjected to in vitro non-enzymatic glycosylation. Age-related increases reported for the fluorescence of Maillard products and the concentration of the sugar-derived cross-link pentosidine are similar to the trends in X-ray parameters reported here. Our results support the cumulative nature of non-enzymatic glycosylation in connective tissues during the human lifespan and indicate that structural changes accompany the chemical alterations. The X-ray parameters show a large degree of scatter for ages older than 60 years, suggesting other complicating factors are present. Studies on a small number of diabetic tendons show small, but not significant, increases compared to age-matched controls.

Adolescent↗

PubMed1992

Nuclear magnetic resonance and circular dichroism studies of a triple-helical peptide with a glycine substitution.

The triple-helical conformation has the stringent amino acid sequence constraint that every third residue must be a glycine, (X-Y-Gly)n. We use nuclear magnetic resonance and circular dichroism to quantify the consequences of a substitution in the glycine position of a triple-helical peptide, and to enhance our understanding of interactions in this basic structural motif. A 30-residue peptide with a Gly----Ala change forms a stable trimer at a folding rate somewhat less than that of the unsubstituted peptide, and the substitution results in a marked decrease in thermal stability and a conformational perturbation of about 30% of the triple-helical structure. Two models were generated for this peptide, one with the alanine residues packed inside the triple helix and one with a looping out of the chain at the substitution site. Studies on the Gly----Ala peptide are useful in understanding connective tissue diseases which result from the substitution of one glycine residue in the triple-helix of fibrillar collagens.

Circular Dichroism↗

PubMed1992

NMR and CD studies of triple-helical peptides.

Triple-helix formation of the peptide (Pro-Hyp-Gly)10 was monitored by nmr and CD spectroscopy. The two-dimensional nmr spectra indicated that the Gly C alpha H and Pro C delta H proton resonances shift upfield in going from the nonhelical to helical form, while hydroxy-proline resonances are unchanged. The integrated areas of the helical and nonhelical resonances could be monitored in the one-dimensional nmr spectrum, and indicate that in the (Pro-Hyp-Gly)10 about 90% of the residues are in a defined triple-helical conformation. The introduction of a glycine to alanine substitution or the deletion of a single hydroxyproline residue in the stable triple-helical peptide (Pro-Hyp-Gly)10 still allows trimers to be formed, but the trimers show a substantial loss of triple helix and decreased thermal stability compared with (Pro-Hyp-Gly)10. Two computer models were generated for the Gly----Ala peptide, one with the Ala side chains packed inside the helix and the other with the region containing the alanines forming a beta-bend that loops out from the helix. The nmr data is more consistent with the latter model.

Amino Acid Sequence↗

PubMed1992

Psychosocial factors influencing non-urgent use of the emergency room: a review of the literature and recommendations for research and improved service delivery.

Despite dramatic increases in use of hospital emergency rooms (ERs) since the 1950s, an estimated 85% of ER visits are made for non-life-threatening reasons. Using a modified version of the Andersen and Newman model of health care utilization, this paper reviews the research literature on ER use to examine what is known about factors that influence three stages of the help-seeking process: (1) problem recognition; (2) the decision to seek help; (3) the decision to use the ER. While predisposing factors other than race are not generally significant, enabling factors such as income, insurance coverage, having a usual source of care, and geographic proximity affect use of the ER, both alone and in interaction with race and other factors. The most common reason for non-urgent ER use was 'other care not available'. In addition to the absence of primary care, non-urgent use of the ER is linked to need factors arising from socioeconomic stress, psychiatric co-morbidities, and a lack of social support. Recommendations for future studies include examining prospectively all 3 stages of decision-making leading to ER use, and considering interactive effects among predictors. Implications for service delivery are discussed, including the need to re-structure health care delivery systems to provide greater access to primary care and provide more attention to psychosocial aspects of patient care in clinical settings.

Emergencies↗

PubMed1992

Studies of collagen in bone and dentin matrix of a Columbian mammoth (late Pleistocene) of central Utah.

A Columbian mammoth, Mammuthus columbi, was excavated at an elevation of 9000 feet in Huntington Canyon, Emery County, Utah. Radiocarbon dates on the skeleton indicated death approximately 11,200 years ago. The skeleton was removed from postglacial, Late Quaternary, lake sediments deposited as glacial runoff approximately 9500 years ago. The bones and teeth were especially well preserved in a saturated lake bed. After excavation the bones and teeth were preserved by controlled desiccation, without hardeners, over a period of 9 months. Microradiography, light and electron microscopy, medium and high angle X-ray diffraction, amino acid analysis and cyanogen bromide peptide mapping were undertaken to evaluate the packing, organization, and preservation of collagen in bone and dentin of this mammoth. Microradiography and light microscopy showed that the bone consisted of especially well preserved compact and trabecular bone, and electron microscopy of demineralized bone and tusk showed that the matrix consisted of lamellae of densely packed cylindrical collagen fibrils. Cell remnants with intact nuclei, with or without a nucleolus, as well as variable lengths of plasma membrane were occasionally present on the surface of bony trabecula. Remnants of odontoblast processes were present in some dentin tubules. High and low angle X-ray diffraction demonstrated that the demineralized matrix contained native collagen molecules and amino acid analysis showed that the composition was comparable to that of type I collagen. Cyanogen bromide peptide mapping indicated that the major peptides of type I collagen were present and had the same electrophoretic mobility as that of type I collagen of demineralized Asian elephant bone and rat tail tendon.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed1992

Glycation alters collagen fibril organization.

Incubation of rat tail tendon in 0.2M ribose results in accelerated non-enzymatic glycosylation of collagen, with the formation of fluorescent cross-links between molecules and decreased solubility. Electron micrographs of tendon cross-sections show an increased fibril packing density with increasing degrees of glycation. After a one-week incubation in ribose, every fibril appears in close contact with all of its neighbors, and the packing density has increased to 76%, from a value of 62% in controls. Irregular diameters and fusion of fibrils also are seen. All of the fibrils in a bundle appear to become cross-linked together, creating a larger stress bearing unit. This model is consistent with stress-strain curves showing a large increase in tensile stress and stiffness after a one-week incubation period in ribose. The diameters of the collagen fibrils increase in size in glycated tendon. We hypothesize larger diameters result from an increased resistance to shrinkage during the specimen preparation process, as a result of the rigid sugar derived cross-links. Closer fibril packing, increased fibril diameters, and irregular diameters have been reported in diabetic tissues, and may result from decades of glycation induced cross-link accumulation.

Animals↗