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B Brodsky

Publications and source records attributed to B Brodsky.

At least 19 recordsLinked to original sources
PubMed2000

Site-specific NMR monitoring of cis-trans isomerization in the folding of the proline-rich collagen triple helix.

Understanding the folding of the proline-rich collagen triple helix requires consideration of the effects of proline cis-trans isomerization and may shed light on the misfolding of collagen in connective tissue diseases. Folding was monitored in real time by heteronuclear 2D NMR spectroscopy for the (15)N labeled positions in the triple-helical peptide T1-892 [GPAGPAGPVGPAGARGPAGPOGPOGPOGPOGV]. In the equilibrium unfolded monomer form, each labeled residue showed multiple peaks with interconversion rates consistent with cis-trans isomerization of Gly-Pro and Pro-Hyp bonds. Real-time NMR studies on the folding of T1-892 showed slow decay of monomer peaks and a concomitant increase in trimer peaks. Gly25 in the C-terminal rich (Gly-Pro-Hyp)(4) domain folds first, consistent with its being a nucleation domain. Analysis of the kinetics indicates that the folding of Gly25 is biphasic and the slower step represents cis-trans isomerization of imino acids. This illustrates that nucleation is limited by cis-trans isomerization. Monitoring Gly6, Gly10, Ala12, and Gly13 monomer and trimer peaks captures the C- to N-terminal propagation of the triple helix, which is also limited by Gly-Pro cis-trans isomerization events. The zipper-like nature of the propagation process is confirmed by the slower rate of folding of Ala6 compared to Gly13, reflecting the larger number of isomerization events encountered by the more N-terminal Ala6. The cis-trans isomerization events at multiple proline residues is a complex statistical process which can be visualized by these NMR studies.

Amino Acid Sequence↗
PubMed2000

Destabilization of osteogenesis imperfecta collagen-like model peptides correlates with the identity of the residue replacing glycine.

Mutations resulting in replacement of one obligate Gly residue within the repeating (Gly-Xaa-Yaa)(n) triplet pattern of the collagen type I triple helix are the major cause of osteogenesis imperfecta (OI). Phenotypes of OI involve fragile bones and range from mild to perinatal lethal. In this study, host-guest triple-helical peptides of the form acetyl-(Gly-Pro-Hyp)(3)-Zaa-Pro-Hyp-(Gly-Pro-Hyp)(4)-Gly-Gly-amide are used to isolate the influence of the residue replacing Gly on triple-helix stability, with Zaa = Gly, Ala, Arg, Asp, Glu, Cys, Ser, or Val. Any substitution for Zaa = Gly (melting temperature, T(m) = 45 degrees C) results in a dramatic destabilization of the triple helix. For Ala and Ser, T(m) decreases to approximately 10 degrees C, and for the Arg-, Val-, Glu-, and Asp-containing peptides, T(m) < 0 degrees C. A Gly --> Cys replacement results in T(m) < 0 degrees C under reducing conditions but shows a broad transition (T(m) approximately 19 degrees C) in an oxidizing environment. Addition of trimethylamine N-oxide increases T(m) by approximately 5 degrees C per 1 M trimethylamine N-oxide, resulting in stable triple-helix formation for all peptides and allowing comparison of relative stabilities. The order of disruption of different Gly replacements in these peptides can be represented as Ala </= Ser < CPO(red) < Arg < Val < Glu </= Asp. The rank of destabilization of substitutions for Gly in these Gly-Pro-Hyp-rich homotrimeric peptides shows a significant correlation with the severity of natural OI mutations in the alpha1 chain of type I collagen.

Amino Acid Substitution↗
PubMed1999

Sequence dependence of the folding of collagen-like peptides. Single amino acids affect the rate of triple-helix nucleation.

The refolding of thermally denatured model collagen-like peptides was studied for a set of 21 guest triplets embedded in a common host framework: acetyl-(Gly-Pro-Hyp)3-Gly-Xaa-Yaa-(Gly-Pro-Hyp)4-Gly-Gly-amide. The results show a strong dependence of the folding rate on the identity of the guest Gly-Xaa-Yaa triplet, with the half-times for refolding varying from 6 to 110 min (concentration = 1 mg/ml). All triplets of the form Gly-Xaa-Hyp promoted rapid folding, with the rate only marginally dependent on the residue in the Xaa position. In contrast, triplets of the form Gly-Pro-Yaa and Gly-Xaa-Yaa were slower and showed a wide range of half-times, varying with the identity of the residues in the triplet. At low concentrations, the folding can be described by third-order kinetics, suggesting nucleation is rate-limiting. Data on the relative nucleation ability of different Gly-Xaa-Yaa triplets support the favorable nature of imino acids, the importance of hydroxyproline, the varying effects of the same residue in the Xaa position versus the Yaa position, and the difficulties encountered when leucine or aspartic acid are in the Yaa position. Information on the relative propensities of different tripeptide sequences to promote nucleation of the triple-helix in peptides will aid in identification of nucleation sites in collagen sequences.

Circular Dichroism↗
PubMed1999

Structural consequences of D-amino acids in collagen triple-helical peptides.

The effects of racemization of aspartic acid on triple-helical formation have been studied using a "host-guest" peptide approach where selected guest Gly-Xaa-Yaa triplets were included within a common acetyl-(Gly-Pro-Hyp)3-Gly-Xaa-Yaa-(Gly-Pro-Hyp)4-Gly-Gly-amide frame-work. Four guest triplets, Gly-Asp-Hyp and Gly-Asp-Ala where Asp is either L-Asp or D-Asp were studied. Thermal stability data indicated that incorporation of D-Asp residues prevented triple-helix formation in phosphate buffered saline, although triple-helical structures were formed in a stabilizing solvent, 67% aqueous ethylene glycol. In this solvent the melting temperatures of D-Asp containing peptides were more than 30 degrees C lower than the corresponding peptides containing L-Asp. For Gly-Asp-Ala peptides, but not Gly-Asp-Hyp, peptides, melting profiles indicated that a mixture of the D- and L-Asp containing peptides were able to form heterotrimer triple-helical molecules. These studies illustrate the dramatic destabilizing effect of D-amino acids on the triple-helix stability, but indicate that they can be accommodated in this conformation.

Amino Acid Sequence↗
PubMed1999

Folding of peptide models of collagen and misfolding in disease.

The misfolding of the triple helix has been shown to play a critical role in collagen diseases. Normal and mutated collagen triple helices can be modeled by short, synthetic peptides of varying design. NMR spectroscopy and circular dichroism studies on the assembly of these peptide models have recently been used to isolate specific steps in the folding pathway and have provided information on the alterations resulting from mutations.

Amino Acid Sequence↗
PubMed1998

Nuclear magnetic resonance shows asymmetric loss of triple helix in peptides modeling a collagen mutation in brittle bone disease.

To investigate a human folding disease, NMR studies were carried out on collagen-like peptides to define the structural consequences of a single amino acid change found in patients with osteogenesis imperfecta (OI), a disease characterized by fragile bones. One peptide included a normal collagen sequence, while a second peptide included a Gly --> Ser substitution as found in a nonlethal case of OI. Residue specific internal dynamics and conformational studies indicate that the normal collagen-like sequence forms a triple helix which is rigid along its entire length. The introduction of a Gly --> Ser substitution induces an asymmetric disruption of the uniform triple helix. While the C-terminal end of the peptide retains the triple helix, the Ser substitution site and residues N-terminal to it exhibit the mobility of a random chain. This equilibrium state indicates that a Gly substitution can terminate the C to N propagation of the triple helix and suggests that renucleation is required for folding to continue. Defective folding has been implicated in brittle bone disease, and these results begin to characterize the folding process in OI collagens. OI collagen studies may also provide insights about defective protein folding, assembly, and aggregation in other human diseases.

Amino Acid Sequence↗
PubMed1998

X-ray crystallographic determination of a collagen-like peptide with the repeating sequence (Pro-Pro-Gly).

The crystal structure of the triple-helical peptide (Pro-Pro-Gly)10 has been re-determined to obtain a more accurate description for this widely studied collagen model and to provide a comparison with the recent high-resolution crystal structure of a collagen-like peptide containing Pro-Hyp-Gly regions. This structure demonstrated that hydroxyproline participates extensively in a repetitive hydrogen-bonded assembly between the peptide and the solvent molecules. Two separate structural studies of the peptide (Pro-Pro-Gly)10 were performed with different crystallization conditions, data collection temperatures, and X-ray sources. The polymer-like structure of one triple-helical repeat of Pro-Pro-Gly has been determined to 2.0 A resolution in one case and 1.7 A resolution in the other. The solvent structures of the two peptides were independently determined specifically for validation purposes. The two structures display a reverse chain trace compared with the original structure determination. In comparison with the Hyp-containing peptide, the two Pro-Pro-Gly structures demonstrate very similar molecular conformation and analogous hydration patterns involving carbonyl groups, but have different crystal packing. This difference in crystal packing indicates that the involvement of hydroxyproline in an extended hydration network is critical for the lateral assembly and supermolecular structure of collagen.

Collagen↗
PubMed1998

Supercoiled protein motifs: the collagen triple-helix and the alpha-helical coiled coil.

The collagen triple-helix and the alpha-helical coiled coil represent the two basic supercoiled multistranded protein motifs. Originally they were characterized in fibrous proteins, but have been found more recently in a number of other proteins containing rod-shaped domains. Coiled-coil domains are responsible for the oligomerization of proteins, as well as other specific functions, while the triple-helix domains associate to form supramolecular structures and bind a variety of ligands. Both structures were originally solved by fiber diffraction, and recent crystallographic studies on small proteins and peptide models have confirmed the structure and provided molecular details. The differences in the molecular conformations of these two motifs and the interactions stabilizing these conformations are discussed. The molecular structures of both motifs constrain the amino acid sequence to recognizable patterns, requiring the (Gly-X-Y)n repeating sequence for the collagen triple-helix and a less stringent heptad repeat requirement (h-x-x-h-x-x-x)n for the coiled-coil domains, where h represents hydrophobic residues. The features and roles of these supercoiled domains in proteins are considered when they are found adjacent in the same protein.

Animals↗
PubMed1998

Gly-X-Y tripeptide frequencies in collagen: a context for host-guest triple-helical peptides.

The collagen triple-helix consists of a repeating (Gly-X-Y)n sequence. In theory, there are more than 400 possible Gly-X-Y triplets, but analysis of sequences from fibrillar and nonfibrillar collagens shows that only a limited set of triplets are found in significant numbers, and many are never observed. The nonrandom frequency of Gly-X-Y triplets makes it practical to experimentally approach the stability of much of the collagen sequence through the study of a limited set of host-guest peptides. In these peptides, individual Gly-X-Y triplets constitute the guest, while the host consists of Gly-Pro-Hyp tripeptides. A set of host-guest peptides was designed to contain the most common nonpolar and charged triplets found in collagen. All formed stable triple-helices, with their melting temperature depending on the identity of the guest triplet. While including less than 10% of all possible triplets, the data set covers 50-60% of collagen sequences and provides a starting point for establishing a stability scale to predict the relative stability of important collagen regions, such as the matrix metalloproteinase cleavage site or binding sites.

Amino Acid Sequence↗
PubMed1998

Skin surface proteolytic activity. Partial characterization and identification.

Skin surface proteolytic activity in the living animal was determined by a sensitive, non-invasive methodology developed in our laboratory. A non-leaky well was constructed on the shaved back of an anesthetized guinea pig. The well contained the reaction mixture including the substrate 125I-S-carboxymethylated insulin B-chain (ICMI). The proteolytic activity was shown to be time-dependent. The activity was strongly inhibited by pepstatin A, indicating the involvement of aspartic proteinase(s) such as cathepsin D and/or E. Pretreatment of the skin with propylene glycol blocked the proteolytic activity. The present study demonstrates the presence of proteolytic activity located on skin surface using a unique, non-invasive method for in situ proteinase determination in the living animal.

Animals↗
PubMed1997

Positional preferences of ionizable residues in Gly-X-Y triplets of the collagen triple-helix.

Collagens contain a high amount of charged residues involved in triple-helix stability, fibril formation, and ligand binding. The contribution of charged residues to stability was analyzed utilizing a host-guest peptide system with a single Gly-X-Y triplet embedded within Ac(Gly-Pro-Hyp)3-Gly-X-Y-(Gly-Pro-Hyp)4-Gly-Gly-NH2. The ionizable residues Arg, Lys, Glu, and Asp were incorporated into the X position of Gly-X-Hyp; in the Y position of Gly-Pro-Y; or as pairs of oppositely charged residues occupying X and Y positions. The Gly-X-Hyp peptides had similar thermal stabilities, only marginally less stable than Gly-Pro-Hyp, whereas Gly-Pro-Y peptides showed a wide thermal stability range (Tm = 30-45 degrees C). The stability of peptides with oppositely charged residues in the X and Y positions appears to reflect simple additivity of the individual residues, except when X is occupied by a basic residue and Y = Asp. The side chains of Glu, Lys, and Arg have the potential to form hydrogen bonds with available peptide backbone carbonyl groups within the triple-helix, whereas the shorter Asp side chain does not. This may relate to the unique involvement of Asp residues in energetically favorable ion pair formation. These studies clarify the dependence of triple-helix stability on the identity, position, and ionization state of charged residues.

Amino Acids↗
PubMed1997

Gly-Pro-Arg confers stability similar to Gly-Pro-Hyp in the collagen triple-helix of host-guest peptides.

A set of host-guest peptides of the form Ac(Gly-Pro-Hyp)3-Gly-X-Y-(Gly-Pro-Hyp)4-Gly-Gly-NH2 has been designed to evaluate the propensity of different Gly-X-Y triplets for the triple-helix conformation (Shah, N. K., Ramshaw, J. A. M., Kirkpatrick, A., Shah, C., and Brodsky, B. (1996) Biochemistry 35, 10262-10268). All Gly-X-Y guest triplets led to a decrease in melting temperature from the host (Gly-Pro-Hyp)8 peptide except for Gly-Pro-Arg. In this Gly-Pro-Hyp-rich environment, Gly-Pro-Arg was found to be as stabilizing as Gly-Pro-Hyp. Decreased stability of host-guest peptides containing Gly-Pro-Lys, Gly-Pro-homo-Arg, and Gly-Arg-Hyp compared with Gly-Pro-Arg indicated a stabilization that is optimal for Arg and specific to the Y-position. Arg was found to have a similar stabilizing effect when residues other than Pro are in the X-position. Both Arg and Hyp stabilize the triple-helix preferentially in the Y-position in a stereospecific manner and occupy largely Y-positions in collagen. However, contiguous Gly-Pro-Hyp units are highly stable and promote triple-helix folding, whereas incorporation of multiple Gly-Pro-Arg triplets was destabilizing and folded slowly due to charge repulsion. In collagen, Gly-Pro-Arg may contribute maximally to local triple-helix stability while also having the potential for electrostatic interactions in fibril formation and binding.

Amino Acid Sequence↗
PubMed1997

Altered collagen structure in mouse tail tendon lacking the alpha 2(I) chain.

Type I collagen is the most prevalent member of the fibril forming family of collagens in higher vertebrates and its heterotrimeric form is comprised of two alpha 1(I) chains and one alpha2(I) polypeptide chain. The functional importance of having two distinct chain types in type I collagen is largely undefined. The existence of a mouse model with a Cola-2 gene mutation (termed oim) that results in non-functional pro alpha 2(I) chains presents a unique opportunity to explore changes in collagen structure resulting from the complete (oim/oim mice) and partial (oim/+ mice) loss of functional alpha 2(I) chains. Tail tendon is a tissue with a well-defined, hierarchical organization of type I collagen. X-ray diffraction studies on oim/oim versus control tendons indicate that the total absence of alpha 2(I) chains results in a decrease in the order of axial packing and a loss of crystalline lateral packing. This suggests that the non-equivalence of three chains is an important determinant of lateral interactions between adjacent molecules and may be involved in the long-range axial order in type I collagen-containing tissues. Both homotrimeric and heterotrimeric type I collagen molecules are found in heterozygous oim mice and these appear to be present in the same co-polymeric fibrils, preventing crystalline lateral packing. In addition to these changes at a fibrillar level, the absence of the alpha 2(I) chain results in an increased enzymatic susceptibility at one site. The oim/oim mice are observed to have reduced body size and smaller tendon bundles, which may be a consequence of these molecular and fibrillar changes in collagen. Furthermore, it is likely that a similar alteration in the molecular packing of collagen in bone fibrils contributes to the osteopenia and decreased bone strength in mice with the oim mutation that are also characteristic of human osteogenesis imperfecta.

Animals↗
PubMed1997

Amino acid sequence environment modulates the disruption by osteogenesis imperfecta glycine substitutions in collagen-like peptides.

Ostoegenesis imperfecta (OI) or "brittle bone" disease is associated with mutations in the genes for type I collagen chains and produces variable phenotypes, ranging from lethal cases at birth to mild cases with increased bone fractures. The most common OI mutations are single base substitutions leading to replacement of Gly by another residue, breaking the typical (Gly-X-Y)n repeating sequence pattern of the collagen triple-helix. Triple-helical peptides were designed to focus on residues 892-921 of the alpha1 chain of type I collagen, where two OI Gly-->Ser mutations are found in close proximity, a mild mutation at site 901 and a lethal mutation at site 913. Peptides were designed to include amino acid sequences around these mutation sites, and were synthesized with the normal sequence or with the Gly-->Ser mutated sequence. The peptide including the normal sequence residues 892-909 with four Gly-Pro-Hyp triplets at the C-terminus formed a stable triple-helix, and introduction of a Ser residue for Gly at the 901 mutation site led to a 50% loss of triple-helix content and a decrease in thermal stability, with little effect on folding. A peptide including residues 904-921 again formed a stable triple-helix, but the introduction of the Gly-->Ser substitution at site 913 led to a much greater decrease in thermal stability. These studies demonstrate the impact of local sequences flanking the Gly substitution on structural consequences and support the concept of variability and regional effects along the collagen molecule.

Amino Acid Sequence↗
PubMed1997

Gly-Gly-containing triplets of low stability adjacent to a type III collagen epitope.

Collagens, in addition to their structural role in the extracellular matrix, possess a number of functional binding domains. In this study, the binding to collagen of a monoclonal antibody is used as a model to define the molecular features involved in triple-helix interactions with other proteins. Here we report the thermal stability of an overlapping set of triple-helical peptides that includes the epitope recognized by a monoclonal antibody to type III collagen. Although the sequences of these peptides are very closely related, by a translation of a single triplet along the collagen chain, substantial variations in the melting temperatures were observed. These variations in thermal stability could not be readily explained by differences in imino acid content, or in numbers of charged or hydrophobic residues. The results indicate that Gly-Gly-Y triplets, which are adjacent to the epitope, have a strong influence in reducing the thermal stability of triple-helical peptides. Further studies, which were carried out on a set of "host-guest" triple-helical peptides containing different Gly-Gly-Y guest triplets, confirm the destabilizing effect of such tripeptides. The presence of Gly-Gly-Y triplets may play an important role in specific functions of type III collagen by modulating the local triple-helical structure or dynamics.

Amino Acid Sequence↗
PubMed1997

Protective effect of povidone-iodine ointment against skin lesions induced by sulphur and nitrogen mustards and by non-mustard vesicants.

Mustard gas (sulphur mustard, SM) is a powerful vesicant employed as a chemical weapon. The present study demonstrates the effect of povidone iodine (PI) ointment against skin toxicity caused by SM. Gross and histopathological examinations showed that application of PI up to 20 min following exposure to the vesicant resulted in marked skin protection. The shorter the interval between exposure and treatment the better was the protection achieved. PI was also effective against other mustards such as carboxybutyl chloroethyl sulphide (CBCS) and mechlorethamine. The fact that PI protected the skin against agents which cannot be oxidized such as iodoacetic acid, divinylsulphone and cantharidine showed that the antidotal effect of PI was unrelated to oxidation of the nitrogen and sulphur atoms of the mustards. PI ointment is proposed as an efficient protective agent against skin toxicity caused by mustards and other alkylators.

Animals↗
PubMed1997

Noninvasive procedure for in situ determination of skin surface aspartic proteinase activity in animals; implications for human skin.

The present study demonstrated a noninvasive procedure for in situ determination of stratum corneum aspartic proteinase in the living animal. A non-leaky well, containing [125I]S-carboxymethylated insulin B-chain (ICMI) as a substrate, was constructed on the shaved back of anesthetized guinea pigs and rats. The enzymatic activity was determined by measuring the radiolabeled trichloroacetic acid soluble material. We demonstrated pepstatin-sensitive proteinase activity bound to the skin surface indicating the involvement of aspartic proteinase(s) such as cathepsin D and/or E. Aged rats had about six fold lower activity than young animals. The proteinase activity was inhibited by the alkylating agent mechlorethamine and by the cosmetic propylene glycol. A similar procedure was carried out with intact human skin pieces obtained during plastic surgery. The activity was inhibited by antihuman cathepsin D antibodies. Cathepsin D was immunohistochemically localized in the corneal and granular layers of the epidermis. Skin surface aspartic proteinase/cathepsin D activity may serve as a marker for skin aging or for certain skin disorders leading to a new approach to their medical treatments.

Aging↗