14 research outputs found
Design, Structure and Applications of Collagen-Mimetic Peptides
The collagen triple helix is a unique protein fold found in all domains of life where is has diverse
roles from imparting structure and strength to tissue, to initiating an immune response. While
many factors affecting the structure and stability of the triple helix have previously been
elucidated, much remains unknown about collagen. Using collagen-mimetic peptides, it is
possible to investigate the molecular structure of the triple helix, determine new pairwise
interactions of amino acids, characterize disease models and also create designer collagens that
will preferentially hybridize to natural collagen-rich tissue. First a selective labeling scheme is
used to thoroughly characterize a well-folded triple helical region, and then to determine the
degree of localized unfolding at the N- and C-termini. Though terminal fraying extends farther
than previously shown, small sequence alterations at the N-terminus have a drastic influence on
local stability (~15C). Next, a single register heterotrimeric mimic of the type I collagen disease
Osteogenesis Imperfecta is used to investigate single point glycine mutations in the B chain, the
A chain or both chains. Unlike past reports, a combination of NMR analysis and molecular
modelling is used to generate structures of the mutated helices and visualize the underlying
mechanisms of helix destabilization in OI. For the first time it is proven that these mutations
cause compositional as well as structural disruptions. Additionally, while several hundred
pairwise interactions are possible in the triple helix, to date only two interactions are wellunderstood
and commonly incorporated into CMP design. To expand on the library of known interactions, the structure and stability of helices containing serine, threonine, phospho-serine and phospho-threonine were investigated. Notably, when phospho-serine is paired with lysine a new highly stabilizing (49.5 C) axial interaction is possible. Finally, the design of a collagen type II targeting peptide is described, and NMR, CD and confocal microscopy are used to investigate
the hybridization of the synthetic peptide with the natural partner strands
Control of collagen triple helix stability by phosphorylation
The phosphorylation of the collagen triple helix plays an important role in collagen synthesis, assembly, signaling, and immune response, although no reports detailing the effect this modification has on the structure and stability of the triple helix exist. Here we investigate the changes in stability and structure resulting from the phosphorylation of collagen. Additionally, the formation of pairwise interactions between phosphorylated residues and lysine is examined. In all tested cases, phosphorylation increases helix stability. When charged-pair interactions are possible, stabilization via phosphorylation can play a very large role, resulting inasmuch as a 13.0 °C increase in triple helix stability. Two-dimensional NMR and molecular modeling are used to study the local structure of the triple helix. Our results suggest a mechanism of action for phosphorylation in the regulation of collagen and also expand upon our understanding of pairwise amino acid stabilization of the collagen triple helix
Synthetic, register-specific, AAB heterotrimers to investigate single point glycine mutations in osteogenesis imperfecta
Osteogenesis imperfecta (OI) is a disease caused primarily by mutations of glycine in the standard (Xaa-Yaa-Gly)n repeat of a type I collagen triple helix. Type I collagen is an AAB heterotrimer which means that, depending on whether the A or B chain is mutated, the glycine substitution will appear once or twice. In this work we use designed axial charged pairs to self-assemble an AAB triple helix with controlled composition and register. We then substitute a single glycine of the B chain with alanine, serine, valine, aspartate, or arginine and assess the impact on the structure and folding of this OI mimic by CD, NMR, and restraint-guided modeling. We find that alanine and serine substitutions are tolerated, resulting in localized disruptions to the triple helix structure, while bulkier amino acids result in alternatively folded structures. This work demonstrates the potential of axial charged pairs to control the structure of low stability triple helices and also helps to elucidate the structure and folding challenges associated with OI-type mutations in collagen
Comparative NMR analysis of collagen triple helix organization from N- to C-termini
The collagen triple helix consists of three supercoiled solvent-exposed polypeptide chains, and by dry weight it is the most abundant fold in mammalian tissues. Many factors affecting the structure and stability of collagen have been determined through the use of short synthetically prepared peptides, generally called collagen-mimetic peptides (CMPs). NMR (nuclear magnetic resonance spectroscopy) investigations into the molecular structure of CMPs have suffered from large amounts of signal overlap and degeneracy because of collagen’s repetitive primary sequence, the close and symmetric packing of the three chains and the identical peptide sequences found in homotrimers. In this paper a peptide library is prepared in which a single isotopic 15N-Gly label is moved sequentially along the peptide backbone. Our approach allows for a more explicit examination of local topology than available in past reports. This reveals larger regions of disorder at the C-terminus than previously detected by crystallographic or NMR studies, and here C-terminal fraying is seen to extend for six amino acids in a (POG)10 sequence. Furthermore, small sequence changes at the N-terminus greatly influence the degree of this localized disorder and may be useful in the future design of CMPs to maximize collagen’s interstrand hydrogen bonding pattern. Our approach and data serves as a reference for future CMP characterizations to determine the quality and extent of folding
Synthetic, Register-Specific, AAB Heterotrimers to Investigate Single Point Glycine Mutations in Osteogenesis Imperfecta
Osteogenesis imperfecta (OI) is a
disease caused primarily by mutations
of glycine in the standard (Xaa-Yaa-Gly)<sub><i>n</i></sub> repeat of a type I collagen triple helix. Type I collagen is an
AAB heterotrimer which means that, depending on whether the A or B
chain is mutated, the glycine substitution will appear once or twice.
In this work we use designed axial charged pairs to self-assemble
an AAB triple helix with controlled composition and register. We then
substitute a single glycine of the B chain with alanine, serine, valine,
aspartate, or arginine and assess the impact on the structure and
folding of this OI mimic by CD, NMR, and restraint-guided modeling.
We find that alanine and serine substitutions are tolerated, resulting
in localized disruptions to the triple helix structure, while bulkier
amino acids result in alternatively folded structures. This work demonstrates
the potential of axial charged pairs to control the structure of low
stability triple helices and also helps to elucidate the structure
and folding challenges associated with OI-type mutations in collagen
Glycine Substitutions in Collagen Heterotrimers Alter Triple Helical Assembly
Osteogenesis imperfecta
typically results from missense mutations
in the collagen genome where the required glycine residues are replaced
with another amino acid. Many models have attempted to replicate the
structure of mutated collagen on the triple helix level. However,
composition and register control of the triple helix is complicated
and requires extreme precision, especially when these destabilizing
mutations are present. Here we present mutations to a composition-
and register-controlled AAB helix where one of the requisite glycines
in the A chain of the triple helix is changed to serine or alanine.
We see a loss of compositional control when the A chain is mutated,
resulting in an A′BB composition that minimizes the number
of mutations included in the triple helix. However, when both A and
B chains are mutated and no nonmutated peptide chains are available,
the designed A′A′B′ composition is reestablished.
Our work shows the ability of the mutations to influence and alter
the composition and register of the collagen triple helix
AT-CuAAC synthesis of mechanically interlocked oligonucleotides
We present a simple strategy for the synthesis of main chain oligonucleotide rotaxanes with precise control over the position of the macrocycle. The novel DNA-based rotaxanes were analyzed to assess the effect of the mechanical bond on their properties
Data set in support of AT-CuAAC synthesis of mechanically interlocked oligonucleotides
Dataset supports: Acevedo-Jake, A., Ball, A. T., Galli, M., Kukwikila, N. M., Denis, M. AS., Singleton, D., Tavassoli, A., & Goldup, S. (2020). AT-CuAAC synthesis of mechanically interlocked oligonucleotides. Journal of the American Chemical Society, 142(13), 5985-5990. https://doi.org/10.1021/jacs.0c01670</span
CCDC 738969: Experimental Crystal Structure Determination
Related Article: E.A.Smith, C.Potter, Z.C.Kennedy, A.J.Puciaty, A.M.Acevedo-Jake, S.D.Hersey, C.R.Metz, W.T.Pennington, D.G.VanDerveer, C.F.Beam|2010|J.Heterocycl.Chem.|47|147|doi:10.1002/jhet.285,An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures
CCDC 689213: Experimental Crystal Structure Determination
Related Article: A.C.Dawsey, C.Potter, J.D.Knight, Z.C.Kennedy, E.A.Smith, A.M.Acevedo-Jake, A.J.Puciaty, C.R.Metz, C.F.Beam, W.T.Pennington, D.G.VanDerveer|2009|J.Heterocycl.Chem.|46|231|doi:10.1002/jhet.50,An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures