NMR and CD Spectroscopy Show that Imino Acid Restriction of the Unfolded State Leads to Efficient Folding

Abstract

Protein folding is determined by molecular features in the unfolded state, as well as the native folded structure. In the unfolded state, imino acids both restrict conformational space and present cis-trans isomerization barriers to folding. Because of its high proline and hydroxyproline content, the collagen triple-helix offers an opportunity to characterize the impact of imino acids on the unfolded state and folding kinetics. Here, NMR and CD spectroscopy are used to characterize the role of imino acids in a triple-helical peptide, T1-892, which contains an 18-residue sequence from type I collagen and a C-terminal (Gly-Pro-Hyp)(4) domain. The replacement of Pro or Hyp by an Ala in the (Gly-Pro-Hyp)(4) region significantly decreases the folding rate at low but not high concentrations, consistent with less efficient nucleation. To understand the molecular basis of the decreased folding rate, changes in the unfolded as well as the folded states of the peptides were characterized. While the trimer states of the peptides are all similar, NMR dynamics studies show monomers with all trans (Gly-Pro-Hyp)(4) are less flexible than monomers containing Pro --> Ala or Hyp --> Ala substitutions. Nucleation requires all trans bonds in the (Gly-Pro-Hyp)(4) domain and the constrained monomer state of the all trans nucleation domain in T1-892 increases its competency to initiate triple-helix formation and illustrates the impact of the unfolded state on folding kinetics.

Sequence Dependence of Renucleation after a Gly Mutation in Model Collagen Peptides

“…Experiments performed on a set of host-guest peptides with different tripeptide sequences have shown that GPO sequences fold faster than GAO sequences, but both are good nucleation sequences (35). Further studies have shown that nucleation propensity may also be related to the dynamics of the monomer state with a highly constrained monomer state related to the efficiency of folding (36). The CD and NMR results on peptides T1-892 and T1-892(GAO) 3 indicate that imino acid-rich sequences of the form (GPO) 4 or GPO(GAO) 3 can act as nucleation domains when placed at the C-terminal end of the peptide.…”

The Thermodynamics and Kinetics of Collagen Folding

“…The importance of the restriction of imino acids in the unfolded state for the efficient folding of the triple helix has recently been described [96].…”

Computational design of self-assembling register-specific collagen heterotrimers

“…S7), the chemical shift of O14(Hα) proton, O14(Hβ1), O14(Hβ2) can be identified using the sequential NOE to the labelled G15(NH) in the 1 H, 1 H-NOESY-15 N-HSQC spectrum as well as the intra-residue NOEs and TOCSY cross-peaks arising from the unlabelled hydroxyproline residue, O14(Hβ1)-O14(Hα) and O14(Hβ2)-O14(Hα). The chemical shift of D16(NH) can be identified from the sequential D16(NH)-G15(Hα1) and D16(NH)-G15(Hα2) NOEs in the 1 H, 1 H-NOESY spectrum, these are necessary to differentiate sequences β(O 14 G 15 D 16 ) and γ(O 14 G 15 P 16 ). Finally, in the case of peptide-γ ( Supplementary Fig.…”

“…Such peptides, usually referred to as collagen mimetic peptides (CMPs), have been widely used to study the relationship between amino-acid composition and triple helical stability [12][13][14] , folding rate [15][16][17] and super-helical symmetry [18][19][20] , as well as to identify ligand-binding motifs [21][22][23] and provide the structural basis for their recognition [24][25][26] .…”