A Synthetic Resilin Is Largely Unstructured

Abstract

Proresilin is the precursor protein for resilin, an extremely elastic, hydrated, cross-linked insoluble protein found in insects. We investigated the secondary-structure distribution in solution of a synthetic proresilin (AN16), based on 16 units of the consensus proresilin repeat from Anopheles gambiae. Raman spectroscopy was used to verify that the secondary-structure distributions in cross-linked AN16 resilin and in AN16 proresilin are similar, and hence that solution techniques (such as NMR and circular dichroism) may be used to gain information about the structure of the cross-linked solid. The synthetic proresilin AN16 is an intrinsically unstructured protein, displaying under native conditions many of the characteristics normally observed in denatured proteins. There are no apparent alpha-helical or beta-sheet features in the NMR spectra, and the majority of backbone protons and carbons exhibit chemical shifts characteristic of random-coil configurations. Relatively few peaks are observed in the nuclear Overhauser effect spectra, indicating that overall the protein is dynamic and unstructured. The radius of gyration of AN16 corresponds to the value expected for a denatured protein of similar chain length. This high degree of disorder is also consistent with observed circular dichroism and Raman spectra. The temperature dependences of the NH proton chemical shifts were also measured. Most values were indicative of protons exposed to water, although smaller dependences were observed for glycine and alanine within the Tyr-Gly-Ala-Pro sequence conserved in all resilins found to date, which is the site of dityrosine cross-link formation. This result implies that these residues are involved in hydrogen bonds, possibly to enable efficient self-association and subsequent cross-linking. The beta-spiral model for elastic proteins, where the protein is itself shaped like a spring, is not supported by the results for AN16. Both the random-network elastomer model and the sliding beta-turn model are consistent with the data. The results indicate a flat energy landscape for AN16, with very little energy required to switch between conformations. This ease of switching is likely to lead to the extremely low energy loss on deformation of resilin.

Physics of engineered protein hydrogels

“…106 The unstructured feature of resilin is further confirmed by using NMR to study the confirmation of a synthetic RLP derived from Anopheles gambiae proresilin sequence. 107 SAXS experiments show a power-law exponent of two in the high q regime, also consistent with a Gaussian random coil chain configuration. It is hypothesized that the high content of Gly and Pro in resilin might be responsible for the highly disordered structure.…”

The development of photochemically crosslinked native fibrinogen as a rapidly formed and mechanically strong surgical tissue sealant

“…The formation of dityrosine in photochemically crosslinked fibrinogen (5% dityrosine) is lower than that found in photochemically crosslinked resilin (25% dityrosine) due to both the higher molar content of tyrosine in resilin (8%) compared to fibrinogen and to the likely differences in molecular association that occurs in the two proteins. While resilin is essentially an unstructured protein [9], fibrinogen chains are highly structured and relatively constrained in the degree of molecular associations possible between a-, b-, and g-subunits in the disulphide-crosslinked native protein. Total dityrosine analysis measures both inter-and intra-molecular dityrosine crosslinks formed in the photochemical crosslinking reaction, however, only the intermolecular crosslinks contribute to the mechanical properties of the material.…”

Molecular and functional characterisation of resilin across three insect orders

“…Previous studies of the secondary structure of resilin-like proteins suggest that they are unstructured with a high degree of disorder dominated by random coil configurations (Lyons et al, 2009;Nairn et al, 2008;Qin et al, 2009). To test that Cf-resB and Hi-resB have similar structural characteristics, we gathered secondary structure information using CD spectra and prediction of intrinsically-unstructured regions of proteins based on estimated energy content (Dosztanyi et al, 2005) (http://iupred.enzim.hu/).…”