Starmaker Exhibits Properties of an Intrinsically Disordered Protein

Abstract

Fish otoliths composed of calcium carbonate and an organic matrix play a primary role in gravity sensing and the perception of sound. Starmaker (Stm) was the first protein found to be capable of influencing the process of biomineralization of otoliths. Stm dictates the shape, size, and selection of calcium carbonate polymorphs in a concentration-dependent manner. To facilitate exploration of the molecular basis of Stm function, we have developed and optimized a protocol for efficient expression and purification of the homogeneous nontagged Stm. The homogeneous nontagged Stm corresponds to its functional form, which is devoid of a signal peptide. A comprehensive biochemical and biophysical analysis of recombinant Stm, along with in silico examinations, indicate for the first time that Stm exhibits the properties of intrinsically disordered proteins. The functional significance of Stm having intrinsically disordered protein properties and its possible role in controlling the formation of otoliths is discussed.

Intrinsically Disordered Proteins in Biomineralization

“…Mammals DMP1 4.0 CD, DLS, FTIR, SAXS (Gericke et al, 2010;He et al, 2003aHe et al, , 2003b DPP 2.8 CD, NMR, SAXS (Cross et al, 2005;Evans et al, 1994;Fujisawa & Kuboki, 1998;George & Hao, 2005;He et al, 2005b;Lee et al, 1977) BSP 4.1 CD, NMR, SAXS (Fisher et al, 2001;Tye et al, 2003Tye et al, , 2005Wuttke et al, 2001) OPN 4.4 CD, NMR, FTIR (Fisher et al, 2001;Gorski et al, 1995) amelogenin 6.6 CD, NMR (Buchko et al, 2010;Delak et al, 2009b;Ndao et al, 2011;Shaw et al, 2008) statherin 8.0 CD, NMR (Long et al, 2001;Naganagowda et al, 1998;Raj et al, 1992) lithostathine 5.7 CD (Gerbaud et al, 2000) Haliotis rufescens AP7 5.2 CD, NMR (Kim et al, 2004(Kim et al, , 2006aMichenfelder et al, 2003;Wustman et al, 2004) Atrina rigita Asprich 2.7-3.5 CD, NMR (Collino et al, 2006;Delak et al, 2009aDelak et al, , 2008Kim et al, 2008;Ndao et al, 2010) Procambrus clarkii CAP-1 3.9 CD (Inoue et al, 2007) Strongylocentrotus purpuratus SM50 10.8 CD, NMR (Xu & Evans, 1999;Zhang et al, 2000) PM27 8.1 CD, NMR Danio rerio Stm 4.1 CD, gel filtration (Kaplon et al, 2008(Kaplon et al, , 2009 Table 1. IDPs involved in biomineralization of calcium carbonate and phosphate for which a disordered structure has been confirmed...…”

“…It has been demonstrated by several methods that Stm is an IDP (Tab. 1) (Kaplon et al, 2008(Kaplon et al, , 2009. Far UV CD spectrum of Stm yielded the signatures typical for a disordered protein.…”

“…Interestingly, calcium ions led to a lower hydrodynamic radius, probably by decreasing electrostatic repulsion. Also, solvent-induced denaturation of Stm indicated the lack of an ordered structure in the molecule (Kaplon et al, 2008(Kaplon et al, , 2009. Recently, another homolog of Starmaker has been identified: Starmaker-like from Oryzias latipes.…”

Enigmatic ear stones: what we know about the functional role and evolution of fish otoliths

“…OMP‐1 and/or Stm might be potential candidates that act (alone or together) as inducers of aragonite mineralization that were lost in tetrapods either due to genetic drift or to selection related to the transition from an aquatic to a terrestrial lifestyle (Amemiya et al, ). OMP‐1 binds calcium and is important for the recruitment of other proteins into the otolith matrix (Petko et al, ), while Starmaker is a so‐called ‘intrinsically disordered’ protein which potentially interacts with multiple other proteins (Kaplon et al, ). Future transcriptomic and proteomic studies are likely to identify new otolith matrix proteins and to elucidate the interplay between proteins involved in otolith formation and biomineralization (see Thomas et al, ).…”

In vivoandin vitroanalysis of starmaker activity in zebrafish otolith biomineralization

“…The protein induces nucleation and then inhibits calcite crystal growth (18). Moreover, Stm belongs to a group of intrinsically disordered proteins (20,21).…”