title: Ca2+ binding enhanced mechanical stability of an archaeal crystallin.
abstract: Structural topology plays an important role in protein mechanical stability. Proteins with β-sandwich topology consisting of Greek key structural motifs, for example, I27 of muscle titin and (10)FNIII of fibronectin, are mechanically resistant as shown by single-molecule force spectroscopy (SMFS). In proteins with β-sandwich topology, if the terminal strands are directly connected by backbone H-bonding then this geometry can serve as a "mechanical clamp". Proteins with this geometry are shown to have very high unfolding forces. Here, we set out to explore the mechanical properties of a protein, M-crystallin, which belongs to β-sandwich topology consisting of Greek key motifs but its overall structure lacks the "mechanical clamp" geometry at the termini. M-crystallin is a Ca(2+) binding protein from Methanosarcina acetivorans that is evolutionarily related to the vertebrate eye lens β and γ-crystallins. We constructed an octamer of crystallin, (M-crystallin)8, and using SMFS, we show that M-crystallin unfolds in a two-state manner with an unfolding force ∼ 90 pN (at a pulling speed of 1000 nm/sec), which is much lower than that of I27. Our study highlights that the β-sandwich topology proteins with a different strand-connectivity than that of I27 and (10)FNIII, as well as lacking "mechanical clamp" geometry, can be mechanically resistant. Furthermore, Ca(2+) binding not only stabilizes M-crystallin by 11.4 kcal/mol but also increases its unfolding force by ∼ 35 pN at the same pulling speed. The differences in the mechanical properties of apo and holo M-crystallins are further characterized using pulling speed dependent measurements and they show that Ca(2+) binding reduces the unfolding potential width from 0.55 nm to 0.38 nm. These results are explained using a simple two-state unfolding energy landscape.
authors.affiliation: Department of Chemical Sciences, Tata Institute of Fundamental Research, Colaba, Mumbai, India.
authors.forename: Venkatraman
authors.initials: V
authors.lastname: Ramanujam
authors.affiliation: Department of Chemical Sciences, Tata Institute of Fundamental Research, Colaba, Mumbai, India.
authors.forename: Hema Chandra
authors.initials: HC
authors.lastname: Kotamarthi
authors.affiliation: Department of Chemical Sciences, Tata Institute of Fundamental Research, Colaba, Mumbai, India.
authors.forename: Sri Rama Koti
authors.initials: SR
authors.lastname: Ainavarapu
mesh_terms: D020816:Amino Acid Motifs; D001105:Archaea / Q000378:metabolism*; D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002942:Circular Dichroism; D003198:Computer Simulation; D003459:Crystallins / Q000737:chemistry* / Q000378:metabolism*; D055550:Protein Stability; D058767:Protein Unfolding; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: D019843:Archaeal Proteins; D003459:Crystallins; D002118:Calcium
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