Ca2+ binding enhanced mechanical stability of an archaeal crystallin

PLoS One. 2014 Apr 11;9(4):e94513. doi: 10.1371/journal.pone.0094513. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Archaea / metabolism*
  • Archaeal Proteins / chemistry*
  • Archaeal Proteins / metabolism*
  • Calcium / metabolism*
  • Circular Dichroism
  • Computer Simulation
  • Crystallins / chemistry*
  • Crystallins / metabolism*
  • Protein Stability
  • Protein Unfolding
  • Spectrometry, Fluorescence
  • Thermodynamics

Substances

  • Archaeal Proteins
  • Crystallins
  • Calcium

Grants and funding

This work was funded by Tata Institute of Fundamental Research, India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.