Direct Measurements of the Mechanical Stability of Zinc-Thiolate Bonds in Rubredoxin by Single-Molecule Atomic Force Microscopy

Abstract

Zinc (Zn) is one of the most abundant metals and is essential for life. Through ligand interactions, often with thiolate from cysteine residues in proteins, Zn can play important structural roles in organizing protein structure and augmenting protein folding and stability. However, it is difficult to separate the contributions of Zn-ligand interactions from those originating from intrinsic protein folding in experimental studies of Zn-containing metalloproteins, which makes the study of Zn-ligand interactions in proteins challenging. Here, we used single-molecule force spectroscopy to directly measure the mechanical rupture force of the Zn-thiolate bond in Zn-rubredoxin. Our results show that considerable force is needed to rupture Zn-thiolate bonds (~170 pN, which is significantly higher than the force necessary to rupture the coordination bond between Zn and histidines). To our knowledge, our study not only provides new information about Zn-thiolate bonds in rubredoxin, it also opens a new avenue for studying metal-ligand bonds in proteins using single-molecule force spectroscopy.

The physics of pulling polyproteins: a review of single molecule force spectroscopy using the AFM to study protein unfolding

“…Electrostatic interactions within a protein can be affected by changes in the environment. Tuning the pH of a solvent has been shown to "switch-on" or "switch-off" electrostatic attractions and repulsions within a protein [81]. Furthermore, these electrostatic interactions can be screened by the introduction of salt [81,219].…”

“…For example, glycerol has been shown to increase the mechanical stability of the protein GB1 [216], while denaturing osmolytes such as GdnHCl and urea have been found to decrease the mechanical stability of proteins [215,217]. The mechanical properties of a protein can be tuned by a change in pH or ionic strength of a buffer [81,81,218]. Electrostatic interactions within a protein can be affected by changes in the environment.…”

“…[8] However, in this approach the substitution of single DNA fragments occurs sequentially, making the process both laborious and time-intensive. An alternative method is based on the chemical coupling of identical protein monomers or dimers [81,83,83,84]. Here, proteins can be linked through either disulfide bridge formation between cysteine pairs at designed locations or maleimide coupling of sulfhydryl groups within the protein.…”

“…Polyproteins provide a unique pattern in force extension traces. Over the last two decades a number of different techniques have been employed to engineer such polyproteins [8,[79][80][81]. A classical approach is based on the assembly of polymerase chain reaction (PCR)-generated DNA cassettes that together encode the full-length polyprotein [8,82].…”

Direct measurement and modulation of single-molecule coordinative bonding forces in a transition metal complex

“…In terms of transition metal complexes, scanning tunnelling microscopy (STM), particularly electrochemical STM (ECSTM), as an advanced tool has offered detailed mapping of single-molecule conductivity patterns in chemical or/and electrochemical environments [10][11][12][13] . Atomic force microscopy (AFM) imaging and spectroscopy have the power to uncover chemically hidden information that would not be revealed by traditional chemical approaches, for example, mechanical activation of chemical bonds (bond rupture) as well as the influence of stretching forces on chemical reactions [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] . A very recent report shows that molecular conductance and Van der Waals forces can also be measured simultaneously by conducting AFM 29 .…”