Exploration of Metal-Ligand Coordination Bonds in Proteins by Single-molecule Force Spectroscopy
TL;DR: It is shown that metalloprotein plays an essential role in many different biological processes and represents an indispensable protein subgroup and the knowledge of this subgroup is essential for the development of new drugs to treat cancer.
Abstract: Thanks to the binding of various metal ions, metalloprotein plays an essential role in many different biological processes and represents an indispensable protein subgroup. Thus, the knowledge of t...
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TL;DR: Local mode analysis as discussed by the authors has been widely used in the field of IR and Raman spectroscopy, and it has been shown that the vibrational motions in polyatomic molecules are delocalized.
Abstract: This Feature Article starts highlighting some recent experimental and theoretical advances in the field of IR and Raman spectroscopy, giving a taste of the breadth and dynamics of this striving field. The local mode theory is then reviewed, showing how local vibrational modes are derived from fundamental normal modes. New features are introduced that add to current theoretical efforts: (i) a unique measure of bond strength based on local mode force constants ranging from bonding in single molecules in different environments to bonding in periodic systems and crystals and (ii) a new way to interpret vibrational spectra by pinpointing and probing interactions between particular bond stretching contributions to the normal modes. All of this represents a means to work around the very nature of normal modes, namely that the vibrational motions in polyatomic molecules are delocalized. Three current focus points of the local mode analysis are reported, demonstrating how the local mode analysis extracts important information hidden in vibrational spectroscopy data supporting current experiments: (i) metal-ligand bonding in heme proteins, such as myoglobin and neuroglobin; (ii) disentanglement of DNA normal modes; and (iii) hydrogen bonding in water clusters and ice. Finally, the use of the local mode analysis by other research groups is summarized. Our vision is that in the future local mode analysis will be routinely applied by the community and that this Feature Article serves as an incubator for future collaborations between experiment and theory.
7 citations
TL;DR: In this article , the authors used atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) to directly measure the rupture force of Co(II)-thiolate bond in Co•substituted rubredoxin (CoRD), revealing that it is a mechanostable chemical bond.
Abstract: Cobalt is a trace transition metal. Although it is not abundant on earth, tens of cobalt‐containing proteins exist in life. Moreover, the characteristic spectrum of Co(II) ion makes it a powerful probe for the characterization of metal‐binding proteins through the formation of cobalt‐ligand bonds. Since most of these natural and artificial cobalt‐containing proteins are stable, we believe that these cobalt‐ligand bonds in the protein system are also mechanically stable. To prove this, we used atomic force microscopy‐based single‐molecule force spectroscopy (AFM‐SMFS) to directly measure the rupture force of Co(II)‐thiolate bond in Co‐substituted rubredoxin (CoRD). By combining the chemical denature/renature method for building metalloprotein and cysteine coupling‐based polyprotein construction strategy, we successfully prepared the polyprotein sample (CoRD)n suitable for single‐molecule studies. Thus, we quantified the strength of Co(II)‐thiolate bonds in rubredoxin with a rupture force of ∼140 pN, revealing that it is a mechanostable chemical bond. In addition, the Co−S bond is more labile than the Zn−S bond in proteins, similar to the result from the metal‐competing titration experiment.
3 citations
TL;DR: Enzymatic protein ligation has become the most powerful and widely used method for high-precision atomic force microscopy single-molecule force spectroscopy study of protein mechanics.
Abstract: Enzymatic protein ligation has become the most powerful and widely used method for high-precision atomic force microscopy single-molecule force spectroscopy (AFM-SMFS) study of protein mechanics. However, this methodology typically requires the functionalization of the glass surface with a corresponding peptide sequence/tag for enzymatic recognition and multiple steps are needed. Thus, it is time-consuming and a high level of experience is needed for reliable results. To solve this problem, we simplified the procedure using two strategies both based on asparaginyl endopeptidase (AEP). First, we designed a heterobifunctional peptide-based crosslinker, GL-peptide-propargylglycine, which links to an N3-functionalized surface via the click reaction. Then, the target protein with a C-terminal NGL sequence can be immobilized via the AEP-mediated ligation. Furthermore, we took advantage of the direct ligation between primary amino in a small molecule and protein with C-terminal NGL by AEP. Thus, the target protein can be immobilized on an amino-functionalized surface via AEP in one step. Both approaches were successfully applied to the AFM-SMFS study of eGFP, showing consistent single-molecule results.
2 citations
TL;DR: To understand the molecular design of titin, six representative Ig domains in titin were chosen and nine glycine residues were added as an artificial interdomain linker between these Ig domains and results showed that the linker affected the mechanical stability of Ig domains.
Abstract: Titin is the largest protein in humans, composed of more than one hundred immunoglobulin (Ig) domains, and plays a critical role in muscle’s passive elasticity. Thus, the molecular design of this giant polyprotein is responsible for its mechanical function. Interestingly, most of these Ig domains are connected directly with very few interdomain residues/linker, which suggests such a design is necessary for its mechanical stability. To understand this design, we chose six representative Ig domains in titin and added nine glycine residues (9G) as an artificial interdomain linker between these Ig domains. We measured their mechanical stabilities using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) and compared them to the natural sequence. The AFM results showed that the linker affected the mechanical stability of Ig domains. The linker mostly reduces its mechanical stability to a moderate extent, but the opposite situation can happen. Thus, this effect is very complex and may depend on each particular domain’s property.
1 citations
TL;DR: In this paper , the outer mitochondrial membrane protein mitoNEET (mNT) and its [2Fe2S] cluster with a single Fe−N(His87) coordinating bond were analyzed using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS).
Abstract: Abstract The outer mitochondrial membrane protein mitoNEET (mNT) is a recently identified iron‐sulfur protein containing a unique Fe2S2(His)1(Cys)3 metal cluster with a single Fe−N(His87) coordinating bond. This labile Fe−N bond led to multiple unfolding/rupture pathways of mNT and its cluster by atomic force microscopy‐based single‐molecule force spectroscopy (AFM‐SMFS), one of most common tools for characterizing the molecular mechanics. Although previous ensemble studies showed that this labile Fe−N(His) bond is essential for protein function, they also indicated that the protein and its [2Fe2S] cluster are stable under acidic conditions. Thus, we applied AFM‐SMFS to measure the stability of mNT and its cluster at pH values of 6, 7, and 8. Indeed, all previous multiple unfolding pathways of mNT were still observed. Moreover, single‐molecule measurements revealed that the stabilities of the protein and the [2Fe2S] cluster are consistent at these pH values with only ≈20 pN force differences. Thus, we found that the behavior of the protein is consistent in both weakly acidic and basic solutions despite a labile Fe−N bond.
1 citations
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TL;DR: In this article, the authors describe a simple, nondestructive procedure for measuring the force constant, resonant frequency, and quality factor of an AFM cantilever spring and the effective radius of curvature of an AU tip.
Abstract: Images and force measurements taken by an atomic‐force microscope (AFM) depend greatly on the properties of the spring and tip used to probe the sample’s surface. In this article, we describe a simple, nondestructive procedure for measuring the force constant, resonant frequency, and quality factor of an AFM cantilever spring and the effective radius of curvature of an AFM tip. Our procedure uses the AFM itself and does not require additional equipment.
3,975 citations
TL;DR: Single-molecule atomic force microscopy was used to investigate the mechanical properties of titin, the giant sarcomeric protein of striated muscle, and refolding of immunoglobulin domains was observed.
Abstract: Single-molecule atomic force microscopy (AFM) was used to investigate the mechanical properties of titin, the giant sarcomeric protein of striated muscle. Individual titin molecules were repeatedly stretched, and the applied force was recorded as a function of the elongation. At large extensions, the restoring force exhibited a sawtoothlike pattern, with a periodicity that varied between 25 and 28 nanometers. Measurements of recombinant titin immunoglobulin segments of two different lengths exhibited the same pattern and allowed attribution of the discontinuities to the unfolding of individual immunoglobulin domains. The forces required to unfold individual domains ranged from 150 to 300 piconewtons and depended on the pulling speed. Upon relaxation, refolding of immunoglobulin domains was observed.
2,959 citations
TL;DR: These techniques are described and illustrated with examples highlighting current capabilities and limitations of single-molecule force spectroscopy.
Abstract: Single-molecule force spectroscopy has emerged as a powerful tool to investigate the forces and motions associated with biological molecules and enzymatic activity. The most common force spectroscopy techniques are optical tweezers, magnetic tweezers and atomic force microscopy. Here we describe these techniques and illustrate them with examples highlighting current capabilities and limitations.
2,155 citations
TL;DR: Iron-sulfur clusters now rank with such biological prosthetic groups as hemes and flavins in pervasive occurrence and multiplicity of function.
Abstract: Iron-sulfur proteins are found in all life forms. Most frequently, they contain Fe2S2, Fe3S4, and Fe4S4 clusters. These modular clusters undergo oxidation-reduction reactions, may be inserted or removed from proteins, can influence protein structure by preferential side chain ligation, and can be interconverted. In addition to their electron transfer function, iron-sulfur clusters act as catalytic centers and sensors of iron and oxygen. Their most common oxidation states are paramagnetic and present significant challenges for understanding the magnetic properties of mixed valence systems. Iron-sulfur clusters now rank with such biological prosthetic groups as hemes and flavins in pervasive occurrence and multiplicity of function.
1,677 citations
TL;DR: The robust reaction conditions and irreversible linkage of SpyTag shed light on spontaneous isopeptide bond formation and should provide a targetable lock in cells and a stable module for new protein architectures.
Abstract: Protein interactions with peptides generally have low thermodynamic and mechanical stability. Streptococcus pyogenes fibronectin-binding protein FbaB contains a domain with a spontaneous isopeptide bond between Lys and Asp. By splitting this domain and rational engineering of the fragments, we obtained a peptide (SpyTag) which formed an amide bond to its protein partner (SpyCatcher) in minutes. Reaction occurred in high yield simply upon mixing and amidst diverse conditions of pH, temperature, and buffer. SpyTag could be fused at either terminus or internally and reacted specifically at the mammalian cell surface. Peptide binding was not reversed by boiling or competing peptide. Single-molecule dynamic force spectroscopy showed that SpyTag did not separate from SpyCatcher until the force exceeded 1 nN, where covalent bonds snap. The robust reaction conditions and irreversible linkage of SpyTag shed light on spontaneous isopeptide bond formation and should provide a targetable lock in cells and a stable module for new protein architectures.
1,086 citations