Folding and Unfolding of Proteins
Proteins are molecules that are composed of a sequence of amino acids. This sequence of amino acids determines the complex helical shape that the protein will assume. The protein helix can be denatured under special conditions (chemical or thermal) and refold to its native state upon the removal of the denaturing source.’70- Each protein has a unique structure and a specific folding pattern for its polypeptide chain that is required for proper biological function.’71,72- Understanding the process of folding and unfolding of proteins can help in controlling protein function for many applications.
The force-extension curves measured with AFM can characterize protein folding and unfolding.’73,74- For example, the mechanical properties of titin immuno-globulin (a giant sarcomeric protein of striated muscle) were studied by repetitive stretching of an individual titin molecule with AFM.[71] The stretching force was recorded as a function of molecule elongation. At large extensions, the force extensions exhibited a sawtooth-like pattern, with a periodicity that varied between 25 and 28 nm. These peaks were attributed to unfolding of individual domains of the protein. The forces required to unfold these domains were 150-300 pN and were dependent on the pulling speed. Refolding of protein was observed upon relaxation.
Fig. 10 The entropic elasticity of proteins and domain unfolding. a) The entropic elasticity of proteins can be described by the WLC model, which expresses the relationship between force (F) and extension (x) of proteins using its persistence length (p) and its contour length (Lc); k is Boltzmann constant and T is temperature. b) The sawtooth pattern of peaks observed when force was applied to extend the protein corresponds to sequential unraveling of individual domains of modular proteins. As the distance between the substrate and the cantilever increases (from state 1 to state 2), the protein elongates, generating a restoring force that bends the cantilever. When a domain unfolds (state 3), the free length of the protein increases, returning the force of the cantilever to near zero. Further extension results in force on the cantilever (state 4). The last peak represents the final extension of the unfolded protein prior to detachment from the AFM tip. c) Consecutive unfolding peaks of recombinant human tenascin-C were fit using WLC model. The contour length (Lc) for each fit is shown; the persistence length (p) was fixed at 0.56 nm.
In a similar study, the folding and unfolding of fibronectin (a modular extracellular matrix protein) was investigated via AFM.[75] Statistical analysis of the force-extension curves clearly revealed the unfolding of three different types of fibronectin. The unfolding of fibronectin was irreversible within the timescale of one extension-relaxation cycle. The folding and unfolding of other proteins such as human tenascin-C[72] (Fig. 10), titin Ig,[76] and barnase[77] were also studied with AFM.
Probing Ligand-Receptor Interactions
Ligand-receptor interactions can occur during the formation of double-stranded DNA, in enzymatic reactions, and in antigen-antibody recognition.[78] To measure these interactions, the AFM tip is functionalized with either the ligand or the receptor and the surface is functionalized with the other component.[79] In the first AFM study to measure the interactions between ligands and receptors, the interactions between a model receptor, streptavidin, and its ligand, biotin, were probed under physiological condi-tions.[80] The adhesion forces between the two function-alized surfaces were 3-8 times higher than the nonspecific interactions measured between blocked streptavidin and biotinylated surfaces. Statistical analysis of the adhesive forces revealed the maximum number of streptavidin-biotin interactions and the force required to rupture the ligand-receptor bond. In another study of the interactions between avidin and biotin, the unbinding forces of discrete complexes were proportional to the enthalpy change of the complex formation, but independent of the free energy.[81] As another example, the interactions between P-selectin and P-selectin glycoprotein ligand-1 were probed with SMFS.[82] By modeling the resulting intermolecular forces, knowledge of the rupture forces, elasticity, and kinetics of the interactions of the P-selectin/P-selectin glycoprotein ligand-1 interactions were obtained.
PROBING POLYSACCHARIDES WITH ATOMIC FORCE MICROSCOPY
Polysaccharides are a large group of molecules that exist as components of plant, animal, algal, bacterial, and yeast cells. They provide structural support and act as an energy reservoirs in plants and animals.[23] Polysaccharides also play an important role in microbial activity, including evasion of host defense systems and attachment to host tissue in infections. Polysaccharides are important in biofilm formation, which affects such diverse microbial processes as the uptake of trace metals in soil,’83- failure of medical implants such as artificial heart valves,’84-the success of bioremediation,’85- and the virulence of pathogenic infections.’86- Polysaccharides are also linked with cancer pathology.’87- In many of these fields, the mechanisms by which polysaccharides control the biological processes are not well known. A better understanding of the properties of polysaccharides and their subsequent biological functions can be obtained by high-resolution studies using AFM imaging and force measurements.
Quantitative Characterization of Polysaccharide Morphology by Atomic Force Microscopy Imaging
Atomic force microscopy imaging of polysaccharides can be used to obtain quantitative information on the molecule’s height,’88- thickness,’88- width,’89- contour length,’33- persistence length,’34- end-to-end distance,’88-and the polydispersity and distribution of polysaccharides on the surfaces of living microbial cells.’90-
Conformational transitions of polysaccharides can be investigated with AFM imaging. For example, the denaturation/renaturation process for the xanthan triple helix was observed with tapping mode AFM imaging under ambient conditions. The triple helix denatured upon heating and renatured when cooled only if sufficient salt was present in solution.’88- The effect of different solvent chemistries (pH and ionic strength) on polysaccharide conformation was also investigated with tapping mode AFM. For example, the conformation of succinoglycan deposited on mica was observed in the presence and absence of salt. When there was no salt in the aqueous solution, a combination of rigid and flexible chains was observed, while only flexible single chains were observed in the presence of 0.01 M KCl.’89-
Fig. 11 Conceptual representation of the conformation of bacterial surface biopolymers at low and high salt concentrations.
Fig. 12 A comparison between average approach curves (each curve is an average of 25 individual force measurements) for P. putida KT2442 in various solutions. Slopes of the compliance region of these curves are — 0.014, — 0.010, – 0.054, — 0.035, — 0.109, and — 0.114 nN/nm, from water to 1 M KCl, respectively.
Another exciting application of AFM with respect to polysaccharide research is the imaging of dynamic biological processes. Gunning et al.’91- imaged molecular motion of a water-soluble wheat pentosan polysaccharide extracted from wheat flour. Parts of the molecules desorbed and readsorbed onto the mica surface during tapping mode imaging in 10-mM HEPES buffer. Loops, trains, and tails were directly observed, confirming that polymer chains were desorbing and readsorbing. The use of AFM to study other dynamic biological processes, including enzymatic breakdown of polysaccharides, is therefore not far from being realizable.
Force Microscopy for Mechanical Characterization of Polysaccharides
Studying the force spectra recorded on polysaccharides (pure polysaccharides or polysaccharides on a microbial surface) provides useful information on the elastic, mechanical, and sometimes chemical nature of the macro-molecules. Examples of the types of information that can be deduced from SMFS are the following: quantitative information about polysaccharide elasticity, estimated by applying polymer statistical models;’92- identifying the components of a mixture of pure polysaccharides;’93-probing the elasticity of polysaccharides on microbial cells;’7,36,94- and qualitative prediction of the conformation of microbial biopolymers.’95- We will discuss some of these examples in detail.
In the first study to quantify the elasticity of macro-molecules on a microbial surface, surface macromolecules of dormant spores of Aspergillus oryzae were probed via SMFS.[94] The elongation forces were well described by the FJC + model, with estimated values of the Kuhn length and the segment elasticity in agreement with reported values for the structural properties of the polysaccharides dextran and amylose.
Single molecule force spectroscopy was also used to qualitatively predict the conformation of biopolymers, predominately polysaccharides, on the surface of the bacterium P. putida KT2442. Forces were measured on individual bacterial cells in solutions with varying added salt concentrations (water-1 M KCl). The biopolymers on the microbial surface adopted a more flexible conformation with increasing solution salt concentration.[95] The flexibility of the molecules was quantified using the FJC model for polymer elasticity. Because of the increased flexibility of the biopolymers in high salt solutions, the biopolymers collapsed onto the surface of the bacterium, leading to a more rigid surface when in high salt (Fig. 11). The transition in polysaccharide conformation was related to the slope of the compliance region of the approach curves measured between the bacterial cells and the AFM silicon nitride tip (Fig. 12). Changes in biopolymer conformation and the biomechanical properties of the bacterium were related to bioadhesion.[95]
Fig. 13 Force-extension curves for single-pectin molecules reveal a two-step transition. A) The shape of the curves for molecules with varying lengths reveals two enthalpic extensions at ~ 300 and ~ 800-900 pN. B) High-resolution normalized plot of the force-extension relationship for a single-pectin molecule. The thin lines are fits of the FJC model modified to include the extensibility of the monomers.
Atomic force microscopy can also be used as a spectroscopic technique for chemical fingerprinting of polysaccharides. Transitions in the flexibility of xanthan, amylose, and dextran upon the cleavage of the pyranose ring were observed by AFM and linked with the chemical structure of the molecules.[96] Specifically, the pyranose ring was identified as the structural unit controlling the molecules’ elasticities. Cleavage of the pyranose ring with 5 mM sodium metaperiodate eliminated the extra enthal-pic component of the elasticity and made the force transition disappear. The transitions were attributed to force-induced elongations of the ring structure and, for some molecules, to transitions in the pyranose ring from a chair to boat structure. These transitions produced fingerprints in the extension-force spectrum that were characteristic of the ground-energy conformations of the pyranose ring and the type of glycosidic linkage present in each polysaccharide[93,97] (Fig. 13).
CONCLUSION
The use of AFM and SMFS to characterize the physico-chemical properties of biopolymers was reviewed. Atomic force microscopy is preferred over other surface characterization techniques because of several unique advantages, including 1) high lateral and vertical resolutions, 2) high signal-to-noise ratio, 3) ability to probe biopolymers in their native environment with minimal sample preparation, 4) ability to measure interaction forces at interfaces, and 5) ability to obtain quantitative information about the chemical structure of macromolecules.
High-resolution imaging of single molecules of DNA, proteins, and polysaccharides, including polysaccharides on microbial surfaces, can now be routinely performed with AFM. Images can provide quantitative information on molecular properties and can be used to elucidate biopolymer conformation. Among the more interesting examples of probing biopolymers with SMFS are DNA sequencing, quantifying ligand-receptor interactions between proteins and lipids, protein folding and unfolding, identifying polysaccharide components from mixtures, and probing the distribution of macromolecules on living cells.
New advancements continue to be made in the formulation of more sensitive AFM probes, improved instrumentation, less-invasive sample preparation techniques, and facilitated data analysis. Undoubtedly, AFM will continue to be a key tool for probing the properties of delicate biopolymers at the nanoscale. Knowledge of the relationships between molecular structure and function of biopolymers will benefit such diverse fields as biotechnology, food safety, environmental science, pharmaceutics, and medicine.
