Browsing by Subject "Single molecule force spectroscopy"

Just as heat, light and electricity do, mechanical forces can also stimulate reactions. Conventionally, these processes - known as mechanochemistry - were viewed as comprising only destructive events, such as bond scission and material failure. Recently, Moore and coworkers demonstrated that the incorporation of mechanophores, i.e., mechanochemically active moieties, can bring new types of chemistry. This demonstration has inspired a series of fruitful works, at both the molecular and material levels, in both theoretical and experimental aspects, for both fundamental research and applications. This dissertation evaluates mechanochemical behavior in all of these contexts.

At the level of fundamental reactivity, forbidden reactions, such as those that violate orbital symmetry effects as captured in the Woodward-Hoffman rules, remain an ongoing challenge for experimental characterization, because when the competing allowed pathway is available, the reactions are intrinsically difficult to trigger. Recent developments in covalent mechanochemistry have opened the door to activating otherwise inaccessible reactions. This dissertation describes the first real-time observation and quantified measurement of four mechanically activated forbidden reactions. The results provide the experimental benchmarks for mechanically induced forbidden reactions, including those that violate the Woodward-Hoffmann and Woodward-Hoffmann-DePuy rules, and in some cases suggest revisions to prior computational predictions. The single-molecule measurement also captured competing reactions between isomerization and bimolecular reaction, which to the best of our knowledge, is the first time that competing reactions are probed by force spectroscopy.

Most characterization for mechanochemistry has been focused on the reactivity of mechanophores, and investigations of the force coupling efficiency are much less reported. We discovered that the stereochemistry of a non-reactive alkene pendant to a reacting mechanophore has a dramatic effect on the magnitude of the force required to trigger reactivity on a given timescale (here, a 400 pN difference for reactivity on the timescale of 100 ms). The stereochemical perturbation has essentially no measurable effect on the force-free reactivity, providing an almost perfectly orthogonal handle for tuning mechanochemical reactivity independently of intrinsic reactivity.

Mechanochemical coupling is also applied here to the study of reaction dynamics. The dynamics of reactions at or in the immediate vicinity of transition states are critical to reaction rates and product distributions, but direct experimental probes of those dynamics are rare. The s-trans, s-trans 1,3-diradicaloid transition states are trapped by tension along the backbone of purely cis-substituted gem-difluorocyclopropanated polybutadiene using the extensional forces generated by pulsed sonication of dilute polymer solutions. Once released, the branching ratio between symmetry-allowed disrotatory ring closing (of which the trapped diradicaloid structure is the transition state) and symmetry-forbidden conrotatory ring closing (whose transition state is nearby) can be inferred. Net conrotatory ring closing occurred in 5.0 ± 0.5% of the released transition states, as compared to 19 out of 400 such events in molecular dynamics simulations.

On the materials level, the inevitable stress in materials during usage causes bond breakage, materials aging and failure. A strategy for solving this problem is to learn from biological materials, which are capable to remodel and become stronger in response to the otherwise destructive forces. Benzocyclobutene has been demonstrated to mechanically active to ortho-quinodimethide, an intermediate capable for [4+4] dimerization and [4+2] cycloaddition. These features make it an excellent candidate for and synthesis of mechanochemical remodeling. A polymer containing hundreds of benzocyclobutene on the backbone was synthesized. When the polymer was exposed to otherwise destructive shear forces generated by pulsed ultrasound, its molecular weight increased as oppose to other mechanophore-containing polymers. When a solution of the polymer with bismaleimide was subjected to pulsed ultrasonication, crosslink occurred and the modulus increased by two orders of magnitude.

The insertion of force-sensitive motifs (mechanophores) into polymer backbones provides a mechanism to induce forbidden reactions, stabilize transition state, and build intrinsic stress-responsive materials. Although polymer mechanochemistry has provided the basis for a variety of stress-responsive materials (e.g., those that are mechanochromic, mechanoluminescent, and mechanocatalytic, or that release small molecules or generate novel chemical reactions), many desirable stress-responsive behaviors have yet to be realized. We applied molecular-level design and synthesis to engineer stress-responsive materials that address several gaps in the prior polymer mechanochemistry toolbox:

Chapter 2 presents four studies of structure-reactivity relationships. First, regiochemical effects on mechanophore reactivity is quantified in the context of three spiropyran (SP) derivatives that are incorporated into polydimethylsiloxane (PDMS) elastomers. Under thermodynamic control, we find that the relative activation of the regioisomers is well correlated with the extent of mechanochemical coupling between the equilibrium reaction coordinate and the applied force, as quantified by computational modeling. Second, subtle differences in stereochemistry between two gem-monochlorocyclopropane (gMCC) stereoisomers (i.e., syn and anti, relative to the polymer attachment points through which force is delivered) lead to dramatic differences in reactivity and reactivity outcomes. The two gMCCs were embedded along a polymer backbone and their mechanochemical reactivities were quantified using single molecule force spectroscopy (SMFS). The mechanical ring-opening of syn-gMCC proceeds along an anti-Woodward-Hoffmann-Depuy pathway and exhibits significantly lower reactivity than anti-gMCC. Further, under tension applied through ultrasonication, the syn-gMCC isomer generates about 0.25 equivalents of HCl per ring-opening event, whereas ring opening of the anti-gMCC led to no detectable HCl under identical conditions. Third, we report the dependence of the mechanical strength of a C-S bond on the oxidation state of the S atom (i.e., the relative mechanical strength of sulfide, sulfoxide and sulfone). Ultrasonication of gem-dichlorocyclopropane (gDCC) copolymers of each sulfur-containing group reveals that their relative mechanical strengths follow the order: polysulfide ~ polysulfone > polysulfoxide. Finally, we demonstrate the effect of cyclic polymer structure/architecture on mechanophore activation along a polymer backbone. A multi-mechanophore, cyclic gDCC copolymer was prepared via ring expansion metathesis polymerization (REMP), and its mechanochemical response to ultrasonication was compared to a linear analog prepared using ring opening metathesis polymerization (ROMP). The cyclic polymer experiences less gDCC activation per fragmentation along its backbone than does the linear analog. This observation suggests conformational memory effects in the nascent cyclic polymer during elongation and fragmentation.

Chapter 3 introduces two new mechanophores that convert mechanical input into potentially useful chemical signals, enriching the available toolkit of stress-responsive behaviors. The first mechanophore is a 1,2-diaitidinone (DAO) based four-member ring that generates reactive isocyanate upon mechanical activation via pulsed ultrasonication; evidence for the generation of isocyanate is acquired by 1H NMR analysis and trapping experiments. We anticipate that this latent reactive isocyanate might lead to materials that heal or strengthen in response to a mechanical load. A second mechanophore is a new thermally stable and nonscissile mechanoacid that is based on a methoxy-substituted gDCC and that overcomes drawbacks present in previously reported mechanoacids. The introduction of the methoxy substituent not only facilitates the release of HCl as a result of gDCC ring opening (0.58 equivalents per activation), it significantly lowers the force necessary to trigger rapid ring opening, as evidenced by SMFS studies. The utility of this new mechanoacid is demonstrated in PDMS elastomers, where its mechanical activation leads to a strain-triggered color change in a pH-sensitive dye prior to fracture of the elastomer. The post-activation kinetics of coloration are used to demonstrate a new concept in mechanochromism, namely not only a spectroscopic indicator of whether and where a mechanical event has occurred, but when it occurred.

Chapter 4 describes how the well-known photoswitch azobenzene, when embedded into PDMS elastomers, can be used as a mechanochromic probe of the molecular forces present in strained bulk materials. Specifically, the cis-to-trans isomerization of azobenzene is accelerated under uniaxial tension. The kinetics are cleanly described by a single exponential first-order process (k = 2.7 × 10-5 s-1) in the absence of tension, but they become multi-exponential under constant strains of 40-90%. The complex kinetics can be reasonably modeled as a two-component process. The majority (~92%) process is slower and occurs with a rate constant that is similar to that of the unstrained system (k = 2.3–2.7 × 10-5 s-1), whereas the rate constant of the minority (~8%) process increases from k = 10.1 × 10-5 s-1 at 40% strain to k = 21.3 × 10-5 s-1 at 90% strain. Simple models of expected force-rate relationships suggest that the average force of tension per strand in the minority component ranges from 28 pN to 44 pN across strains of 40-90%.

Finally, in Chapter 5, polymer mechanochemistry is integrated into two degradable polymers to demonstrate new concepts in mechanically coupled degradable polymers. The unintentional scission of chemical degradable functionalities on the polymer backbone can diminish polymer properties, and we report a strategy that combats unintended degradation in polymers by combining two common degradation stimuli—mechanical and acid triggers—in an “AND gate” fashion. A cyclobutane (CB) mechanophore is used as a mechanical gate to regulate an acid-sensitive ketal that has been widely employed in acid degradable polymers. This gated ketal is further incorporated into the polymer backbone. In the presence of acid trigger alone, the pristine polymer retains its backbone integrity, and delivering high mechanical forces alone by ultrasonication degrades the polymer to an apparent limiting molecular weight of 28 kDa. The sequential treatment of ultrasonication followed by acid, however, leads to a further 11-fold decrease in molecular weight to 2.5 kDa. Experimental and computational evidence further indicate that the ungated ketal possesses mechanical strength that is commensurate with the conventional polymer backbones. Single molecule force spectroscopy (SMFS) reveals that the force necessary to activate the CB molecular gate on the timescale of 100 ms is approximately 2 nN. With this success in hand, we noted that mechanical-only polymer degradation is intrinsically limited to one chain scission per stretching event. To overcome this drawback, we integrate multiple copies of a [4.2.0]bicyclooctene (BCOE) based mechanophore into the polymer backbone. Mechanochemical remodeling of the polymer backbone occurs through the force-promoted forbidden ring-opening of BCOE, the product of which undergoes a subsequent, slower cascade lactonization that leads to a spontaneous, force-free decrease in average molecular weight to 4.4 kDa (from an initial molecular weight of over 120 kDa) over the course of 9 days.

Large multi-domain proteins, which are ubiquitous in the proteomes in eukaryotic and prokaryotic organisms, still lack intensive studies on their folding mechanisms due to their complicated interactions between and inside of their domains. My work is broadly aimed at characterize folding behaviors and mechanisms of large, multi-domain proteins.

In the first part of my thesis work, I developed a novel mechanical folding polypeptide probe based on the anti-parallel coiled-coil domain of a natural protein (Archeal Box C/D sRNP Core Protein) to enable us to capture the progress of the unfolding front along the host protein structure. In this way, the structural origin of the signal from single molecule force spectroscopy (SMFS) based on atomic force microscopy (AFM) can be directly identified. Beyond this published work, I also characterized and compared the unfolding pathway of two homologous multi-domain proteins – yeast phosphoglycerate kinase (PGK) and its E.coli homolog. I found that yeast PGK has much higher mechanical stability than E.coli PGK although previous literature reported that they had similar thermodynamics stability determined by bulk measurements. In collaboration with Mr. Zackary N. Scholl, we characterized another multi-domain protein, protein S, and its two isolated domains (protein S N terminal domain and C terminal domain). By matching the statistical distributions of the unfolding forces from the truncated domains with the distributions of forces from full protein S, we solved the problem of assigning force peaks in the force-extension (FE) curves to individual domains. However, accurate determination of the structural correspondence to the force peaks still needs insertion of CC probes into the loop regions of these multi-domain proteins.

In the second part of my thesis work, I focused on integrating our AFM-SMFS with fluorescence microscopy methods. In particular, I have focused on constructing and mounting a home-made AFM force spectrometer on a high magnification inverted microscope to monitor fluorescence signal changes when stretching a single protein molecule. I am also setting up a total internal reflection fluorescence microscope (TIRF) for combination of Förster resonance energy transfer (FRET) and SMFS measurements in the future.

In Chapter 1, I will discuss background of multi-protein unfolding, SMFS method principles and coarse-grained (CG) steered molecular dynamics (SMD) simulation. This chapter will focus on the importance of understanding multi-protein unfolding pathway, the advantage of using SMFS as an experimental way of characterizing multi-domain protein unfolding behavior. As a theoretical way to analyze what event happened during the forced unfolding of multi-domain protein, CG-SMD simulations usually support our SMFS results, and providing details for interpreting FE curves obtained in SMFS measurements.

In Chapter 2, I present my recently completed work on developing a mechanical force probe based on an anti-parallel coiled-coil polypeptide chain. This work has just been published by Angewandte Chemie International Edition. The probe I developed provides a new way to determine the structural relation of the peaks shown in protein unfolding FE curves.

In Chapter 3, I report my recently started work on characterization of yeast PGK. I will show the preliminary data we obtained, (this work is also in collaboration with Mr. Zackary N. Scholl), and discuss differences in the FE traces of the two homologs and propose future work directions.

In Chapter 4, I briefly mention another work, Ca2+dependence of protein S unfolding pathway. The NPS and CPS domains were also individually studied by AFM, and their unfolding data statistics helped exploring comprehensively the structural origin of full protein S unfolding data.

In Chapter 5, I will introduce my completed work on combination of AFM and inverted Zeiss microscope. The completion of this work will enable simultaneous recording of the fluorescence signal in an SMFS experiment performed on a single molecule in the future.

In Chapter 6, I will describe my recent work on building a TIRF microscope to realize TPM measurement on single molecule multi-domain protein in our lab. We hope that the integrated instrument could enable us to detect FRET signal coming from unzipping of the CC probe when stretching by AFM.

Ankyrin repeats (ARs) are polypeptide motifs identified in thousands of proteins. Many AR proteins play a function as scaffolds in protein-protein interactions which may require specific mechanical properties. Also, a number of AR proteins have been proposed to mediate mechanotransduction in a variety of different functional settings. The folding and stability of a number of AR proteins have been studied in detail by chemical and temperature denaturation experiments, yet the mechanic of AR proteins remain largely unknown. In this dissertation, we have researched the mechanical properties of AR proteins by using protein engineering and a combination of atomic force microscopy (AFM)-based single-molecule force spectroscopy and steered molecular dynamics (SMD) simulations. Three kinds of AR proteins were investigated: NI6C (synthetic AR protein), D34 (of ankyrin-R) and gankyrin (oncoprotein). While the main focus of this research was to characterize the response of AR proteins to mechanical forces, our results extended beyond the protein nanomechanics to the understanding of protein folding mechanisms.