Browsing by Subject "Atomic force microscopy"

This study is focused on designing and characterizing protein-based building blocks in order to construct self-assembled nano-structured biomaterials. In detail, this research aims to: (1) investigate a new class of proteins that possess nanospring behaviors at a single-molecule level, and utilize these proteins along with currently characterized elastomeric proteins as building blocks for nano-structured biomaterials; (2) develop a new method to accurately measure intermolecular interactions of self-assembling two or more arbitrary (poly)peptides, and select some of them which have appropriate tensile strength for crosslinking the proteins to construct elastomeric biomaterials; (3) construct well-defined protein building blocks which are composed of elastomeric proteins terminated with self-oligomerizing crosslinkers, and characterize self-assembled structures created by the building blocks to determine whether the elasticity of proteins at single-molecule level can be maintained.

Primary experimental methods of this research are (1) atomic force microscope (AFM) based single-molecule force spectroscopy (SMFS) that allows us to manipulate single molecules and to obtain their mechanical properties such as elasticity, unfolding and refolding properties, and force-induced conformational changes, (2) AFM imaging that permits us to identify topology of single molecules and supramolecular structures, and (3) protein engineering that allows us to genetically connect elastomeric proteins and self-assembling linkers together to construct well-defined protein building blocks.

Nanospring behavior of á-helical repeat proteins: We revealed that á-helical repeat proteins, composed of tightly packed á-helical repeats that form spiral-shaped protein structures, unfold and refold in near equilibrium, while they are stretched and relaxed during AFM based SMFS measurements. In addition to minimal energy dissipation by the equilibrium process, we also found that these proteins can yield high stretch ratios (>10 times) due to their packed initial forms. Therefore, we, for the first time, recognized a new class of polypeptides with nanospring behaviors.

Protein-based force probes for gauging molecular interactions: We developed protein-based force probes for simple, robust and general AFM assays to accurately measure intermolecular forces between self-oligomerization of two or more arbitrary polypeptides that potentially can serve as molecular crosslinkers. For demonstration, we genetically connected the force probe to the Strep-tag II and mixed it with its molecular self-assembling partner, the Strep-Tactin. Clearly characterized force fingerprints by the force probe allowed identification of molecular interactions of the single Strep-tag II and Strep-Tactin complex when the complex is stretched by AFM. We found a single energy barrier exists between Strep-tag II and Strep-Tactin in our given loading rates. Based upon our demonstration, the use of the force probe can be expanded to investigate the strength of interactions within many protein complexes composed of homo- and hetero-dimers, and even higher oligomeric forms. Obtained information can be used to choose potential self-assembling crosslinkers which can connect elastomeric proteins with appropriate strength in higher-order structures.

Self-assembled nano-structured biomaterials with well-defined protein-based building blocks: We constructed well-defined protein building blocks with tailored mechanical properties for self-assembled nano-structured materials. We engineered protein constructs composed of tandem repeats of either a I27-SNase dimer or a I27 domain alone and terminated them with a monomeric streptavidin which is known to form extremely stable tetramers naturally. By using molecular biology and AFM imaging techniques, we found that these protein building blocks transformed into stable tetrameric complexes. By using AFM based SMFS, we measured, to our knowledge for the first time, the mechanical strength of the streptavidin tetramer at a single-molecule level and captured its mechanical anisotropy. Using streptavidin tetramers as crosslinkers offers a unique opportunity to create well-defined protein based self-assembled materials that preserve the molecular properties of their building blocks.

This research describes the development of a state-of-the-art atomic force microscope (AFM) for improved force spectroscopy. Although the AFM has been used extensively in this field of research, the performance of the instrument has been limited by inefficient operation techniques, incorrect experimental assumptions, and inadequate controller design. This research focuses on overcoming these deficiencies by providing precise control over the instrument for specialized research in a manner that is conducive to the natural science researcher.

To facilitate this research, a custom, multi-axis AFM system was constructed. The instrument was designed primarily for AFM-based force spectroscopy and as a result a substantial amount of research focused on the development of a wide variety of approach/retraction methods for the instrument. Defining research in this area included the development of methods to minimize potentially damaging compressive forces, form polymer bridges at different tip-sample gap widths, produce clean, deconvoluted force-extension curves, and limit single molecule force spectroscopy pulling geometry errors. In an effort to increase the efficiency of the instrument, the programs developed during this research were fully automated, allowing autonomous operation of the instrument for long periods of time. To compliment the data collection programs, both manual and automated analysis programs with force-volume imaging capabilities were also developed.

By studying the AFM from a dynamic systems, measurements, and controls approach, the resulting controllers were tailored to meet the process requirements of the intended applications. In doing so, the sensitivity of the instrument was improved for applications of interest. By incorporating control over the environment, contact force, loading rate, and pulling angle, the research has increased the accuracy of the AFM such that molecules and receptor-ligand binding events can be investigated with greater detail. Furthermore, the incorporation of a graphical user interface and automated data collection and analysis tools has made the AFM a more user-friendly, efficient instrument for the natural science researcher.

Virtually all biologically relevant functions and processes are mediated by non-covalent, molecular recognition events, demonstrating astonishingly diverse affinities and specificities. Despite extensive research, the origin of affinity and specificity in aqueous solution - specifically the relationship between ligand binding thermodynamics and structure - remains remarkably obscure and is further complicated in the context of multivalent interactions. Multivalency describes the combinatorial interaction of multiple discrete epitopes across multiple binding surfaces where the association is considered as the sum of contributions from each epitope and the consequences of multivalent ligand assembly. Gaining the insight necessary to predictably influence biological processes with novel therapeutics begins with an understanding of the molecular basis of solution-phase interactions, and the thermodynamic parameters that follow from those interactions. Here we continue our efforts to understand the basis of aqueous affinity and the nature of multivalent additivity.

Multivalent additivity is the foundation of fragment-based drug discovery, where small, low affinity ligands are covalently assembled into a single high affinity inhibitor. Such systems are ideally suited for investigating the thermodynamic consequences of multivalent ligand assembly. In the first part of this work, we report the design and synthesis of a fragment-based ligand series for the Grb2-SH2 protein and thermodynamic evaluation of the low affinity ligand fragments compared to the intact, high affinity inhibitor by single and double displacement isothermal titration calorimetry (ITC). Interestingly, our investigations reveal positively cooperative multivalent additivity - a binding free energy of the full ligand greater than the sum of its constituent fragments - that is largely enthalpic in origin. These results contradict the most common theory of multivalent affinity enhancement arising from a "savings" in translational and rotational entropy. The Grb2-SH2 system reported here is the third distinct molecular system in which we have observed enthalpically driven multivalent enhancement of affinity.

Previous research by our group into similar multivalent affinity enhancements in protein-carbohydrate systems - the so-called "cluster glycoside effect" - revealed that evaluation of multivalent interactions in the solution-phase is not straightforward due to the accessibility of two disparate binding motifs: intramolecular, chelate-type binding and intermolecular, aggregative binding. Although a number of powerful techniques for evaluation of solution-phase multivalent interactions have been reported, these bulk techniques are often unable to differentiate between binding modes, obscuring thermodynamic interpretation. In the second part of this work, we report a competitive equilibrium approach to Molecular Recognition Force Microscopy (MRFM) for evaluation of ligand binding at the single-molecule level with potential to preclude aggregative associations. We have optimized surface functionalization strategies and MRFM experimental protocols to evaluate the binding constant of surface- and tip-immobilized single stranded DNA epitopes. Surprisingly, the monovalent affinity of an immobilized species is in remarkable agreement with the solution-phase affinity, suggesting the competitive equilibrium MRFM approach presents a unique opportunity to investigate the nature of multivalent additivity at the single molecule level.

Primary OAG (POAG) is the second leading cause of irreversible blindness in the US and its prevalence is expected to worsen in the coming years. Major pathological changes have been attributed to the Juxtacanicular Tissue (JCT) and Inner Wall (IW) of Schlemm’s Canal (SC) within the human conventional outflow pathway. Biological tissues like the JCT/IW are viscoelastic in nature with both intra-cellular and extra-cellular mechanisms by which to store and dissipate applied forces. How these events contribute towards regulation of the local mechanobiology in the dynamic reciprocitybetween cells, their extracellular matrix (ECM), and in ultimately regulating outflow resistance, is poorly understood. Furthermore, the viscoelastic properties of the human trabecular meshwork (hTM) tissue are poorly understood. As a first step to develop more insight into the role of viscoelasticity, it was our goal to determine the localized dynamic mechanical properties of different regions of the hTM as a function of dexamethasone treatment. To explore the viscoelastic properties of the different tissue regions in the hTM comprising the hTM, we applied our co-located AFM-based rheometer/CLSM method to frontal sections of hTM under control and early ocular hypertensive conditions across a broad frequency range (1 Hz-1 kHz). We specifically considered the storage and loss moduli in the ocular pulse-relevant frequency range (1-10 Hz) and their changes across regions under dex treatment for two donors.

The role of articular cartilage in diarthrodial joints is primarily mechanical as the tissue provides a nearly frictionless, load-bearing surface that supports and distributes forces generated during joint loading. Embedded within the extensive cartilage extracellular matrix (ECM), chondrocytes are surrounded by a narrow, distinct pericellular matrix (PCM) that is thought to regulate the biomechanical microenvironment of the cell and influence chondrocyte metabolism, cartilage homeostasis, and overall joint health. While previous studies of PCM mechanical properties required physical extraction of the cell and PCM from the tissue, atomic force microscopy (AFM) provides a means for high resolution microindentation testing that can be used to measure local mechanical properties in situ. This dissertation develops and applies AFM microindentation techniques to 1) evaluate the microscale elastic properties of the cartilage PCM and ECM in situ and 2) correlate site-specific biochemical composition with biomechanical properties of the PCM and ECM.

An AFM-based stiffness mapping technique was experimentally validated and applied to cartilage sections to evaluate ECM and PCM properties in situ with minimal disruption of native matrix integration. As expected, PCM elastic moduli were significantly less than ECM moduli, uniform with depth, and mechanically isotropic. ECM moduli exhibited distinct depth-dependent anisotropy and unexpectedly, were found to decrease with depth from the articular surface. Both the PCM and ECM exhibited alterations in microscale moduli and their spatial distributions when evaluated in cartilage presenting early degenerative changes associated with osteoarthritis (OA) as compared to healthy tissue.

The ability to correlate site-specific biochemical composition with local biomechanical properties provides a more complete characterization of the chondrocyte microenvironment. To this end, we developed novel immunofluorescence (IF)-guided AFM stiffness mapping and demonstrated that PCM mechanical properties correlate with the presence of type VI collagen. Extending this technique by using dual IF, we presented new evidence for a defining role of perlecan in the PCM, showing that interior regions of the PCM rich in perlecan and type VI collagen exhibit lower elastic moduli than peripheral PCM and ECM regions lacking perlecan. Furthermore, lower moduli at the PCM interior were significantly influenced by the presence of heparan sulfate. IF-guided AFM stiffness mapping was combined with enzymatic digestion to demonstrate that the micromechanical properties of the PCM exhibit high resistance to specific enzymatic digestion of aggrecan and aggrecan-associated glycosaminoglycans but are vulnerable to proteolytic degradation by leukocyte elastase.

Overall, this research generates new insights into the complex structural, compositional, and functional relationships between the cartilage ECM and PCM and provides the tools and framework for further studies to continue to investigate their importance in regulating chondrocyte physiology in health and disease.

One promising HIV-1 vaccine target is the membrane-proximal external region (MPER) of viral gp41. MPER is poorly immunogenic, however, the two rare neutralizing antibodies (NAbs), 2F5 and 4E10, bind to MPER with great neutralizing ability. Although their neutralizing mechanism represents a promising framework for the design of new HIV-1 liposomal vaccine candidates, this mechanism remains poorly understood. It is known that 2F5 and 4E10 are required to first associate with HIV-1 lipids before binding to the target MPER antigen, however, little is known about how lipid membranes contribute to NAb-antigen binding. To this end we have developed model membrane systems to study NAb and antigen lipid interactions.

We first created a surface plasmon resonance (SPR) spectroscopy based assay that monitors antibody binding to thiol monolayers, which mimic the surface chemical properties of lipid membranes. Next, we focused on mimicking the lipid phase organization (i.e., domain formation) of native membranes by using supported lipid bilayers (SLBs). We used simple SLB compositions to model the liquid-disordered (Ld) and gel phases. To model the HIV-1 envelope, we used a complex SLB composition that contains an Ld and liquid-ordered (Lo) phase. To reliably create model HIV-1 SLBs, we developed an SLB formation technique that uses amphipathic, α-helical peptides as a catalyst to generate complex SLBs that have a high cholesterol content and contain multiple lipid types. For all SLB surfaces we used atomic force microscopy (AFM) to visualize membrane domains, antigen presentation, and antibody-membrane interactions.

Results from experiments using thiol surfaces showed that NAb binding to hydrophobic thiol surfaces was significantly greater than that of control monoclonal antibodies. This supports the hypothesis that these NAbs embed into the hydrophobic membrane core. Our results demonstrate that 2F5/4E10 do not interact with the highly ordered gel and Lo domains in the SLB but exclusively bind to the Ld phase. This suggests that 2F5/4E10 require low membrane order and weak lateral lipid-lipid interactions to insert into the hydrophobic membrane interior. Thus, vaccine liposomes that primarily contain an Ld phase are more likely to elicit the production of lipid reactive, 2F5- and 4E10-like antibodies, compared to liposomes that contain an Lo or gel phase. In the context of liposomal antigen presentation, our results show that the presence of the MPER656 antigen can severely limit the Ld area available for antibody interactions. Subsequently, this reduces the amount of MPER656 that is accessible for 2F5/4E10 binding, since MPER656 preferentially localizes to the Ld area. If Ld forming lipid components are used in vaccine liposomes, it is important to ensure that the presence of antigen does not inhibit large-scale Ld formation.

Proteins are specialized molecules that catalyze most of the reactions that can sustain life, and they become functional by folding into a specific 3D structure. Despite their importance, the question, "how do proteins fold?" - first pondered in in the 1930's - is still listed as one of the top unanswered scientific questions as of 2005, according to the journal Science. Answering this question would provide a foundation for understanding protein function and would enable improved drug targeting, efficient biofuel production, and stronger biomaterials. Much of what we currently know about protein folding comes from studies on small, single-domain proteins, which may be quite different from the folding of large, multidomain proteins that predominate the proteomes of all organisms.

In this thesis I will discuss my work to fill this gap in understanding by studying the unfolding and refolding of large, multidomain proteins using the powerful combination of single-molecule force-spectroscopy experiments and molecular dynamic simulations.

The three model proteins studied - Luciferase, Protein S, and Streptavidin - lend insight into the inter-domain dependence for unfolding and the subdomain stabilization of binding ligands, and ultimately provide new insight into atomistic details of the intermediate states along the folding pathway.