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The efficacy of many cancer drugs is hindered by P-glycoprotein (Pgp), a cellular pump that removes drugs from cells. To improve chemotherapy, drugs capable of evading Pgp must be developed. Despite similarities in structure, vinca alkaloids (VAs) show disparate Pgp-mediated efflux ratios. ATPase activity and binding affinity studies show at least two binding sites for the VAs: high- and low-affinity sites that stimulate and inhibit the ATPase activity rate, respectively. The affinity for ATP from the ATPase kinetics curve for vinblastine (VBL) at the high-affinity site was 2- and 9-fold higher than vinorelbine (VRL) and vincristine (VCR), respectively. Conversely, VBL had the highest Km (ATP) for the low-affinity site. The dissociation constants (KDs) determined by protein fluorescence quenching were in the order VBL < VRL< VCR. The order of the KDs was reversed at higher substrate concentrations. Acrylamide quenching of protein fluorescence indicate that the VAs, either at 10 mM or 150 mM, predominantly maintain Pgp in an open-outward conformation. When 3.2 mM AMPPNP was present, 10 mM of either VBL, VRL, or VCR cause Pgp to shift to an open-outward conformation, while 150 mM of the VAs shifted the conformation of Pgp to an intermediate orientation, between opened inward and open-outward. However, the conformational shift induced by saturating AMPPNP and VCR condition was less than either VBL or VRL in the presence of AMPPNP. At 150 mM, atomic force microscopy (AFM) revealed that the VAs shift Pgp population to a predominantly open-inward conformation. Additionally, STDD NMR studies revealed comparable groups in VBL, VRL, and VCR are in contact with the protein during binding. Our results, when coupled with VAs-microtubule structure-activity relationship studies, could lay the foundation for developing next-generation VAs that are effective as anti-tumor agents. A model that illustrates the intricate process of Pgp-mediated transport of the VAs is presented. 

Kymograph analysis is employed across the biological atomic force microscopy (AFM) community to boost temporal resolution. The method is well suited for revealing protein dynamics at the single molecule level in near-native conditions. Yet, kymograph analysis comes with limitations that depend on several factors including protein geometry and instrumental drift. This work focuses on conformational dynamics of difficult-to-study sparse distributions of membrane proteins. We compare and contrast AFM kymograph analysis for two proteins, one of which (SecDF) exhibits conformational dynamics primarily in the vertical direction (normal to the membrane surface) and the other (Pgp) exhibits a combination of lateral dynamics and vertical motion. Common experimental issues are analyzed including translational and rotational drift. Conformational transition detection is evaluated via kymograph simulations followed by state detection algorithms. We find that kymograph analysis is largely robust to lateral drift. Displacement of the AFM line scan trajectory away from the protein center of mass by a few nanometers, roughly half of the molecule diameter, does not significantly affect transition detection nor generate undue dwell time errors. On the other hand, for proteins like Pgp that exhibit significant azimuthal maximum height dependence, rotational drift can potentially produce artifactual transitions. Measuring the height of a membrane protein protrusion is generally superior to measurement of width, confirming intuition based on vertical resolution superiority. In low signal-to-noise scenarios, common state detection algorithms struggle with transition detection as opposed to infinite hidden Markov models. AFM kymography represents a valuable addition to the membrane biophysics toolkit; continued hardware and software improvements are poised to expand the method’s impact in the field. 

Candida albicans is a commensal fungus that can cause epithelial infections and life-threatening invasive candidiasis. The fungus secretes candidalysin (CL), a peptide that causes cell damage and immune activation by permeation of epithelial membranes. The mechanism of CL action involves strong peptide assembly into polymers in solution. The free ends of linear CL polymers can join, forming loops that become pores upon binding to membranes. CL polymers constitute a therapeutic target for candidiasis, but little is known about CL self-assembly in solution. Here, we examine the assembly mechanism of CL in the absence of membranes using complementary biophysical tools, including a new fluorescence polymerization assay, mass photometry, and atomic force microscopy. We observed that CL assembly is slow, as tracked with the fluorescent marker C-laurdan. Single-molecule methods showed that CL polymerization involves a convolution of four processes. Self-assembly begins with the formation of a basic subunit, thought to be a CL octamer that is the polymer seed. Polymerization proceeds via the addition of octamers, and as polymers grow they can curve and form loops. Alternatively, secondary polymerization can occur and cause branching. Interplay between the different rates determines the distribution of CL particle types, indicating a kinetic control mechanism. This work elucidates key physical attributes underlying CL self-assembly which may eventually evoke pharmaceutical development. 

Abstract The fundamental molecules of life are polymers. Prominent examples include nucleic acids and proteins, both of which exhibit a large array of mechanical properties and three-dimensional shapes. The bending rigidity of individual polymers is quantified by the persistence length. The shape of a polymer, dictated by the topology of the polymer backbone, a line trace through the center of the polymer along the contour path, is also an important characteristic. Common biomolecular architectures include linear, cyclic (ring-like), and branched structures; combinations of these can also exist, as in complex polymer networks. Determination of persistence length and shape are largely informative to polymer function and stability in biological environments. Here we demonstratePersistence lengthShapePolymer (PS Poly), a near-fully automated algorithm designed to obtain polymer persistence length and shape from single molecule images obtained in physiologically relevant fluid conditions via atomic force microscopy. The algorithm, which involves image reduction via skeletonization followed by end point and branch point detection, is capable of rapidly analyzing thousands of polymers with subpixel precision. Algorithm outputs were verified by analysis of deoxyribonucleic acid, a very well characterized macromolecule. The method was further demonstrated by application to candidalysin, a recently discovered and complex virulence factor fromCandida albicans. Candidalysin forms polymers of highly variable shape and contour length and represents the first peptide toxin identified in a human fungal pathogen. PS Poly is a robust and general algorithm. It can be used to extract fundamental information about polymer backbone stiffness, shape, and more generally, polymerization mechanisms. 

Abstract Membrane proteins play critical roles in disease and in the disposition of many pharmaceuticals. A prime example is P-glycoprotein (Pgp) which moves a diverse range of drugs across membranes and out of the cell before a therapeutic payload can be delivered. Conventional structural biology methods have provided a valuable framework for comprehending the complex conformational changes underlying Pgp function, which also includes ATPase activity, but the lack of real-time information hinders understanding. Atomic force microscopy (AFM) is a single-molecule technique that is well-suited for studying active membrane proteins in bilayers and is poised to advance the field beyond static snapshots. After verifying Pgp activity in surface-support bilayers, we used kymograph analysis in conjunction with AFM imaging and simulations to study structural transitions at the 100 ms timescale. Though kymographs are frequently employed to boost temporal resolution, the limitations of the method have not been well characterized, especially for sparse non-crystalline distributions of pharmaceutically relevant membrane proteins like Pgp. Common experimental challenges are analyzed, including protein orientation, instrument noise, and drift. Surprisingly, a lateral drift of 75% of the protein dimension leads to only a 12% probability of erroneous state transition detection; average dwell time error achieves a maximum value of 6%. Rotational drift of proteins like Pgp, with azimuthally-dependent maximum heights, can lead to artifactual transitions. Torsional constraints can alleviate this potential pitfall. Confidence in detected transitions can be increased by adding conformation-altering ligands such as non-hydrolysable analogs. Overall, the data indicate that AFM kymographs are a viable method to access conformational dynamics for Pgp, but generalizations of the method should be made with caution. 

P-glycoprotein (Pgp) plays a pivotal role in drug bioavailability and multi-drug resistance development. Understanding the protein’s activity and designing effective drugs require insight into the mechanisms underlying Pgp-mediated transport of xenobiotics. In this study, we investigated the drug-induced conformational changes in Pgp and adopted a conformationally-gated model to elucidate the Pgp-mediated transport of camptothecin analogs (CPTs). While Pgp displays a wide range of conformations, we simplified it into three model states: ‘open-inward’, ‘open-outward’, and ‘intermediate’. Utilizing acrylamide quenching of Pgp fluorescence as a tool to examine the protein’s tertiary structure, we observed that topotecan (TPT), SN-38, and irinotecan (IRT) induced distinct conformational shifts in the protein. TPT caused a substantial shift akin to AMPPNP, suggesting ATP-independent ‘open-outward’ conformation. IRT and SN-38 had relatively moderate effects on the conformation of Pgp. Experimental atomic force microscopy (AFM) imaging supports these findings. Further, the rate of ATPase hydrolysis was correlated with ligand-induced Pgp conformational changes. We hypothesize that the separation between the nucleotide-binding domains (NBDs) creates a conformational barrier for substrate transport. Substrates that reduce the conformational barrier, like TPT, are better transported. The affinity for ATP extracted from Pgp-mediated ATP hydrolysis kinetics curves for TPT was about 2-fold and 3-fold higher than SN-38 and IRT, respectively. On the contrary, the dissociation constants (KD) determined by fluorescence quenching for these drugs were not significantly different. Saturation transfer double difference (STDD) NMR of TPT and IRT with Pgp revealed that similar functional groups of the CPTs are accountable for Pgp-CPTs interactions. Efforts aimed at modifying these functional groups, guided by available structure-activity relationship data for CPTs and DNA-Topoisomerase-I complexes, could pave the way for the development of more potent next-generation CPTs. 

The fungus Candida albicans is the most common cause of yeast infections in humans. Like many other disease-causing microbes, it releases several virulent proteins that invade and damage human cells. This includes the peptide candidalysin which has been shown to be crucial for infection. Human cells are surrounded by a protective membrane that separates their interior from their external environment. Previous work showed that candidalysin damages the cell membrane to promote infection. However, how candidalysin does this remained unclear. Similar peptides and proteins cause harm by inserting themselves into the membrane and then grouping together to form a ring. This creates a hole, or ‘pore’, that weakens the membrane and allows other molecules into the cell’s interior. Here, Russell, Schaefer et al. show that candidalysin uses a unique pore forming mechanism to impair the membrane of human cells. A combination of biophysical and cell biology techniques revealed that the peptide groups together to form a chain. This chain of candidalysin proteins then closes in on itself to create a loop structure that can insert into the membrane to form a pore. Once embedded within the membrane, the proteins within the loops rearrange again to make the pores more stable so they can cause greater damage. This type of pore formation has not been observed before, and may open up new avenues of research. For instance, researchers could use this information to develop inhibitors that stop candidalysin from forming chains and harming the membranes of cells. This could help treat the infections caused by C. albicans.