Folding and hydrodynamics of a DNA i-motif from the c-MYC promoter determined by fluorescent cytidine analogs.

The four-stranded i-motif (iM) conformation of cytosine-rich DNA has importance to a wide variety of biochemical systems that range from their use in nanomaterials to potential roles in oncogene regulation. The iM structure is formed at slightly acidic pH, where hemiprotonation of cytosine results in a stable C-C(+) basepair. Here, we performed fundamental studies to examine iM formation from a C-rich strand from the promoter of the human c-MYC gene. We used a number of biophysical techniques to characterize both the hydrodynamic properties and folding kinetics of a folded iM. Our hydrodynamic studies using fluorescence anisotropy decay and analytical ultracentrifugation show that the iM structure has a compact size in solution and displays the rigidity of a double strand. By studying the rates of circular dichroism spectral changes and quenching of fluorescent cytidine analogs, we also established a mechanism for the folding of a random coil oligo into the iM. In the course of determining this folding pathway, we established that the fluorescent dC analogs tC° and PdC can be used to monitor individual residues of an iM structure and to determine the pKa of an iM. We established that the C-C(+) hydrogen bonding of certain bases initiates the folding of the iM structure. We also showed that substitutions in the loop regions of iMs give a distinctly different kinetic signature during folding compared with bases that are intercalated. Our data reveal that the iM passes through a distinct intermediate form between the unfolded and folded forms. Taken together, our results lay the foundation for using fluorescent dC analogs to follow structural changes during iM formation. Our technique may also be useful for examining folding and structural changes in more complex iMs.

Effect of basic cell-penetrating peptides on the structural, thermodynamic, and hydrodynamic properties of a novel drug delivery vector, ELP[V5G3A2-150].

Elastin-like polypeptides (ELPs) are large, nonpolar polypeptides under investigation as components of a novel drug delivery system. ELPs are soluble at low temperatures, but they desolvate and aggregate above a transition temperature (TT). This aggregation is being utilized for targeting systemically delivered ELP-drug conjugates to heated tumors. We previously examined the structural, thermodynamic, and hydrodynamic properties of ELP[V5G3A2-150] to understand its behavior as a therapeutic agent. In this study, we investigate the effect that adding basic cell-penetrating peptides (CPPs) to ELP[V5G3A2-150] has on the polypeptide's solubility, structure, and aggregation properties. CPPs are known to enhance the uptake of ELP into cultured cells in vitro and into tumor tissue in vivo. Interestingly, the asymmetric addition of basic residues decreased the solubility of ELP[V5G3A2-150], although below the TT we still observed a low level of self-association that increased with temperature. The ΔH of the aggregation process correlates with solubility, suggesting that the basic CPPs stabilize the aggregated state. This is potentially beneficial as the decreased solubility will increase the fraction aggregated and enhance drug delivery efficacy at a heated tumor. Otherwise, the basic CPPs did not significantly alter the biophysical properties of ELP. All constructs were monomeric at low temperatures but self-associate with increasing temperature through an indefinite isodesmic association. This self-association was coupled to a structural transition to type II ß-turns. All constructs reversibly aggregated in an endothermic reaction, consistent with a reaction driven by the release of water.

Biochemical evidence that human EB1 does not bind preferentially to the microtubule seam.

EB1 is a highly conserved microtubule (MT) plus end tracking protein (+TIP) involved in regulating MT dynamics, but the mechanisms of its effects on MT polymerization remain undefined. Resolving this question requires understanding how EB1 interacts with MTs. Previous electron microscopy of the S. pombe EB1 homolog Mal3p suggested that Mal3p binds specifically to the MT seam, implying that EB1 family members promote MT polymerization by stabilizing the seam. However, more recent electron microscopy indicates that Mal3p binds everywhere except the seam. Neither set of experiments investigated the behavior of human EB1, or provided an explanation for why these studies arrived at different answers. To resolve these questions, we have used a combination of MT-binding assays and theoretical modeling with MTBindingSim. Our results indicate that human EB1 binds to the lattice, consistent with the recent Mal3p results, and show that Mal3p-binding assays that were previously interpreted as evidence for preferential seam binding are equally consistent with weak lattice binding. In addition, we used analytical ultracentrifugation to investigate the possibility that the EB1 monomer-dimer equilibrium might contribute to EB1 binding behavior, and determined that the EB1 dimerization dissociation constant is approximately 90 nM. We and others find that the cellular concentration of EB1 is on the order of 200 nM, suggesting that a portion of EB1 may be monomeric at physiological concentrations. These observations lead us to suggest that regulation of EB1 dimerization might play a role in controlling EB1 function.

Structural and hydrodynamic analysis of a novel drug delivery vector: ELP[V5G3A2-150].

The therapeutic potential of elastin-like polypeptide (ELP) conjugated to therapeutic compounds is currently being investigated as an approach to target drugs to solid tumors. ELPs are hydrophobic polymers that are soluble at low temperatures and cooperatively aggregate above a transition temperature (TT), allowing for thermal targeting of covalently attached drugs. They have been shown to cooperatively transition from a disordered structure to a repeating type II ß-turn structure, forming a ß-spiral above the TT. Here we present biophysical measurements of the structural, thermodynamic, and hydrodynamic properties of a specific ELP being investigated for drug delivery, ELP[V5G3A2-150]. We examine the biophysical properties below and above the TT to understand and predict the therapeutic potential of ELP-drug conjugates. We observed that below the TT, ELP[V5G3A2-150] is soluble, with an extended conformation consisting of both random coil and heterogeneous ß structures. Sedimentation velocity experiments indicate that ELP[V5G3A2-150] undergoes weak self-association with increasing temperature, and above the TT the hydrophobic effect drives aggregation entropically. These experiments also reveal a previously unreported temperature-dependent critical concentration (Cc) that resembles a solubility constant. Labeling ELP[V5G3A2-150] with fluorescein lowers the TT by 3.5°C at 20 µM, whereas ELP[V5G3A2-150] dissolution in physiological media (fetal bovine serum) increases the TT by ∼2.2°C.

Are fluorescence-detected sedimentation velocity data reliable?

Sedimentation velocity analytical ultracentrifugation is a classical biophysical technique that is commonly used to analyze the size, shape, and interactions of biological macromolecules in solution. Fluorescence detection provides enhanced sensitivity and selectivity relative to the standard absorption and refractrometric detectors, but data acquisition is more complex and can be subject to interference from several photophysical effects. Here, we describe methods to configure sedimentation velocity measurements using fluorescence detection and evaluate the performance of the fluorescence optical system. The fluorescence detector output is linear over a concentration range of at least 1 to 500nM fluorescein and Alexa Fluor 488. At high concentrations, deviations from linearity can be attributed to the inner filter effect. A duplex DNA labeled with Alexa Fluor 488 was used as a standard to compare sedimentation coefficients obtained using fluorescence and absorbance detectors. Within error, the sedimentation coefficients agree. Thus, the fluorescence detector is capable of providing precise and accurate sedimentation velocity results that are consistent with measurements performed using conventional absorption optics, provided the data are collected at appropriate sample concentrations and the optics are configured correctly.

Renin-dependent hypertension caused by nonfocal stenotic aberrant renal arteries: proof of a new syndrome.

We have identified 2 relatively young patients with significant hypertension, an elongated single aberrant renal artery supplying blood to a renal segment, and evidence for localization of the elevated plasma renin activity to the side and vein draining the affected kidney. Furosemide-induced diuresis and acute oral captopril stimulated the renal vein/contralateral renin ratios to 4.3:1 and 6.5:1 in patients 1 and 2, respectively. These renal vein ratios are significantly higher than normal (>3:1 under similar conditions). Partial resection of the portion of the kidney affected by the aberrant tortuous artery led to a marked reduction in blood pressure in patient 1. Patient 2, not an operative candidate, responded satisfactorily to use of a converting enzyme inhibitor, which helped to confirm the dependency of the blood pressure on the abnormal flow relationship existing within that aberrant artery and the kidney. We believe these 2 patients are representative of a small but distinct subgroup within the larger number of patients with elongated single or multiple renal aberrant arteries. Each aberrant artery had no focal stenosis, although a decrease in flow relative to the tissue perfusion demands was apparent from the marked activation of the renin-angiotensin system in the venous system draining that artery. The increased length of such vessels may contribute to their decreased flow, although their average diameter may reside just above such a critical value for a normal length vessel. This new syndrome, involving more than one component of the flow/resistance relationship, has been overlooked when renin-dependent forms of hypertension are considered.