Lisa A. Kueltzo

Structure-Function Analysis of Invasion Plasmid Antigen C (IpaC) from Shigella flexneri

Shigella flexneri causes a self-limiting gastroenteritis in humans, characterized by severe localized inflammation and ulceration of the colonic mucosa. Shigellosis most often targets young children in underdeveloped countries. Invasion plasmid antigen C (IpaC) has been identified as the primary effector protein for Shigella invasion of epithelial cells. Although an initial model of IpaC function has been developed, no detailed structural information is available that could assist in a better understanding of the molecular basis for its interactions with the host cytoskeleton and phospholipid membrane. We have therefore initiated structural studies of IpaC, IpaC I′, (residues 101–363 deleted), and IpaC ΔH (residues 63–170 deleted). The secondary and tertiary structure of the protein was examined as a function of temperature, employing circular dichroism and high resolution derivative absorbance techniques. ANS (8-anilino-1-napthalene sulfonic acid) was used to probe the exposure of the hydrophobic surfaces under different conditions. The interaction of IpaC and these mutants with a liposome model (liposomes with entrapped fluorescein) was also examined. Domain III (residues 261–363) was studied using linker-scanning mutagenesis. It was shown that domain III contains periodic, sequence-dependent activity, suggesting helical structure in this section of the protein. In addition to these structural studies, investigation into the actin nucleation properties of IpaC was conducted, and actin nucleation by IpaC and some of the mutants was exhibited. Structure-function relationships of IpaC are discussed.

Probing protein structure and dynamics by second-derivative ultraviolet absorption analysis of cation–π interactions

We describe an alternate approach for studying protein structure using the detection of ultraviolet (UV) absorbance peak shifts of aromatic amino acid side chains induced by the presence of salts. The method is based on the hypothesis that salt cations (Li+, Na+, and Cs+) of varying sizes can differentially diffuse through protein matrices and interact with benzyl, phenyl, and indole groups through cation–π interactions. We have investigated the potential of this method to probe protein dynamics by measuring high resolution second‐derivative UV spectra as a function of salt concentration for eight proteins of varying physical and chemical properties and the N‐acetylated C‐ethyl esterified amino acids to represent totally exposed side chains. We show that small shifts in the wavelength maxima for Phe, Tyr, and Trp in the presence of high salt concentrations can be reliably measured and that the magnitude and direction of the peak shifts are influenced by several factors, including protein size, charge, and the local environment and solvent accessibility of the aromatic groups. Evaluating the empirical UV spectral data in light of known protein structural information shows that probing cation–π interactions in proteins reveals unique information about the influence of structure on aromatic side chain spectroscopic behavior.

Conformational lability of herpesvirus protein VP22.

The herpesvirus protein VP22 traffics between cells, being exported from expressing cells in a non-Golgi-dependent manner and localizing in the nuclei of surrounding cells. This transport is retained in certain VP22 fusion proteins, making VP22 a candidate for use in macromolecular drug delivery. In an effort to understand the physical basis for this activity, we have initiated structural studies of VP22.C1, the C-terminal half of VP22, which possesses the full transport activity of the native protein. CD and Fourier transform infrared analyses indicate a secondary structure consisting of approximately 30% α-helix, 17% β-sheet, and 51% disordered and turn structure. Unfolding studies conducted by CD, differential scanning calorimetry, and fluorescence reveal a series of discrete structural transitions in the range of 20–60 °C. CD and fluorescence studies of interactions between VP22.C1 and divalent cations and model polyanions indicate that Mg2+, Zn2+, oligonucleotides, and heparin interact with the protein, causing changes in secondary structure and thermal stability. Additionally, the interaction of VP22.C1 with model lipids was examined. Fluorescence titrations of the protein withtrans-parinaric acid at various temperatures suggest the binding of one to two molecules of parinaric acid to VP22.C1 at temperatures >40 °C, suggesting the possibility of conformation dependent membrane interaction under physiological conditions.

Ultraviolet Absorption and Circular Dichroism Spectroscopy of Nonviral Gene Delivery Complexes

A powerful, yet often underutilized tool available to probe the structure of macromolecules is absorption spectroscopy. The first of the various spectroscopic techniques to be widely developed, data from absorption spectroscopy is commonly viewed as low in overall information content compared to modern forms of X-ray diffraction, nuclear magnetic resonance, fourier-transformed infrared, and circular dichroism (CD) spectroscopies. With the development of diode array spectrophotometers and analysis software capable of quickly and simply producing high-definition derivative spectra, however, ultraviolet-visible spectroscopy remains a useful analytical technique, especially when applied to nucleic acids. This approach offers significant advantages in terms of flexibility, accuracy, speed, low expense, and instrumental availability. Thus, the technique can be productively applied to the analysis of nonviral gene delivery vectors, focusing primarily on the oligonucleotide component. Here are presented applications of derivative absorbance spectroscopy to the analysis of nucleic acid-cationic polymer complexes. Additionally, a brief overview of the theory, instrumentation, and general applications of zero and second-derivative absorption spectroscopy is presented, followed by a discussion of the complementary absorptive technique of circular dichroism in the same context.

Potential use of non-classical pathways for the transport of macromolecular drugs.

Since an increasing number of drug delivery strategies utilising proteins and peptides exhibiting ‘non-classical’ transport activities have been proposed, studies have begun to establish underlying functional relationships between different vectors. These attempts to find common factors have been hampered by a lack of biophysical data for the various potential protein and peptide transporters, as well as by the structural and functional diversity of the group as a whole. We describe the various types of vectors being considered for use and the preliminary therapeutic successes that have been achieved. Additionally, the various models that have been proposed for non-classical import and export are outlined and discussed in relation to therapeutic delivery. Possible future developments are also discussed.

Characterization of Synthetic Gene Delivery Vectors by Infrared Spectroscopy

For many decades, infrared (IR) spectroscopy has been used to characterize the structure of molecules. In IR spectroscopy, absorption of light, corresponding to vibrational and rotational transitions of a molecule, is measured. For a transition to be IR-active, a change in the dipole moment of a particular bond must occur upon excitation. This vibrational energy is not only dependent on the chemical nature of the particular covalent bonds, but also on the environment of these coupled atoms and bonds. IR spectroscopy has been previously employed in the study of the structure of nucleic acids, producing not only information about the individual bases, sugars, and phosphate backbone, but also providing information about the helical conformation of polynucleotides (1-3). IR spectroscopy has also been successfully applied to the analysis of lipids, as well as to numerous other polymers (4). Thus, IR spectroscopy potentially possesses the ability to obtain structural information about all of the components of most synthetic gene delivery complexes, as well as changes in the structure of polymeric or lipid components upon complex formation. In addition to the ability to gather detailed structural information, there are also some practical advantages to the use of IR spectroscopy for the study of plas-mid DNA and DNA complexes compared to other techniques, including the availability of a variety of sampling techniques, permitting the analysis of samples in a wide variety of physical states including solutions, solids, and gels. There is also no upper limit to the size of the sample molecule examined, allowing both short oligonucleotides and higher molecular weight DNA to be studied. IR spectroscopy is not a destructive technique, and requires only small amounts of material, making it ideal for the analysis of valuable samples.