Sarah Rozinek

Biophysical characterization of the interaction of human albumin with an anionic porphyrin

The manuscript describes the characterization of the interaction between meso-tetrakis(p-sulfonatophenyl)porphyrin (TSPP) and human serum albumin (HSA). TSPP is a candidate for the photosensitization of structural and functional changes in proteins while HSA provides both an excellent protein model and binding and functional characteristics that could be explored in future applications of the approach. A combination of optical spectroscopic techniques (e.g., fluorescence spectroscopy, fluorescence lifetime, circular dichroism, etc.) and computational docking simulations were applied to better characterize the TSPP/HSA interaction. Recent advances have revealed that the complex formed by TSPP and HSA has become potentially relevant to biomedical applications, biomaterials research and protein photosensitized engineering. The study has determined a likely location of the binding site that places TSPP at a site that overlaps partially with the low affinity site of ibuprofen and places one of the SO3− groups of the ligand in proximity of the Trp214 residue in HSA. The characterization will enable future studies aimed at photosensitizing non-native functions of HSA for biomedical and biomaterial applications.

Photoinduced structural changes to protein kinase A

The importance of porphyrins in organisms is underscored by the ubiquitous biological and biochemical functions that are mediated by these compounds and by their potential biomedical and biotechnological applications. Protoporphyrin IX (PPIX) is the precursor to heme and has biomedical applications such as its use as a photosensitizer in phototherapy and photodetection of cancer. Among other applications, our group has demonstrated that low-irradiance exposure to laser irradiation of PPIX, Fe-PPIX, or meso-tetrakis (4-sulfonatophenyl) porphyrin (TSPP) non-covalently docked to a protein causes conformational changes in the polypeptide. Such approach can have remarkable consequences in the study of protein structure/function relationship and can be used to prompt non-native protein properties. Therefore we have investigated protein kinase A (PKA), a more relevant protein model towards the photo-treatment of cancer. PKA’s enzymatic functions are regulated by the presence of cyclic adenosine monophosphate for intracellular signal transduction involved in, among other things, stimulation of transcription, tumorigenesis in Carney complex and migration of breast carcinoma cells. Since phosphorylation is a necessary step in some cancers and inflammatory diseases, inhibiting the protein kinase, and therefore phosphorylation, may serve to treat these diseases. Changes in absorption, steady-state fluorescence, and fluorescence lifetime indicate: 1) both TSPP and PPIX non-covalently bind to PKA where they maintain photoreactivity; 2) absorptive photoproduct formation occurs only when PKA is bound to TSPP and irradiated; and 3) PKA undergoes secondary structural changes after irradiation with either porphyrin bound. These photoinduced changes could affect the protein’s enzymatic and signaling capabilities.

Photoinduced conformational changes of human serum albumin bound to protoporphyrin IX and hemin

Irradiation of porphyrins bound to proteins applies to photodynamic cancer therapy, photo-reduction of water, and the possibility of modifying proteins to impart new functions. Upon binding Protoporphyrin IX (PPIX) and hemin, respectively, to human albumin (HSA), the bound products’ response to low-dose irradiation at pH 7.4 is examined in this study. Spectroscopic data suggests that irradiation of PPIX when bound to HSA causes small secondary and tertiary protein conformational changes. Alternately, sizeable alterations are not seen when hemin bound to HSA is irradiated. This difference indicates a different photophysical mechanism for PPIX than for hemin.

Protein-protein binding before and after photo-modification of albumin

Bioeffects of directed-optical-energy encompass a wide range of applications. One aspect of photochemical interactions involves irradiating a photosensitizer with visible light in order to induce protein unfolding and consequent changes in function. In the past, irradiation of several dye-protein combinations has revealed effects on protein structure. Beta lactoglobulin, human serum albumin (HSA) and tubulin have all been photo-modified with meso-tetrakis(4- sulfonatophenyl)porphyrin (TSPP) bound, but only in the case of tubulin has binding caused a verified loss of biological function (loss of ability to form microtubules) as a result of this light-induced structural change. The current work questions if the photo-induced structural changes that occur to HSA, are sufficient to disable its biological function of binding to osteonectin. The albumin-binding protein, osteonectin, is about half the molecular weight of HSA, so the two proteins and their bound product can be separated and quantified by size exclusion high performance liquid chromatography. TSPP was first bound to HSA and irradiated, photo-modifying the structure of HSA. Then native HSA or photo-modified HSA (both with TSPP bound) were compared, to assess loss in HSA’s innate binding ability as a result of light-induced structure modification.