Peptide and protein folding

Following the Early Steps in Protein Folding

One of the major challenges in the field of Biophysical Chemistry is the study of protein folding mechanisms, i.e., how an unstructured polypeptide chain can rapidly adopt a unique, densely packed, three dimensional structure. Erroneous folding is the molecular basis for a wide range of human disorders, such as neurodegenerative diseases including Alzheimer's and Parkinson's disease. The earliest conformational events related to folding, such as the formation of the isolated helical segments, reverse turns and β hairpins, occur within microseconds or less. Our aim is to make the early states of folding experimentally accessible, so we need to initiate those processes in shorter timescale than the fastest structural event of interest. We use ultrafast pH-jump technique to induce peptide and protein unfolding. The conformational changes during unfolding are monitored by time-resolved photoacoustics calorimetry (TR-PAC), enabling the determination of kinetic constants, enthalpy and volume changes accompanying the unfolding process. PAC is used in complement with other fast detection techniques (fluorescence and transient absorption spectroscopy) and structural techniques (NMR, CD, Ultrasound Absorption). We presently focus our study on model peptides with well defined secondary structure and proteins susceptible of pH unfolding.


Pressure wave permeation

Breaching Barriers

This project unfolds an alternative method to breach skin and cell membranes: the use of pressure waves to transiently open channels through those barriers, so that molecules can diffuse rapidly before they reversible close (within few minutes). Photoacoustic pressure waves may approach the ideal method for transdermal drug delivery because they are painless, do not lead to allergies or contaminations, and the skin rapidly recovers after the drug is administered. They may be the only direct method known that permeate the nuclear membrane and offer valuable alternative for in-vivo cell membrane controlled permeation. The efficacy of the acoustic wave depends on its form, that is, intensity, rise-time, duration and peak pressure. The efficient delivery of a relatively large molecule requires that a very high pressure is applied for a very short period of time onto the biological material. The best method to generate such an acoustic wave is by the absorption of very short laser pulses by an appropriate material. The radiative energy transiently heats the material, causes its thermal expansion and, in a confined space, efficiently originates a pressure wave. The physics of this process are known from the technique of Photoacoustic Calorimetry. When the photoacoustic wave generated by an appropriate material reaches the barrier, it produces an "earthquake" that transiently disturbs its structure and increases its permeation to large molecules.


Materials for energy

Nanostructured materials for energy applications

The quest for non-pollutant energy sources and a better use of energy is a worldwide priority, which can only be met investing in knowledge-based energy conversion devices. Presently only a small fraction of the solar radiation is used in energy conversion devices. Photovoltaic systems harvest the UV and visible part of the spectrum, corresponding to 58% of the total solar energy density. The remaining 42% correspond to the IR component, and are waste heat in conventional semiconductor-based cells. Economical collection of the UV-VIS solar radiation may be achieved by dye-sensitized solar cells (DSSC), which are among the most promising solutions for clean energy generation. Waste heat conversion has been focused on nanomaterials with favorable thermal and electron conduction properties for thermoelectric applications. Our project focuses on the molecular understanding of the interfaces existing in such energy-conversion devices, aiming at a chemical description of the processes occurring at interfaces, contributing to the design of more efficient devices. We are preparing new nanomaterials, new dyes that harvest a wider part of the Solar spectrum and optimizing the designed devices. Furthermore, we will develop and apply new techniques to investigate the properties of the materials: Photoacoustic thermal characterization of the nanomaterials will be related to their micro-structural properties. As Photoacoustic waves cross materials and act as sensors of their entire depth, we will perform subsurface analysis and depth-profiling. Our vision is that a tandem system combining solid-state DSSCs and Thermoelectric Materials will efficiently harvest and convert most of the solar spectrum and make economically viable the widespread use of Solar Energy.



The basis of the Photoacoustic (PA) effect is the non-radiative release of part of the light absorbed by the molecules in a given material, resulting in local heating and thus the generation of a pressure (or acoustic) wave propagating away from the heat source.
Since the discovery of the PA effect, in 1880 by A. G. Bell, techniques related to its application have evolved to become very efficient methods for thermal and optical characterization of light absorbing materials. The comprehensive description of the PA effect in solids and liquids, the invention of intense laser sources, and the development of highly sensitive acoustic detectors contributed to those advances.
In our Laboratory we have an accumulated experience of 20 years on time resolved Photoacoustic Calorimetry (TR-PAC). This technique was initially devised for studies in solution, but was recently extended to solid:liquid interfaces. New setups and methodologies have been developed for that purpose, and proved useful for the characterization of photoelectrochemical cells.
The intensity of the PA signal depends on the sample absorption coefficients at the incident light wavelength, on the efficiency of conversion of the incident light into heat, and on how the heat diffuses through the sample. Spectroscopic information can be obtained from the first dependency, and the absorption spectra of opaque solids acquired. The information about the non-radiative decay channels is the basis of TR-PAC, allowing the determination of enthalpic, kinetic and volume changes in photoinduced processes. Over the years we applied this technique to electron and proton transfer reactions, to the photophysical study of triplet states, to energy transfer reactions with singlet oxygen formation and many others. Recently we are using TR-PAC to follow conformational changes in peptides and proteins. Reconstruction of the heat defunded by an irradiated source is the basis of the imaging technique denominated Photoacoustic Tomography. This technique has great potential for applications in Biomedicine as it combines the advantages of acoustic resolution detection with a light absorptive contrast agent.


Photodynamic Therapy

Singlet Oxygen

In Photodynamic Therapy (PDT) a patient is injected with a molecule solution (photosensitizer) that shows some selectivity for photodamaged tumour tissue. After certain time the tumour area is irradiated with visible or near-infrared light. The photosensitizer absorbs light and transfers its energy to ground-state molecular oxygen generating singlet oxygen that attacks the (tumour) tissues. Optimal sensitizers properties can be classified as photochemical or biological. Among the first is included the ability of singlet molecular oxygen O2 sensitisation with a high quantum yield. The most important precursor of singlet oxygen is the triplet state of the sensitizer. Thus, a high singlet oxygen quantum yield requires at least three sensitizer triplet state properties: a near unity quantum yield , an electronic energy at least 20 kJ/mol above that of singlet oxygen (ED=94 kJ/mol) and a long lifetime (tT >5 µs). The desired biological properties of the sensitizers are: i) little or no dark toxicity, ii) selective accumulation and prolonged retention in tumour tissues, iii) controlled photofading to reduce the unwanted skin photosensitivity side effects and increase light penetration during therapy. The singlet molecular oxygen generation can be detected by it's near-infrared electronic emission. So, detection of the 1270 nm emission of singlet oxygen provides direct a way to evaluate the amount of this molecule formed, and therefore, partially of the efficiency of the particular sensitiser used. As part of a comum effort to design new high efficient sensitesers, I'm involved in the direct detection and quantification of singlet oxygen, using Time Resolved Phosporescence Detection. This studies are performed with an adapted Applied Photophysics flash kinetic spectrometer where the singlet oxygen emission at 1270 nm that follows laser excitation of aerated sensitizer solutions is detected by a Hamamatsu R5509-42 photomultiplier, cooled to 193 K in a liquid nitrogen chamber. It's important to emphasize that the photophysical properties of a sensitizer are important determinants for the activity of the photosensitizing drug, but they are not a direct measure for in vivo photodynamic efficiency. PDT efficiency depends upon the intrinsic photobiophysical properties of the sensitizer, the dose of photoactivating light absorbed by the photosensitizer, the oxygenation of the tissue, the selective uptake of the sensitizer into the cells, and the intracellular localization of the sensitizer.


Supercritical Fluids

Reactivity and Spectroscopy in SCF

Supercritical fluids (SCF) are highly compressed gases which combine properties of gases and liquids in an intriguing manner. A fluid is supercritical when it is above its critical temperature and critical pressure, where there is no discontinuous transition between the gas and liquid phases as a function of density. A supercritical fluid behaves like a gas in that it will expand to fill the confines of a container but forms solutions and has solvent power of a liquid. Supercritical fluids exhibit valuable thermodynamic and transport properties that make them particularly useful for chromatography and for a variety of extraction processes. Recently these fluids have been considered as interesting alternatives to organic solvents for the study of chemical reactions. Some properties show why SCF have solvent power similar to liquids, but better mass transfer characteristics: Density, gas 1; SCF 700; liquid 1000 kg/m3. Viscosity, gas 10-5; SCF 10-4; liquid 10-3 N s/m3. Diffusion coefficient, gas 10-1; SCF 10-4; liquid 10-5 cm2/s. A unique feature of fluids in the supercritical regime is their extreme compressibility. Densities ranging all the way from that of a dilute gas to that of a dense liquid can be achieved through modest changes in applied pressure. Since many properties of a fluid vary directly with its density, SCFs provide a convenient continuously tuneable solvent medium for many chemical processes. Environmentally benign separation and reactions processes are emerging to utilize the unique properties of SCF. Supercritical CO2 is, next to water, the cheapest and more ecological solvent available to man. So, carbon dioxide is the supercritical fluid of choice because it is natural, non-toxic, and cheap, having often good selectivity and capacity. Supercritical CO2 is used to extract caffeine from green coffee beans to make decaffeinated coffee or to extract flavour compounds for use in the food, pharmaceutical and cosmetic applications. A better use of supercritical CO2 requires an in-depth characterisation of its properties over a wide range of temperatures and pressures, followed by reactivity studies. The supercritical cell built in our laboratory is used with absorption (NIR/VIS/UV), fluorescence, flash photolysis and single photon counting equipment. Our present work focus on photoinduced reactions in supercritical fluids, namely excited-state proton-transfers and photoinduced electron transfers. Some of the species involved in these reactions are also good probes of solvent properties and may contribute to the characterisation of supercritical media. We study the unusual photophysics behaviour of some substances at high pressure. The specific use of photophysic characteristics of molecules for probing the macro and micromolecular behaviour of SCF is also studied.


Chemical Reactivity

Photoinduced Electron Transfer

Our purpose is the study of chemical reactivity in solution. Distinct theories of chemical reactivity are considered and their forecasts compared. Instruments are developed and new experiences elaborated with the intention to test the different predictions of the theories of chemical reactivity in electron transfer (ET) reactions. Specifically a high-pressure optical cell was developed to make kinetic studies of organic molecules dissolved in supercritical fluids, and test the medium effects. Photoinduced electron transfers between aromatic hydrocarbons and nitriles were measured in n-heptane, tetrahydrofuran and supercritical carbon dioxide. The dynamics of direct and inverse electron transfers as a function of the Gibbs energy of reaction and external pressure were studied. A range of dielectric and transport properties was investigated, and original data was obtained in the interpretation of reaction energy and medium effects on reactivity. A new relation between the recombination ET rates of those reactions and the respective Gibbs energy, that challenges the classical approaches to these reactions, was observed. The origin of the inverted region in charge separations and its end in recombination, giving rise to a new normal region, is explained by the increase of the intrinsic reorganizational barrier in the series of reactions in supercritical carbon dioxide with the reaction energy. The origin and shape of the inverted region is associated with the coupling between the reagents and the medium. The emergence of this new concept must be taken into account and may contribute to important technological developments such as the design of solar cells and of more efficient molecular devices.


Medium Dynamics Effects

Electron Transfer in Proteins

Traditional description of electron transfer (ET) processes, based on a nonadiabatic model, implicitly assumes that the vibrational relaxation dynamics of the medium (solvent and polypeptide in the case of ET in proteins) are fast compared to the rate of ET from donor (D) to acceptor (A). Under conditions where DA coupling is strong and medium relaxation dynamics slow that assumption no longer holds and, in this medium controlled adiabatic limit, rates should be independent of DA coupling and limited by medium dynamics. Experimental determination of ET rates of strongly coupled DA pairs in proteins as Azurin (A=Cu) and Cytochrome c (A=Fe), using different donors as Ru and Os permits access to different reaction energies and can test the theoretical forecasts and makes a contribute to a clear understanding of biological electron flow. Among other things the work specifically involves use of Molecular Biology techniques to conveniently mutate proteins in order to get appropriate labeling sites, synthesis of the inorganic Ru and Os complexes, proteins labeling and experimental kinetics determination using absorption and emission fast-spectroscopy with nano and picoseconds time resolution.