Shed light on the exciting life of proteins!

To begin with

Proteins are substances necessary for our body’s health and well being . However, the shape of a protein is critical for biological processes, such as the transport of oxygen in the body by the blood, the efficiency of the immune system, and its association with the development of diseases such as Alzheimer, Parkinson or even cancer. Scientists try to develop basic knowledge about protein shapes (‘protein folding’), by studying tiny protein parts called peptides.

Keywords

Protein, Protein folding, Amino acid, Electromagnetic radiation, Spectroscopy, Infrared (IR) light, Ultraviolet (UV) light

About

Proteins are mostly known as substances necessary for our body’s health and well being, which can be found in certain types of food, especially red meat but also fish, dairy products, pulses etc. Proteins are produced within cells and are indeed the biological workhorses that carry out vital functions in every organism. An original research approach is applied at Francis Perrin Laboratory providing vital knowledge on proteins, which combines sophisticated methodology, experimental techniques and advanced theoretical models. (watch Dr. Mons talking about the combination of theoretical models and experimental data)

By studying the structure of a small molecule corresponding to specific parts of larger proteins, the scientists try to:
•    understand why a protein molecule adopts specific shapes  and not others
•   predict the probable shapes a protein is going to adopt on the basis of its molecular structure –that prediction model would be extremely beneficial, e.g. in the development of pharmaceutical products (watch Dr. Gloaguen talking about the project goals)

Links with society

Many different people, including biochemists, physical chemists, theoreticians, physicians designing drugs or protocols to cure diseases need to know as much as possible about proteins to get answers to very basic questions. Scientists at Francis Perrin Laboratory focus on fundamental research and try to collect data and answer some of these questions about folded proteins. (watch Dr. Gloaguen talking about the type of their research) Unravelling the forces responsible for the structure of proteins may help in understanding their biological function and the way they are working, and here, lies great hidden potential directly affecting pharmaceutical and biotech research. That’s how basic research increases the chances for improving various aspects of everyday life. (watch Dr. Mons talking about the importance of protein shape)

Research innovation

By studying the structure of peptides which are small molecules corresponding to tiny parts of larger proteins, scientists try to:
•    understand why a protein adopts specific shapes and not others
•    predict the probable shapes a protein is going to adopt on the basis of its structure – that prediction model would be extremely beneficial, e.g. in the development of pharmaceutical products

The method used at Francis Perrin Laboratory includes both, sophisticated methodology and experimental techniques, which use laser light beams and advanced theoretical models.
This novel approach has been successfully tested for several years at the Francis Perrin Laboratory. The main advantages of this method compared to other experimental or theoretical methods in use (e.g. X-ray Crystallography or Nuclear Magnetic Resonance) are the following:

•    the experiments evolve in a very simple environment with no interfering parameters (isolated environment). This allows for a better and more accurate identification of all the internal factors related to protein folding. (watch Dr. Mons talking about the importance of gas phase environment)
•    the central role of lasers (light amplifiers) during the experiments guarantees the acquisition of data as precise as possible. The experimental results are then fed in new computer-based models in order to predict some probable shapes of the protein molecule. The more accurate the data, the more reliable the model predictions.

Achievements so far
Scientists at Francis Perrin Laboratory have successfully introduced a new technique that enables the first experiments revealing the shape of very small parts of proteins called isolated peptides. The detailed study of these tiny portions of proteins give a unique opportunity to work on protein folding at an unprecedented level.


The future
Scientists at the Francis Perrin Laboratory now work closely with theoreticians in order to include their results in elaborate theoretical models able to describe correctly the shape of any peptide. Once implemented in a computer, these models will hopefully be applied to larger proteins giving indications about their shape more reliably than ever before.
Experimental developments are also in progress in order to control the shape of peptides using ultraviolet (UV) laser beams. These new experiments will create new knowledge about another important property of proteins, their flexibility.


Relevant scientific publications
--  Physical Chemistry Chemical Physics (PCCP), M. Mons et al., 2007, 9, 4491

Description of the method

Let’s now follow the researchers of the Francis Perrin Laboratory (FPL) into their lab.

Scientists study peptides structure with the help of spectroscopy. Spectroscopy is a technique which is used to measure the interaction of matter with light in order to reveal various characteristics of this matter. Spectroscopy measurements are based upon the amount of light absorbed, transmitted or scattered by an object. Light is electromagnetic radiation which can be emitted  at specific wavelengths onto the matter using lasers. This technique is extremely useful in the case of studying the shape of proteins, where two kinds of light beams, the infrared (IR) and ultraviolet (UV), can reveal the molecule’s structure. (Watch Dr. Mons talking about the use of IR and UV spectroscopy)

When studying proteins structure, scientists at FPLpeptides experiment with in the gas phase. But, is it wise to do so? Most of chemical compounds can exist in at least three different states of matter, the solid, the liquid and the gas phase (think, for example, of water: ice, liquid and vapor). In the gaseous state, the components of matter (molecules or atoms) can move around randomly and are separated from each other so that there is almost no interaction between them. This environment is very different from the liquid phase where proteins are normally found, but it is also much simpler, making easier the understanding of basic phenomena happening in a “solo” molecule like its folding. (watch Dr. Mons talking about the importance of gas phase environment)


Peptides Interrogation: The research process step by step

(press here to watch cartoon heroes TREL in new adventures:  "A peptide chain folds during the experiment by making bonds between different parts of the chain. Infra-red photons are then used to know what bonds have been created, helping the researchers to identify the shape of the folded peptide")

Proteins are too small to be observed directly in a microscope. (watch Dr. Mons talking about the key point of their experiments) Therefore, scientists try to identify their shape through indirect ways using sophisticated spectroscopic techniques. Some structural characteristics of the peptides, such as chirality (i.e. the orientation of the helical part of a protein’s segment) cannot be easily characterized however it is a crucial piece of information about its 3-dimensional shape. (watch Dr. Mons talking about chirality) The technical details of the research process at FPL are briefly described as follows:

STEP 1 – Experimental preparation
A mixture is prepared using a peptide (few tens of milligrams = 10²⁰ molecules) and graphite. When green laser light is directed on the solid mixture, the graphite absorbs the energy and causes small portion of the mixture to suddenly vaporise. Through this process, the peptide molecules pass quite softly into the gas phase where the experiments are conducted.


STEP 2 – Experimental data collection
Scientists send two kinds of light beams onto the peptide molecules: infrared (IR) and ultraviolet (UV). They are both used in a different but complementary way: IR light provokes the molecules’ vibration. In this specific experiment, a vibration of the chemical bonds that hold together nitrogen and hydrogen atoms is particularly indicative of the peptide shape. Scientists also use UV beams for detection purposes: UV light is absorbed leading to the “ionisation of the molecule”. (watch Dr. Mons talking about ionization) In the end, the combination of UV beams needed to perform the measurement, and IR beams to gather structural pieces of information point towards certain conclusions about the peptide shape.


STEP 3 – Combining theoretical and experimental data
But, how can we tell which really is the shape of the studied part of the protein molecule, when the possible shapes are millions in number? (watch Dr. Gloaguen talking about the problem of detecting the right shape) Computer simulation is needed to figure out which shapes are the most probable depending on their stability. This process demands a compromise between computational time and accuracy. Usually after a few days of approximate calculations only a thousand of shapes remain. Scientists will then start formulating hypotheses about the possible shape, on the basis of the experimental findings. Specific shapes can obviously be removed from the list of candidate shapes as they have no chances to fit these criteria. Hopefully, no more than about 100 shapes among the remaining 1000 will have such a potential. Accurate sophisticated calculations taking several weeks will be then be applied to these candidates in order to directly compare them with the experimental data. (watch Dr. Gloaguen talking about the strong connection between theoreticians and experimentalists)


STEP 4 - Results
In most cases, only one candidate shape "survives" to this last process, meaning that the scientists have indeed identified the shape of the peptide molecule. Some times, though, things do not go so smoothly and more than one probable shape matches the experimental results. (watch Dr. Gloaguen going through a long list of structures) To put it another way, a complete characterisation is not possible due to the limitations of our knowledge, or our scientific equipment, or both. But pushing these limits is the way research progresses! The results become more accurate when more experiments are carried out, better computer aided methods evolve, and more inventive theoretical concepts are proposed by the researchers.

Historical background

Proteins resist understanding for more than 50 years: Three Nobel stories…

Scientists from various disciplines such as biochemistry, molecular biology, biophysics, computing sciences etc. have been struggling to find proteins’ active shape since the 1950’s. Here, are three moments of glory:
Christian Anfinsen and his colleagues tried for the first time to understand the process of protein folding in the early 1960s. About 12 years later, in 1972, Anfinsen received the Nobel Prize in Chemistry for concluding that the amino acid sequence determines the shape of a protein. He showed that the proteins actually tie themselves: if they become unfolded, they fold back into a proper shape of their own accord.
Meanwhile, another Nobel story had come out in 1962. Researchers Max F. Perutz and John C. Kedrew were the first to successfully identify the structures of complex proteins (the so called haemoglobin and myoglobin, respectively) using X-Ray Crystallography.
A major challenge in protein research by means of X-Ray Crystallography was also faced in 1988 by Johann Deisenholer, Robert Huber and Hartmut Michel. The three researchers managed to observe proteins which are naturally buried within cell membranes, and are closely related to photosynthesis – a procedure directly linked with our nourishment and all life on Earth. For this discovery, which also provided the foundation for identifying other important proteins, these scientists won the third Nobel in Chemistry.

The"face" of Science: Let's meet the scientists!

Scientists at the Francis Perrin Laboratory (LFP), had the idea to study the 3-dimensional shape of very small parts of proteins, namely slightly modified peptides, using state of the art methods based on spectroscopy. But, who are these people working behind the scenes?

 
The "face" of science: Dr. Michel Mons, Research Director at CEA

Brief introduction: I started my scientific career by studying how molecules can fall apart once they are excited by sunlight. That’s particularly important for air pollutants like nitrogen dioxide (NO₂). Nitrogen dioxide molecules are commonly found in big cities produced in large quantities by car exhausts. The fragmentation of such molecules links with the complex chemistry of aerosols. Later on, I turned to bigger molecules, called biomolecules, having in mind to use the technical skills and other knowledge that physicists have developed so far in order to study the smaller molecules.

Research interests: I am currently interested in the interactions between light beams and molecular systems (groups of molecules). The question is how one can "interrogate" the molecules in order to learn more about their structure and consequent shape. (watch Dr. Mons talking about proteins' interrogation via spectroscopy) 

Sources of inspiration: Complex biological systems like proteins. The elementary function of each protein stems from its incredible variety and complexity at the molecular level. This complexity is, in turn, deeply rooted in the variety of the chemical interactions between the molecules themselves.

Important moments in his “scientific life”: One of the last scientific achievements of the group, namely, the observation of a common secondary structure in proteins has been isolated and characterized in a small isolated peptide. This structure, called b-hairpin, is commonly found in numerous proteins.

Research team behind the scenes: Such an ambitious research project asks for many skills, eventually gathering people with varried scientific backgrounds. The nucleus of the research group thus is composed of 3 experimentalists (Michel Mons, head of the group, François Piuzzi and Eric Gloaguen), one theoretician (Valérie Brenner) and a technician (Benjamin Tardivel). Yet, many other people have also contributed for some period of time. Among them are students, postdoc researchers, visiting professors etc. Furthermore, the group collaborates with many scientific associates from around the world including countries such as the United States, the United Kingdom, Czech Republic, Germany, Japan, Italy and Spain.

Scientific editor of the "Protein Folding" Digital Exhibit: Eric Gloaguen, CNRS assistant researcher

"I decided to join the group of Michel Mons since 2006 in order to work on the project aiming at understanding how proteins fold. Being the fourth scientific project I am involved in, it also belongs to the field of Chemical Physics. I am very interested in this scientific field because it reveals some fascinating aspects of molecules with the use of powerful physics experiments thus enabling a deep understanding of important processes at microscopic level. For example, I had the opportunity by sending light of different colours on to a molecular object to study how different chemical reactions may be induced, how fast molecules can convert light energy into motion, or how chemical reactions occurring on small particles can affect the atmosphere of the Earth!
I currently concentrate on a new stimulating interface between physics, chemistry and biology, which gives me the opportunity to collaborate with scientists from different fields on an interdisciplinary project on proteins. Beside its role in crucial biological processes including lots of human diseases (Alzheimer’s, cancer etc.), protein folding also constitutes an intriguing and science promising issue related to the molecules’ flexibility. That’s why it is really exciting to design and carry out fruitful experiments, which will provide the scientific community with useful information. Finally, I have to say I am very proud to be part of this adventure taking place in the tiny, but so important world of molecules!"

Editing team

Editing Team of the Digital Exhibit “Shed light on the exciting life of proteins!”

Scientific editors: Michel Mons (leader of this research project); Eric Gloaguen (CNRS assistant researcher)
Research technician: Benjamin Tardivel

Content Coordination: Glykeria Anyfandi
Science Communication Editors: Evlalia Amygdalaki, Glykeria Anyfandi
Content Administration: Christina Troumpetari, John Stoitsis
Technical Development: John Stoitsis
Animation and ICT material: CediS, Free University of Berlin
Photographs, videos & web material: Evlalia Amygdalaki
e-Knownet Live experiments: Interactive Science & Technology Exhibition, Eugenides Foundation