How do I smell? Much the same as you see

As the International Year of Crystallography approaches its end, Dr David von Stetten explains how unique protein analysis has helped us understand the common mechanisms in our senses Every day in Grenoble, France, scientists arrive from all over the world with bags of mysterious, meticulously prepared frozen crystals and a similar aim – to understand how the building blocks of life carry out the functions that keep us and every animal, plant, fungus and feline going day after day. Approximately 10% of the world’s investigations into protein structure take place at the European Synchrotron Radiation Facility (ESRF). Earlier this year results were published from a study led by researchers from the Institute for Medical Physics and Biophysics (IMPB) at the Charité in Berlin who used the ESRF's X-ray techniques to uncover a new common feature in the molecular processes that are responsible for our senses such as taste, smell, and vision. The team discovered that what seem like structurally and functionally different proteins, important in sight and smell, in fact share a common component. This result, published in the journal Nature Communications, provides another clue in the search to understand how our senses work. The study is just the latest in a series of important protein analyses carried out at the ESRF, the largest synchrotron X-ray source in Europe. Its size, coupled with its in house expertise, its investment in world leading technology and the speed at which it can carry out these studies (anywhere between 10s and 100s of protein crystals can be analysed in a day) has made the ESRF the place to go for this type of protein structural analysis. Named X-ray crystallography, the technique is in its centennial year, a landmark celebrated around the world through the International Year of Crystallography (www.iycr2014.org). With the growing research interest in the role and function of protein molecules, the ESRF has been quick to offer the budding structural biology community a range of tools with which to carry out their analysis. ID29, where the latest study on our sense proteins was carried out, was built and optimised exclusively for protein crystallography, but is actually only one of five similar specially developed instruments at the ESRF, with four more under construction. How proteins carry out their function is governed by their structure – what they look like. To investigate structure, ID29 has been fitted with the latest in microdiffractometer technology, allowing users to tailor the beam sizes down anywhere between 75 and 10 microns in diameter, depending on the size of their protein crystals. However the first step in this process starts well away from the synchrotron ring. Users of the ESRF generally begin by growing their protein crystals in their own labs. This is not a trivial task. Proteins are living matter and don't naturally crystallise in this way. To achieve protein crystallisation, the user is required to take a purified protein and essentially dry it out in a slow and controlled manner. If you can stop the sample from completely drying out, it will crystallise, meaning that all of the protein molecules arrange themselves in a regular, ordered manner.

What seem like structurally and functionally different proteins, important in sight and smell, in fact share a common component

The crystals that are produced – which are typically anywhere between 10-100 micrometres in size – are then brought to Grenoble and placed on the ESRF beamline. When the crystal, with its regular structure, is hit by the X-ray beam, the incoming x-rays are scattered, creating a regular pattern of spots, arranged in overlaying circles, which is then recorded on an almost square metre sized X-ray camera, up to 100 times per second. These so-called diffraction images need to be collected at a range of angles, and orientations – this is done by accurately rotating the sample in the beamline. The combination of all of these images is then interpreted via a complicated mathematical algorithm (basically a Fourier transform) which calculates the positions of all the atoms in 3D space. Getting an accurate image requires a very precise approach – you need to know the speed of the sample’s rotation and must avoid moving it outside the beam. ID29 uses an instrument called a goniometer to achieve this. To ensure the best possible results from the experiments run on ID29 the instrument is housed right next to a unique spectroscopic lab called the Cryobench. Completely unrelated to X-rays, the lab's instruments record the visible spectra of protein crystals, i.e. their colour. Fundamentally a protein's colour describes its absorption properties – and these have been shown to vary in a very subtle way, which depends on the protein's state – (e.g. active or inactive) and can be temperature-dependent. In fact, some specific proteins can be identified by their colour, but these differences are too subtle to see through a standard microscope. This is where the Cryobench plays its part, ensuring that the protein hasn't changed during crystallisation. This final check played a vital role in the recent study into the proteins responsible for our senses, as we will see a bit later. In mammals, the process of ‘sensing’ something is defined by a complex interplay between stimulus, proteins and receptors. For example, in order to see something, the protein rhodopsin must be present – it is responsible for the initial light detection in vision. Rhodopsin belongs to a large protein family called G-protein coupled receptors (GPCRs) which are found in the membranes around every living cell. Their role is to sense, for example the presence of molecules outside the membrane, or light, and to amplify and pass on the signal. When light shines on a rhodopsin molecule, the molecule changes shape, creating a new signal pathway. Vision is effectively regulated by the switching on and off of these signal pathways which eventually results in an image being produced in the brain. In this process, switching off the signal is an essential step, which happens when the protein arrestin binds to (previously activated) rhodopsin. Interestingly, the analysis by the team at the Charité in Berlin, performed on 500 crystals at 100 K (-173 °C) using synchrotron X-ray sources at both the ESRF and BESSY II facility in Berlin, showed that several different variants of arrestins share a common sequence motif which binds to the GPCR. This suggests very similar interactions between GPCRs and the interaction partners involved in different senses. Using the instrumentation on ID29, the Berlin team demonstrated clearly that both the G protein and arrestin contain a structurally similar component with a homologous protein sequence, which binds and recognises the receptor in a similar way. The results were confirmed using the complementary Cryobench spectroscopic lab which was able to identify and distinguish between the protein involved in our sense of sight as it passed through the several different steps involved in the vision process, each of which subtly changes its colour. The Cryobench analysis gave the team complete confidence that each crystal was in the correct state prior to X-ray analysis. The findings from the study provide a new, detailed insight into how our sense organs actually ‘sense’. Building on years of work, it furthers our understanding of the mechanisms by which GPCR receptor proteins interact with their partner proteins in the signal transduction chain. It’s well known that GPCRs play a vital role in physiological processes and in the development of diseases in the body. With at least one third of all currently available commercial medications directly acting on GPCRs, this work may have longer-term implications for the detailed understanding of protein receptors. However the applications of the x-ray techniques at ESRF are not restricted to human health, or even to human proteins. In recent years instruments like ID29 has investigated more exotic molecules such as the cyan fluorescent protein originally found in some jellyfish as green fluorescent protein, which has since proved its value in medical research by making certain parts of an organism fluorescent, allowing investigation of living creatures in a more natural way.  At the ESRF, the team was able to reveal how the protein creates its fluorescence, an understanding which allowed the team to propose a more optimally fluorescent design. In the plant kingdom, teams have used X-rays at the ESRF to investigate phytochrome, the light-sensitive protein that tells the plant when to be green in the presence of sunlight, as well as controlling flowering and seed germination. Their analysis revealed vital structural information about the sequence of changes that switch them into their active state. Even this variety of studies barely scratches the surface of the full scope of research around protein crystallography. Everything you see in the living world is related to proteins in some way or another, and there are countless samples just waiting to be investigated. Fortunately for the structural biology community, X-ray sources like the ESRF are in a better position than ever to image and analyse, in unprecedented detail, and on ever-shorter timescales, these crucial building blocks of life. Author Dr David Von Stetten is a physicist with a background in spectroscopy who is now a beamline scientist at the European Synchrotron Radiation Facility. After studying physics in Dresden, Alaska, and Berlin, he then completed his PhD in Berlin, doing Raman spectroscopy on phytochrome. He then came to the Cryobench at the ESRF as a postdoc, and after a beamline scientist