Do you know what sound can tell you about proteins?

Sound is a pretty simple concept. Stuff makes noise and you hear it with your ears. You can even be pipetting on your bench-top while listening to Rihanna if it makes you feel good and energetic. But sound waves can be used to do lots of amazing things apart from that: some have practical applications in science an even in our normal day to day life. In this post I would like to tell you some of the craziest things you didn’t know you could do with sound and how you could use it on your ongoing research.

What is sound?

Sound is made of acoustic waves, which are a type of longitudinal waves that propagate by means of adiabatic compression and decompression. (Longitudinal waves are waves that have the same direction of vibration as their direction of travel). Sound is like light in some ways: it travels out from a definite source just as light travels out from the Sun or a light bulb. But there are some very important differences between light and sound as well. We know light can travel through a vacuum because sunlight has to race through the vacuum of space to reach us on Earth. Sound, however, cannot travel through a vacuum: it always has to have something to travel through (known as a medium), such as air, water, glass or metal. The first person to discover that sound needs a medium was the brilliant English scientist Robert Boyle (1627–1691). He carried out a classic experiment: he set an alarm clock ringing, placed it inside a large glass jar and while the clock was still ringing, sucked all the air out with a pump. As the air gradually disappeared, the sound died out because there was nothing left in the jar for it to travel through.

Surface Acoustic Waves (SAW): the waves that let you use your cell phone and help you park your car

A surface acoustic wave (SAW) is a type of
 mechanical wave motion which travels
along the surface of a solid material. The 
wave was discovered in 1885 by Lord 
Rayleigh and is often named after him.
 Rayleigh showed that SAWs could explain one 
component of the seismic signal due to an earthquake, 
a phenomenon not previously understood. These days,
 these acoustic waves are often used in electronic
 devices. At first sight it seems odd to use an acoustic 
wave for an electronic application, but acoustic waves 
have some particular properties that make them pretty cool for specialized purposes. Have you ever noticed that many wrist watches have a quartz crystal used for accurate frequency generation? Well, this is an acoustic resonator though it uses bulk acoustic waves rather than surface waves. Surface waves on the other hand have lots of applications as well, such as touch screens and parking sensors.

A SAW touch screen work by using ultrasonic waves to detect touch events and the location of the user’s input.

They are made up of a glass sheet with transmitting transducers, receiving transducers and reflectors. The transmitting transducers produce ultrasonic waves that skim over the surface of the screen, get reflected and are captured by the receiving transducers. When a soft matter like human skin, touches the screen, the surface acoustic waves are absorbed and the receiving transducers do not register any input. Based on this, sensors can calculate the location of the touch event.

Already in the 1970s German inventor Rainer Buchmann developed parking sensors, which are proximity sensors for road vehicles designed to alert the driver to obstacles while parking.

The sensors emit acoustic pulses, with a control unit measuring the return interval of each reflected signal and calculating object distances. If you happen to have one of this devices as I do (thanks to my husband who ‘strongly’ recommended it) you would know that the system in turns warns the driver with acoustic tones, the frequency indicating object distance, with faster tones indicating closer proximity and a continuous tone indicating a minimal pre-defined distance.

SAW reaches biochemistry: acoustic waves moving towards biosensors

SAW also have applications in biochemistry, for example in biosensors. A biosensor is an analytical device containing an immobilized biological sensitive material (enzyme, antibody, antigen, organelles, DNA, cells, tissues or organic molecules) in contact with or integrated within a transducer, which converts a biological signal into a quantitatively measurable electrical signal.

SAW-based sensors are systems in which high frequency acoustic waves travel close to the surface of a piezoelectric substrate.

On top of that substrate two transducers are placed, one at each side, whose functions are necessary for generating and receiving acoustic waves (called transmitter and receiver, respectively). The transmitter converts an alternating electrical signal into an acoustic wave which travels along the surface of the piezoelectric substrate and the receiver converts the acoustic wave back to an electrical signal for detection and analysis. The area between the generator and receiver is often coated with a chemically sensitive surface for molecular absorption or adsorption, which is in other words where the ‘sample’ goes.

Upon interaction with molecules on the sensor surface, distinct characteristics of the travelling acoustic waves are altered:

·     Changes in total mass on the biosensor result in a shift of the wave's phase. This phase shift provides information about the on- and off-rates, as well as the stoichiometry of molecular interactions.

·     A change in flexibility of the molecules alters the wave's amplitude. This directly reflects changes in the conformation of the molecules, e.g. after binding to compounds.

Both signal types are detected and quantified separately and can be used to comprehensively characterize the interaction mechanism of the molecules on a kinetic and structural level.

Assessment of protein-small-molecule interactions by acoustic waves

A beautiful example on how SAW can be used to analyze interactions of a protein and its substrate is that of MBP (1). The E. coli maltose binding protein (MBP) is a monomeric  periplasmic protein, that is often used as a fusion protein to either mediate solubility of its fusion partners or as an affinity-tag for amylose affinity purification. In its cellular function, MBP is the corresponding receptor molecule to the bacterial maltose ABC transporter system. Upon traversing the bacterial outer membrane, by means of a specified channel protein, maltose is efficiently captured by MBP and delivered to the inner-membrane ABC transporter system for nutrient uptake. Interestingly, maltose binding to MBP results in a marked conformational change: in a clamp like motion, the MBP N- and C-domain close around the associated maltose molecule.

Using SAW technology, it was able to resolve the structural transition occurring in MBP upon maltose binding. Accurate dissociation constants were determined not only by analysis of the mass-binding induced phase-signal, but also by quantification of the conformational-change induced amplitude response. So far, highly sensitive fluorescence detection was required to quantify conformational changes on such a small scale. Now, Surface Acoustic Wave sensors allow monitoring of intramolecular conformational transitions with impressive signal to noise ratios for immobilized molecules. Therefore, SAW presents an ideal tool to investigate mechanisms of protein small-molecule interactions by separating mass binding and resulting conformational changes.

Conformational changes at protein-protein interaction followed by SAW biosensor

Simple binding parameters, especially stand-alone Kd values have repeatedly been shown to characterize protein interactions, but additional parameters, such as kinetics and the conformational effects on a target protein due to ligand binding are being investigated particularly in drug discovery applications.

In general, binding of proteinaceous biomolecules onto the sensor surface results in both an increase in phase and a decrease in amplitude. The increase in phase is attributed to the mass loading, while the decrease in amplitude can be assigned to the additional viscosity of the bound molecules, as they are not perfectly rigid.

A system previously used for selective measurement of conformational changes is the signal transduction pathway involving the small GTP-binding protein Ras and its effector molecules (2). The use of SAW, besides of retrieving information regarding to binding kinetics, retrieved an interesting insight of conformational changes of their interplayer proteins. For example, the recorded amplitude signal, used as a measure of the conformational changes, showed how salt influenced on protein rigidity and hence on the strength of binding.

SAW chip-based biosensors for detecting cancer cells.

Although the title of this post goes in the protein direction, it is worth noting that SAW has extended applications to any molecule, cells and even viral particles. A good example is its use on the detection of cancer cells.

In a beautiful study, a nanostructured chip surface was fabricated enabling binding via spaced antibodies specifically targeting surface proteins of cancer cells and detection of extremely low numbers of circulating tumor cells (CTC) (3). Human cancer cell lines JEG-3 (lymphoblastic leukemia) and MOLT-17 (placental choriocarcinoma) from cell cultures were successfully detected using a SAW biosensor which showed significant responses on less than 10 cells injected in a single run. Thus SAW biosensors are highly promising tool for medical diagnostics as well.

My final words… ballerinas and conformational changes

If you think about it, proteins can be thought as ballerinas in the way that they fulfill their functions by changing their shapes (conformations) flexibly. This nature’s ‘ballerinas’ undergo conformational changes as part of their interactions with other proteins or drug molecules or in response to changes in their environment. As our appreciation of proteins as dynamic, flexible molecules grows, so does the need for tools to probe the conformational landscape in real time and in physiological conditions. SAW biosensors appear as an excellent and highy sensitive tool for analyzing conformational changes.

As you can see, sound goes beyond music and it has tremendous applications in science. Let’s open our minds and let us be amazed by new technologies that can offer an inner look into our favourite molecules.

1.http://www.nanotempertechnologies.com/uploads/media/Application_Note_NT-SE-001_Conformational_Changes_MBP_01.pdf

2. Klumpers, Ulrich Götz, Tanja Kurtz, Christian Herrmann, Thomas M.A. Gronewold, Conformational changes at protein–protein interaction followed online with an SAW biosensor, Sensors and Actuators B: Chemical, Volume 203, November 2014, Pages 904-908.

3. Patrick Bröker, Klaus Lücke, Markus Perpeet, Thomas M.A. Gronewold, A nanostructured SAW chip-based biosensor detecting cancer cells, Sensors and Actuators B: Chemical, Volume 165, Issue 1, April 2012, Pages 1-6.