Proteins Explained

Proteins, also known as polypeptides, are organic compounds made up of amino acids. They’re large, complex molecules that play many critical roles in the body.

Proteins are made up of hundreds of thousands of smaller units that are arranged in a linear chain and folded into a globular form. There are 20 different types of amino acids that can be combined to make a protein and the sequence of amino acids determines each protein’s unique 3-dimensional structure and its specific function.

Proteins do most of the work in cells and are required for the structure, function, and regulation of the body’s tissues and organs. Essential parts of organisms, they participate in virtually every process within cells. Many proteins are enzymes that catalyse biochemical reactions and are vital to metabolism. The size of a protein is an important physical characteristic and scientists often use particle size analysers in their studies to discuss protein size or molecular weight.

THE STRUCTURE OF PROTEINS

To be able to perform their biological function, proteins fold into one or more specific spatial conformations driven by a number of non-covalent interactions such as hydrogen bonding, ionic interactions, Van der Waals forces, and hydrophobic packing. This understanding is the topic of the scientific field of structural biology, which employs traditional techniques such as X-ray crystallography, NMR spectroscopy, and Circular Dichroism spectrometry to determine the structure of proteins.

Most proteins fold into unique three-dimensional structures. The shape that a protein folds into naturally is known as its native conformation. While most proteins can fold unassisted through the chemical properties of their amino acids, others require the aid of molecular chaperones. There are four distinct aspects of a protein’s structure:

• Primary structure: The amino acid sequence.
• Secondary structure: Regularly repeating local structures stabilised by hydrogen bonds.
• Tertiary structure: The overall shape of a single protein molecule; the spatial relationship of the secondary structures to one another.
• Quaternary structure: The structure formed by several protein molecules which function as a single protein complex.

Protein structures range in size from tens to several thousand amino acids. By physical size, proteins are classified as nanoparticles, between 1 – 100nm. Very large aggregates can be formed from protein subunits. For example, many thousand actin molecules assemble into a microfilament.

TRADITIONAL PROTEIN ANALYSIS TECHNIQUES

Proteins differ from each other according to the type, number and sequence of amino acids that make up the polypeptide backbone. Hence, they have different molecular structures, nutritional attributes and physicochemical properties.

There are three major protein analysis techniques: protein separation, western blotting and protein identification.

1. PROTEIN SEPARATION

Protein electrophoresis is the process of separating or purifying proteins by placing them in a gel matrix and then observing protein mobility in the presence of an electrical field. It’s an important approach to studying protein function and the effect of a particular protein on development or a physical function by introducing it into an organism.

The most commonly used technique for protein separation is sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins can be separated according to solubility, size, charge and binding affinity. SDS-PAGE separates proteins mainly on the basis of molecular weight as opposed to charge or folding. It’s a technique that’s widely used in biochemistry, forensics, genetics and molecular biology.

Other methods include:

• Isoelectric Focussing: In this method, different molecules are separated by their electric charge differences. This technique is a type of zone electrophoresis that is usually performed in a gel and takes advantage of the fact that a molecule’s charge changes with the pH of its surroundings.
• Chromatic Methods: There are two chromatic methods frequently used for protein separation – high-performance liquid chromatography and thin-layer chromatography. Both these methods are particularly useful adjuncts to gel-based approaches. Although chromatography is a common technique in biochemistry laboratories used for purification, identification and quantification of protein mixtures, laser diffraction is traditionally used for pre-column size and polydispersity management.
• Two-dimensional Gel Electrophoresis: This is a powerful gel-based method commonly used to analyse complex samples in the interest of characterising the full range of proteins in the sample, not just a few specific proteins.

2. WESTERN BLOTTING

The western blot technique uses three elements to identify specific proteins from a complex mixture of proteins extracted from cells: separation by size, transfer to a solid support, and marking target protein using a proper primary and secondary antibody to visualise.

The most common version of this method is immunoblotting. This technique is used to detect specific proteins in a given sample of tissue homogenate or extract. The sample of proteins is first electrophoresed by SDS-PAGE to separate the proteins based on molecular weight. The proteins are then transferred to a membrane where they are probed using antibodies specific to the target protein.

3. PROTEIN IDENTIFICATION

There are two methods that are commonly used to identify proteins: Edman Degradation and Mass Spectrometry.

Developed by Pehr Edman, Edman Degradation is a method of sequencing amino acids in a peptide. Here, the amino-terminal residue is labeled and cleaved from the peptide without disrupting the peptide bonds between other amino acid residues.

Protein Mass Spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles for determining masses of particles and the elemental composition of a sample of molecules as well as for elucidating the chemical structure of molecules such as peptides. Protein mass spectrometry is an important method for the accurate mass determination and characterisation of proteins, and a variety of methods and instrumentations have been developed for its many uses. The application of mass spectrometry to study proteins became popularised in the 1980s after the development of MALDI and ESI. These ionisation techniques have played a significant role in the identification of proteins. Identification can be made via:

• Peptide mass fingerprinting uses the masses of proteolytic peptides as input to a search of a database of predicted masses that arise from digestion of a list of known proteins. The main advantage of this method is that it doesn’t depend on protein sequencing for protein identification. The limitation of this method is that it requires the database to have the protein which is already characterised on another organism.
• De novo peptide sequencingis performed without prior knowledge of the amino acid sequence. This method can obtain the peptide sequences without a protein database and uses computational approaches to deduce the sequence of peptide directly from the experimental MS/MS spectra. It can be used for unsequenced organisms, antibodies, peptides with posttranslational modifications (PTMs) and endogenous peptides.

MODERN PROTEIN ANALYSIS TECHNIQUES

Protein complex analysis involves extensive interpretation of the structure and function of proteins, which are present in complex biological samples. Though recent protein complex analysis methods are efficient in identifying the structure of protein complex, there are some limiting factors.

The ever-increasing number of alternative ways to detect protein-protein interactions (PPIs) speaks volumes about the creativity of scientists in hunting for the optimal technique. Modern techniques are continually allowing us to study protein more effectively, efficiently and at reduced costs and include:

1. LIGHT SCATTERING

Light scattering techniques are particularly sensitive to larger molecules in preparations of smaller molecules. Any increase in the size of a protein will most likely be the result of aggregate formation. The sensitivity of the light scattering measurement to larger proteins means that the earliest stages of denaturation, leading to the formation of a few aggregates, will result in changes in the mean hydrodynamic size.

Batch Dynamic Light Scattering (DLS): Size measurement is the primary measurement of proteins that can be performed with batch-mode DLS. Since proteins have a very consistent composition and fold into tight structures, the hydrodynamic size relates predictably with molecular weight. The activity and function of a protein is closely related to correct folding and structure. As such, activity is also directly related to the size of the protein, meaning size can also be used as a predictor of activity. DLS the most sensitive technique for detecting small quantities of aggregates in preparations. Zetasizer software has a model to predict the molecular weight of a protein from its hydrodynamic size by DLS. Request a demo.

Static Light Scattering (SLS): Following on from DLS measurements, SLS measurements can also be made of proteins. Often highly purified, many protein samples should be applicable for batch measurements of molecular weight using SLS, as long as the concentrations are accurately known. By measuring the amount of light scattered at different concentrations of sample, the molecular weight, which is proportional to the amount of light scattered, can be calculated by creating a Debye plot. The slope of the line in the Debye plot is 2x the 2nd virial coefficient (a measure of molecular interaction within a solution) so this technique can also be useful for studying crystallisation conditions. A strongly positive value indicates good solubility while a strongly negative value indicates a propensity to aggregate. Request a demo.

Charge and Zeta Potential: Using a suitably sensitive instruments such as the Zetasizer Ultra, and an appropriate method such as the patented diffusion barrier technique, Zeta-potential measurements of proteins are also possible. A significant number of the functional groups on amino acids can be charged and any combination of these may be in their charged or uncharged states in the protein. This will change depending on the conditions in the local environment and it is important to note that zeta-potential can be different from the calculated net charge based on the likely state of the charged residues in the molecule.

Charge is of particular interest to protein chemists and Zeta potential should be able to compete with iso-electric focusing, currently one of the primary methods for determining protein charge, as it allows the protein to be kept in conditions far nearer to its native state. It should be noted, however, that proteins are subject to being denatured by the applied electric field, which can make zeta-potential measurements difficult. The diffusion barrier technique is a method that’s used to reliably measure the electrophoretic mobility of proteins by reducing the impact of the measurement process. For more information, contact us.

Overall, Zeta potential is a measure of the strength of the repulsive forces between molecules in solution. Conventionally, this has been used as a primary indicator of the stability of a sample preparation. With high Zeta potential, and consequently, high intermolecular repulsive force, a drug or protein preparation can be expected to be stable for longer periods than a similar preparation with low Zeta potential. Request a demo.

2. MULTI-DETECTION GPC/SEC

While DLS can be used to characterise the oligomeric state of a protein, it is unable to resolve a mixture of oligomers. Adding SEC capabilities to a light scattering detector is a way to greatly improve its resolution.

The Malvern OMNISEC Resolve and Reveal system separates molecules based on their size making it an excellent partner for light scattering. By separating the molecules before measuring them, using DLS or SLS, this technique can be used to identify the different components in a mixture. At known concentrations, measured with a refractive index or a UV detector, molecular weight can be related directly to the amount of light scattered by a molecule. This can be combined with data from a viscometer, which measures viscosity allowing size and some structural aspects to be determined. Thus, a large amount of information can be obtained for a single protein sample using this method.

The Malvern OMNISEC also adds another dimension to the detection of aggregates. By separating them from the primary sample, it’s possible to further characterise and quantify them. Manufacturers of protein solutions routinely use SEC as the final step in purification. SEC is used to separate samples in order to remove any aggregates formed in the sample preparation. The same is true when purifying a single protein from biological samples. Request a demo.

3. Circular Dichroism Spectrometry

Circular Dichroism (CD) is an absorption spectroscopy method based on the differential absorption of left and right circularly polarized light. Optically active chiral molecules will preferentially absorb one direction of the circularly polarized light. The difference in absorption of the left and right circularly polarized light can be measured and quantified. UV CD is used to determine aspects of protein secondary structure. Vibrational CD, IR CD, is used to study the structure of small organic molecules, proteins and DNA. UV/Vis CD investigates charge transfer transitions in metal-protein complexes.

JASCO J1000 series CD spectrometers provides maximum signal-to-noise under high absorbing, low light intensity conditions of the far-UV spectral region to explore the structure and stability of biomolecules. It provides unparalleled optical performance and versatile flexibility for advanced biomolecular characterisation and stereochemical analysis. The Dual Polarizing Prism Monochromator covers the entire region required for routine analysis of biomolecules with excellent stray-light rejection for accurate results. Enhanced vacuum UV measurement enables the measurement of a CD spectrum in the vacuum UV region can go down to 163nm which is of critical importance for biomolecules. Request a demo

4. Isothermal Titration Calorimetry

Isothermal titration calorimetry (ITC) is a physical technique used to determine the thermodynamic parameters of interactions in solution. It is most often used to study the binding of small molecules (such as medicinal compounds) to larger macromolecules (proteins, DNA etc.). It consists of two cells which are enclosed in an adiabatic jacket. The compounds to be studied are placed in the sample cell, while the other cell, the reference cell, is used as a control and contains the buffer in which the sample is dissolved.

The MicroCal PEAQ-ITC offers the highest sensitivity for label free measurements of binding affinity and thermodynamics with low sample consumption for the study of biomolecular interactions. It delivers direct measurement of all binding parameters in a single experiment and can analyze weak to high affinity binders, using as little as 10µg sample. Semi-automated maintenance minimizes operator intervention and the system is upgradable to the fully automated MicroCal PEAQ-ITC Automated, making it ideal for laboratories where speed, sensitivity and the ability to accommodate higher workloads in the future are paramount. Request a demo

PROTEIN ANALYSIS MADE EASY

Crystallisation of proteins is a necessary step for elucidating their detailed 3-dimensional structure. Crystallisation is a difficult process that requires a highly purified protein kept in ideal conditions. In DLS measurements, polydispersity is a measure of the purity of a sample. A protein sample with a very low polydispersity indicates that it is highly purified, that all the protein is in one particular oligomeric conformation and that its structure is very well controlled under these conditions, all of which are required for crystallisation. By identifying a protein sample with the lowest polydispersity, a researcher can find the most suitable conditions for crystallisation.

Size can also be used as a predictor of activity and quaternary structure of the protein can also be studied. When proteins oligomerise, their size and molecular weight will increase in discrete increments corresponding to the addition of separate proteins. By measuring the protein under different conditions the oligomeric state of the protein can be assessed. Many proteins rely on correct quaternary structure in order to function, so again, hydrodynamic size can be used as a predictor of activity.

In adverse conditions such as extremes of temperature and pH, a protein will become denatured. By controlling these conditions, and measuring the hydrodynamic radius, the melting point of the protein can be established. This is related to the stability of the protein and can be used as a predictor of shelf life.

Need the right tools for measurement? ATA Scientific offers a comprehensive toolbox for analysing proteins in a number of ways, including Multi detection GPC/SEC, Circular Dichroism (CD), Microcalorimetry (ITC/ DSC), Dynamic and Static light scattering (DLS/ SLS) and more. Contact us today for a free consultation and make protein analysis easy. We provide the instruments and ongoing support so that you can be confident in your results.