Proteins are determinants of cell, tissue and organ structure and physiology. Proteins are a class of macromolecules occurring in biosystems in wide abundance and diversity. Finally, researchers harness the power of proteins to produce metabolites and other products of industrial importance.
The basic experimental approach to how proteins are studied in the lab is:
1. extracting the protein from its biological source.
2. separating or purifying target proteins from other proteins based on its physio-chemical properties.
3. characterizing the protein.
Using our basic understanding of protein structure, in this article we will try to understand why proteins are so important in biological systems. Additionally, we will look at the basic approaches scientists take to study proteins.
In this article:
Proteins play essential roles in cell structure and physiology
Proteins: major components of cell, tissue and organ structure
Proteins control all aspects of physiology
How abundant and diverse are proteins in biological system?
Basic approaches for protein experimentation
Techniques of protein separation
Proteins play essential roles in cell structure and physiology
In living cells and tissues proteins serve two essential purposes:
- proteins are a major structural component for cells, tissues and organs.
- proteins control every part of cell physiology.
Because of this, from a basic science standpoint, researchers use proteins as probes to study these events.
Let’s take a deeper look into each important area proteins are involved in.
Proteins: major components of cell, tissue and organ structure
Proteins are one of the most important basic building blocks of all biological entities starting from the cell.
To illustrate how proteins are involved in structure, let’s think about the cell membrane and its composition.
According to the widely accepted fluid mosaic model of cell membrane composition, proposed by Singer and Nicholson, a cell membrane can be considered protein icebergs in a sea of lipids.
The proteins (icebergs) are floating in a bilayer (sea) of lipids. Since the proteins are floating in the lipid-sea, as opposed to permanently being anchored at one specific location, the cell membrane is fluid-an important structural paradigm of cells.
So, as you can understand, proteins are indeed very important and highly abundant constituents of cells.
Moving from the membrane to within the cell (intracellular), here is an example of how proteins are involved in a structural framework. A type of protein, histone, intricately binds with another biological macromolecule – DNA, to form the chromatin network which make up chromosomes.
Now, moving on to a tissue or whole organismal level, an example of a very abundant protein that supports tissue structural framework is collagen.
Another suitable example is the extracellular matrix in animals. This region is a three-dimensional network of proteins and a type of carbohydrate known as glycosaminoglycans.
So, as we see from the level of a single cell to that of tissues and organs, proteins are important and abundant structural constituents.
Also, in their functions as structural components, proteins are often intricately involved with other biological macromolecules such as carbohydrates and lipids. The cell membrane itself is an example of this.
Proteins control all aspects of physiology
Proteins actively participate and regulate all aspects of physiology because they are metabolically active.
Other than proteins, all other biomolecules (carbohydrates, lipids, nucleic acids) are metabolically inert.
In other words, these biomolecules cannot participate in physiological processes without the involvement of proteins.
Minor exceptions to this rule are some classes of non-coding RNAs such as ribozymes, and some types of hormones such as testosterone and cortisol. They can regulate physiological processes like proteins.
The metabolic potential of proteins, in contrast to other biological macromolecules such as carbohydrates, DNA or lipids, lies in their tertiary and quaternary structures.
As discussed in this article, proteins can fold into intricately defined structures. This gives them the specificity to bind to particular molecules – substrates for enzymes or receptors for cell-signaling proteins or antigens for immunoglobulins for example – which drive various physiological processes.
Figure 1. Illustrates the concepts of enzyme-substrate, antigen-immunoglobulin and receptor-ligand. The specific architecture of enzymes, immunoglobulins and receptors enable high binding specificity.
It is true that the ultimate blueprint of an organism’s physiology is in its genetic material, DNA or RNA.
However, this genetic information is physiologically meaningless unless it is translated to proteins that perform or facilitate the metabolic job.
In any and every domain of bioscience research – disease biology, microbial physiology, cell and molecular biology– whenever the bigger molecular picture needs to be understood, studying proteins is absolutely necessary.
We’ve highlighted some of the important functions proteins play in Table 1 below.
Table 1. Important types of functional proteins
|
Functional Type |
Examples |
|
Enzymes |
Trypsin, Rubisco, DNA polymerases |
|
Immunologic molecules |
antibodies, complement proteins, interferons |
|
Cell signaling |
G-protein coupled receptors, ligand gated ion channels |
|
Transcription factors |
STAT proteins, TATA-binding protein |
|
Hormones |
Insulin, Oxytocin |
|
Chaperones |
Calnexin, Calreticulin |
|
Intracellular trafficking |
Caveolin, Clathrin |
|
Tissue-wide transport |
Hemoglobin |
|
Toxins |
Tetanus toxin, Botulinum toxin |
|
Blood clotting |
Fibrinogen, tissue factor (F3) |
|
Stress-related |
Dehydrin, antifreeze proteins |
How abundant and diverse are proteins in biological system?
Proteins are one of the most abundant molecules scientists encounter in a biological system. More than half of the dry weight of an average cell is proteins. The number of both unique types of proteins as well as that of the total protein molecules in a cell are much more lipids and carbohydrates.
To put things in perspective with respect to protein abundance and diversity, we’ve highlighted 6 really interesting facts:
- The percentage of proteins in dry weight of cells often exceeds 50%.
- The human genome is estimated to encode more than 17,000 polypeptide-coding mRNAs.
- After alternative splicing, the actual number of proteins in humans is predicted to exceed 90,000. This signifies more diversity in the proteome, as compared to the genome. Post translational modifications to the polypeptide, further diversifies the protein pool in a cell.
- In terms of abundance, human cells on an average may contain 1-3 billion protein molecules.
- Even in less complicated single cell organisms such as bacteria, the number of unique protein molecules can be as high as 10,000. The model bacterium Escherichia coli is estimated to have 3-4 million protein molecules in its cell.
- All cells in an organism have identical genomes. However, abundance of a specific protein in the cells of that organism is highly variable. It depends on the cell type, and external and internal conditions prevailing around and inside the cell.
- These make studying proteins important, interesting and complicated all at the same time.
Basic approaches for protein experimentation
The basic experimental approach to how proteins are studied in the lab is as follows:
1. extracting the protein from its biological source.
2. separating or purifying it from other proteins based on its physio-chemical properties.
3. characterizing the protein.
Keeping in mind why scientists study proteins so heavily, let us take a glance at the generic approaches taken when experimenting with proteins.
A schematic outline of the experimental route taken while studying proteins is laid out in Figure 2.
Figure 2. Experimental route taken while studying proteins.
The source of the protein of interest may be a bacterial culture or plants or animal experimental models such as mouse, harvested organs, or from clinical samples or cell culture.
The first step in studying a protein is extracting it from its source.
If the protein of interest is an extracellular one, such as a secreted bacterial toxin, the extraction process may be as simple as pelleting the cells in the culture to obtain the supernatant, where the protein of interest lies in solution.
However, in many cases the extraction is more complicated than this. The protein of interest may be an intracellular protein.
For example, you might be studying a protein that binds to the genome and regulates gene expression.
In these cases, the cellular tissue needs to be lysed to get the protein of interest out.
For cell lysis, a variety of methods can be applied ranging from vigorously stirring with beads using a vortex, mechanical force (mortar and pestle for example), sonication, pressure fluctuations (as in a French pressure press), using enzymes such as lysozyme, etc.
As an additional level of complexity, the protein of interest may also be a structural component of a cell organelle. Membrane proteins are characteristic examples.
In such cases, the protein of interest is insoluble; that is, it does not float around in solution within the cell cytoplasm.
The general approach to extract insoluble proteins is to centrifuge the cell lysate at a certain speed so that the organelle is pelleted from the cell cytosol.
For example, for extracting bacterial cell membrane proteins, the cell lysate is ultracentrifuged at a speed of 27000xg for 30-60mins as illustrated in figure 3 below. This separates the membrane fraction (with the protein of interest in it) as a pellet from the cytoplasmic proteins which remain in the supernatant. This technique is called differential centrifugation.
Figure 3. Illustration showing differential centrifugation
An alternative approach for separating cell fractions and the protein components within, is to use a density gradient. This method is known as density gradient centrifugation.
Please follow figure 4 along with this text for putting things in perspective.
The first few steps are simple.
The whole cell lysate is centrifuged at a relatively low speed (4000xg). This pellets the cell membrane fraction; the desired endoplasmic reticulum (ER) fraction remains in solution as the supernatant.
The next step is a higher speed centrifugation at 15000xg to pellet the cell fraction that contains the ER.
To put things in perspective, both these centrifugations are differential centrifugations.
Consider a eukaryotic cell. Organelles such as endoplasmic reticulum (ER) have a characteristic density. If the cell lysate is put in a medium of differential densities and centrifuged, the cellular subfractions and their constituent proteins will separate based on their densities and will settle at respective regions of the medium matching their corresponding densities.
At this point, after centrifugation, that specific layer of the medium is extracted which matches the density of the ER. This layer contains the ER because of matching densities.
In this way, the ER can be separated out of the remainder of the cell constituents as illustrated in figure 4 below.
Figure 4. How density gradient centrifugation is used to extract the Endoplasmic Reticulum (ER) fraction of cells.
Now this pellet that contains the ER is resuspended in a solution of 1.25M sucrose.
At this point, we’ll talk about a density gradient column, as the one shown in figure 4.
An analogy to explain the density gradient tube may be a layered cocktail drink – one that has multiple different drinks that are layered one above the other in the same glass, but they do not get mixed with each other.
The different layers do not get mixed because of their differing densities.
This is the concept with a density gradient tube. The layers, in this case, are of sucrose solutions of varying concentrations (0.3M, 1.15M, 1.25M, 1.35M and 2M), that give them different densities.
What this means is that the tube is filled with sucrose solutions of varying concentrations (0.3M-2M), and consequently, of varying densities.
This has been depicted in figure 4: if you see, the lower sucrose concentrations are depicted in lighter shades of purple, and as the concentrations (and consequently, the densities) increase, the purple shade is made bolder.
The lowest concentration part (0.3M sucrose) will have the lowest density and will settle at the top most part of the column. This is followed by 1.15M, then the 1.25M solution, and so on; till the 2M solution, which is of the highest density. And so, it settles right at the bottom of the tube.
This is what the sucrose density gradient tube represents.
Now coming to the part of how this tube is used in separating out the ER fraction.
The pellet containing the ER is resuspended in a solution of 1.25M sucrose. So, this resuspension solution (containing the pellet that has the ER) will have a density that matches with the 1.25M sucrose part of the tube. Because of this, when this re-suspension solution is put in the gradient tube, it will layer at the level of the 1.25M part - due to matching densities.
Now after this, when the column is centrifuged at a really high speed (180,000xg), the constituents of that resuspension solution (that had the pellet) get separated into 3 layers (top, middle and bottom; as mentioned in the figure) based on their densities.
The experimenter knows which layer should have the ER, because density of ER of that cell type can be easily known from published literature. He/ she picks that layer from the tube, and gets the ER fraction.If that layer of the medium is extracted which matches the density of the ER, the ER can be separated out of the remainder of the cell constituents.
Once the desired subcellular fraction, such as the ER or cell membrane is obtained and it contains our protein of interest, it is then solubilized, often with detergents, to bring the protein of interest into solution for further experimentation. This completes the extraction procedure.
When studying a protein of interest in a living system such as a leaf, the next step after extracting the protein from its source is to separate it from other proteins.
Techniques of protein separation
Let us take a quick conceptual look at common protein separation methods.
As described in this article, every protein has a specific:
- molecular weight, depending on its amino acid sequence.
- size and shape owing to its tertiary structure.
- overall charge at a certain pH, owing to the charges of its constituent amino acids.
- hydrophilicity (polar nature) or hydrophobicity (non-polar nature) depending on its amino acid side chains.
Any one or multiple of these factors may be used to separate
proteins. The different methods of protein separation commonly employed in
research, along with the underlying separation principle, are listed in Table
2.
Table 2. Techniques of protein separation and the underlying biophysical principle
|
Native gel electrophoresis |
Charge and molecular weight |
|
SDS PAGE |
Molecular weight |
|
Isoelectric focussing |
Charge |
|
Chromatography
|
1. Size and molecular weight 2. Charge and electrostatic interactions 3. Molecular weight 4. Polarity or hydrophobicity 5. Polarity or hydrophobicity |
|
Centrifugation |
Density |
Another approach of protein separation, known as affinity purification, has become very popular with the advent of recombinant DNA technology.
For affinity purification, using genetic engineering, the protein of interest is tagged with a stretch of particular amino acids – most commonly histidine.
Specific amino acids have specific chemical properties because of their structure. In this case, for example, histidine can bind and coordinate with cations Ni2+ and Co2+. So, when a mixture of proteins with the histidine-tagged protein of interest in it is passed through a column that has immobilized Ni2+ ions, the protein of interest gets attached to the column because of its affinity for Ni2+ ions, while the remaining proteins flow through. Subsequently the protein of interest is eluted out of the column.
So, by now you have extracted your protein of interest from its source and have separated or purified it from other proteins in the sample.
The next step is detecting your target protein, and as an extension to that, quantifying it in your sample.
The generic approach for this is to treat the protein sample with a substance, most commonly an antibody that readily binds to the protein of interest. If the protein of interest is in the protein sample, then the antibody binds. If not, the antibody does not bind.
This antibody also must have a feature which lets its presence be detected and its levels quantified, most often by colorimetry or spectrophotometry.
The presence of the antibody in the protein sample even after it is washed away indirectly proves the presence of the protein of interest in the sample.
This type of methodology is employed in protein detection approaches such as ELISA and Western blot.
Now that we have seen the basic approaches to study proteins, let us look at a hypothetical research case study to put everything in perspective.
Imagine a bacterial pathogen produces a certain protein toxin ‘A.’ However, it is hypothesized that by applying a new drug, toxin ‘A’ production will decrease. How can this hypothesis be tested? The easiest approach would be to first extract proteins from two samples of bacteria, one treated with the drug, and one that is untreated. The next step is performing a Western blot using an antibody against toxin ‘A’ to test if the levels of toxin ‘A’ indeed diminish upon treatment with this new drug.
Concluding thoughts
After discussing what proteins are, in this article we have discussed two things: why are proteins so important in research, and the theoretical basics of research on proteins is approached in laboratories.
Building up from these fundamentals, here are a few suggested articles that you can read:
