Macromolecular crowding
Encyclopedia
Solution
In chemistry, a solution is a homogeneous mixture composed of only one phase. In such a mixture, a solute is dissolved in another substance, known as a solvent. The solvent does the dissolving.- Types of solutions :...
when high concentrations of macromolecule
Macromolecule
A macromolecule is a very large molecule commonly created by some form of polymerization. In biochemistry, the term is applied to the four conventional biopolymers , as well as non-polymeric molecules with large molecular mass such as macrocycles...
s such as protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s are present. Such conditions occur routinely in living cells
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
; for instance, the cytosol
Cytosol
The cytosol or intracellular fluid is the liquid found inside cells, that is separated into compartments by membranes. For example, the mitochondrial matrix separates the mitochondrion into compartments....
of Escherichia coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
contains about 300–400 milligrammes per millilitre (mg/ml) of macromolecules. Crowding occurs since these high concentrations of macromolecules reduce the volume of solvent
Solvent
A solvent is a liquid, solid, or gas that dissolves another solid, liquid, or gaseous solute, resulting in a solution that is soluble in a certain volume of solvent at a specified temperature...
available for other molecules in the solution, which has the result of increasing their effective concentrations.
This crowding effect can make molecules in cells behave in radically different ways than in test-tube assays. Consequently, measurements of the properties of enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
s or processes in metabolism
Metabolism
Metabolism is the set of chemical reactions that happen in the cells of living organisms to sustain life. These processes allow organisms to grow and reproduce, maintain their structures, and respond to their environments. Metabolism is usually divided into two categories...
that are made in the laboratory in dilute solutions may be different by many orders of magnitude from the true values seen in living cells. The study of biochemical processes under realistically crowded conditions is very important, since these conditions are a ubiquitous property of all cells and crowding may be essential for the efficient operation of metabolism.
Cause and effects
The interior of cells is a crowded environment. For example, an Escherichia coliEscherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
cell is only about 2 micrometre
Micrometre
A micrometer , is by definition 1×10-6 of a meter .In plain English, it means one-millionth of a meter . Its unit symbol in the International System of Units is μm...
s (μm) long and 0.5 μm in diameter, with a cell volume of 0.6 - 0.7 μm3. However, E. coli can contain up to 4,288 different types of proteins, and about 1,000 of these types are produced at a high enough level to be easily detected. Added to this mix are various forms of RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
and the cell's DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
chromosome, giving a total concentration of macromolecules of between 300 to 400 mg/ml. In eukaryote
Eukaryote
A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...
s the cell's interior is further crowded by the protein filament
Protein filament
In biology, a filament is a "long chain of proteins, such as those found in hair, muscle, or in flagella". They are often bundled together for strength and rigidity. Some cellular examples include:*Actin filaments*Microtubules*Intermediate filaments...
s that make up the cytoskeleton
Cytoskeleton
The cytoskeleton is a cellular "scaffolding" or "skeleton" contained within a cell's cytoplasm and is made out of protein. The cytoskeleton is present in all cells; it was once thought to be unique to eukaryotes, but recent research has identified the prokaryotic cytoskeleton...
, this meshwork divides the cytosol into a network of narrow pores.
Excluded volume
The concept of excluded volume was introduced by Werner Kuhn in 1934 and applied to polymer molecules shortly thereafter by Paul Flory.- In liquid state theory :...
effect increases the effective concentration of macromolecules (increasing their chemical activity
Activity (chemistry)
In chemical thermodynamics, activity is a measure of the “effective concentration” of a species in a mixture, meaning that the species' chemical potential depends on the activity of a real solution in the same way that it would depend on concentration for an ideal solution.By convention, activity...
), which in turn alters the rates
Reaction rate
The reaction rate or speed of reaction for a reactant or product in a particular reaction is intuitively defined as how fast or slow a reaction takes place...
and equilibrium constants of their reactions. In particular this effect alters dissociation constant
Dissociation constant
In chemistry, biochemistry, and pharmacology, a dissociation constant is a specific type of equilibrium constant that measures the propensity of a larger object to separate reversibly into smaller components, as when a complex falls apart into its component molecules, or when a salt splits up into...
s by favoring the association of macromolecules, such as when multiple proteins come together to form protein complex
Protein complex
A multiprotein complex is a group of two or more associated polypeptide chains. If the different polypeptide chains contain different protein domain, the resulting multiprotein complex can have multiple catalytic functions...
es, or when DNA-binding protein
DNA-binding protein
DNA-binding proteins are proteins that are composed of DNA-binding domains and thus have a specific or general affinity for either single or double stranded DNA. Sequence-specific DNA-binding proteins generally interact with the major groove of B-DNA, because it exposes more functional groups that...
s bind to their targets in the genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
. Crowding may also affect enzyme reactions involving small molecules if the reaction involves a large change in the shape of the enzyme.
The size of the crowding effect depends on both the molecular mass
Molecular mass
The molecular mass of a substance is the mass of one molecule of that substance, in unified atomic mass unit u...
and shape of the molecule involved, although mass seems to be the major factor – with the effect being stronger with larger molecules. Notably, the size of the effect is non-linear, so macromolecules are much more strongly affected than are small molecules such as amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
s or simple sugars
Monosaccharide
Monosaccharides are the most basic units of biologically important carbohydrates. They are the simplest form of sugar and are usually colorless, water-soluble, crystalline solids. Some monosaccharides have a sweet taste. Examples of monosaccharides include glucose , fructose , galactose, xylose...
. Macromolecular crowding is therefore an effect exerted by large molecules on the properties of other large molecules.
Importance
Macromolecular crowding is an important effect in biochemistryBiochemistry
Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...
and cell biology
Cell biology
Cell biology is a scientific discipline that studies cells – their physiological properties, their structure, the organelles they contain, interactions with their environment, their life cycle, division and death. This is done both on a microscopic and molecular level...
. For example, the increase in the strength of interactions between proteins and DNA produced by crowding may be of key importance in processes such as transcription
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
and DNA replication
DNA replication
DNA replication is a biological process that occurs in all living organisms and copies their DNA; it is the basis for biological inheritance. The process starts with one double-stranded DNA molecule and produces two identical copies of the molecule...
. Crowding has also been suggested to be involved in processes as diverse as the aggregation of hemoglobin
Hemoglobin
Hemoglobin is the iron-containing oxygen-transport metalloprotein in the red blood cells of all vertebrates, with the exception of the fish family Channichthyidae, as well as the tissues of some invertebrates...
in sickle-cell disease
Sickle-cell disease
Sickle-cell disease , or sickle-cell anaemia or drepanocytosis, is an autosomal recessive genetic blood disorder with overdominance, characterized by red blood cells that assume an abnormal, rigid, sickle shape. Sickling decreases the cells' flexibility and results in a risk of various...
, and the responses of cells to changes in their volume.
The importance of crowding in protein folding
Protein folding
Protein folding is the process by which a protein structure assumes its functional shape or conformation. It is the physical process by which a polypeptide folds into its characteristic and functional three-dimensional structure from random coil....
is of particular interest in biophysics
Biophysics
Biophysics is an interdisciplinary science that uses the methods of physical science to study biological systems. Studies included under the branches of biophysics span all levels of biological organization, from the molecular scale to whole organisms and ecosystems...
. Here, the crowding effect can accelerate the folding process, since a compact folded protein will occupy less volume than an unfolded protein chain. However, crowding can reduce the yield of correctly-folded protein by increasing protein aggregation
Protein aggregation
Protein aggregation is the aggregation of mis-folded proteins, and is thought to be responsible for many degenerative diseases, such as Alzheimer's. It has also been implicated in CAG repeat diseases....
. Crowding may also increase the effectiveness of chaperone proteins such as GroEL
GroEL
GroEL belongs to the chaperonin family of molecular chaperones, and is found in a large number of bacteria. It is required for the proper folding of many proteins. To function properly, GroEL requires the lid-like cochaperonin protein complex GroES...
in the cell, which could counteract this reduction in folding efficiency. It has also been shown that macromolecular crowding affects protein-folding dynamics as well as overall protein shape where distinct conformational changes are accompanied by secondary structure alterations implying that crowding-induced shape changes may be important for protein function and malfunction in vivo.
A particularly striking example of the importance of crowding effects involves the crystallin
Crystallin
In anatomy, a crystallin is a water-soluble structural protein found in the lens and the cornea of the eye accounting for the transparency of the structure. It has also been identified in other places such as the heart, and in aggressive breast cancer tumors....
s that fill the interior of the lens
Lens (anatomy)
The crystalline lens is a transparent, biconvex structure in the eye that, along with the cornea, helps to refract light to be focused on the retina. The lens, by changing shape, functions to change the focal distance of the eye so that it can focus on objects at various distances, thus allowing a...
. These proteins have to remain stable and in solution for the lens to be transparent; precipitation
Protein precipitation
Precipitation is widely used in downstream processing of biological products, such as proteins. This unit operation serves to concentrate and fractionate the target product from various contaminants. For example, in the biotechnology industry protein precipitation is used to eliminate contaminants...
or aggregation
Particle aggregation
Particle aggregation in materials science is direct mutual attraction between particles via van der Waals forces or chemical bonding....
of crystallins causes cataract
Cataract
A cataract is a clouding that develops in the crystalline lens of the eye or in its envelope, varying in degree from slight to complete opacity and obstructing the passage of light...
s. Crystallins are present in the lens at extremely high concentrations, over 500 mg/ml, and at these levels crowding effects are very strong. The large crowding effect adds to the thermal stability of the crystallins, increasing their resistance to denaturation
Denaturation (biochemistry)
Denaturation is a process in which proteins or nucleic acids lose their tertiary structure and secondary structure by application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent , or heat...
. This effect may partly explain the extraordinary resistance shown by the lens to damage caused by high temperatures.
Study
Due to macromolecular crowding enzyme assayEnzyme assay
Enzyme assays are laboratory methods for measuring enzymatic activity. They are vital for the study of enzyme kinetics and enzyme inhibition.-Enzyme units:...
s and biophysical measurements performed in dilute solution may fail to reflect the actual processes that take place in the cytosol. One approach to produce more accurate measurements would be to use highly-concentrated extracts of cells, to try to maintain the cell contents in a more natural state. However, when using such extracts it is very difficult to study one process in isolation. Consequently, the crowded effects may be mimicked in vitro by adding high concentrations of an inert molecule such as polyethylene glycol
Polyethylene glycol
Polyethylene glycol is a polyether compound with many applications from industrial manufacturing to medicine. It has also been known as polyethylene oxide or polyoxyethylene , depending on its molecular weight, and under the tradename Carbowax.-Available forms:PEG, PEO, or POE refers to an...
or ficoll
Ficoll
Ficoll is a neutral, highly branched, high-mass, hydrophilic polysaccharide which dissolves readily in aqueous solutions. Ficoll radii range from 2-7 nm. It is prepared by reaction of the polysaccharide with epichlorohydrin...
to assays containing purified components. However, using such artificial crowding agents can be complicated, as these crowding molecules can sometimes interact in other ways with the process being examined, such as by binding weakly to one of the components.