The phenomenon of
macromolecular crowding alters the properties of molecules in a
solutionIn chemistry, a solution is a homogeneous mixture composed of two or more substances. In such a mixture, a solute is dissolved in another substance, known as a solvent. Gases may dissolve in liquids, for example, carbon dioxide or oxygen in water. Liquids may dissolve in other liquids. Gases can...
when high concentrations of
macromoleculeA macromolecule is a very large molecule most often created by some form of polymerization. In the context of biochemistry, the term may be applied to the four conventional biopolymers , as well as non-polymeric molecules with large molecular mass such as macrocycles...
s such as
proteinProteins are organic compounds made of amino acids arranged in a linear chain and folded into a globular form. The amino acids in a polymer chain are joined together by the peptide bonds between the carboxyl and amino groups of adjacent amino acid residues...
s are present. Such conditions occur routinely in living
cellsThe cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of an organism that is classified as living, 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
cytosolThe cytosol or intracellular fluid is the liquid found inside cells. In eukaryotes this liquid is separated by cell membranes from the contents of the organelles suspended in the cytosol, such as the mitochondrial matrix inside the mitochondrion...
of
Escherichia coliEscherichia coli , is a Gram negative bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E...
contains about 300-400 milligrammes per millilitre (mg/ml) of macromolecules. Crowding occurs since these high concentrations of macromolecules reduce the volume of
solventA solvent is a liquid, solid, or gas that dissolves another solid, liquid, or gaseous solute, resulting in a solution.The most common solvent in everyday life is water. Most other commonly-used solvents are organic chemicals. These are called organic solvents...
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.
The phenomenon of
macromolecular crowding alters the properties of molecules in a
solutionIn chemistry, a solution is a homogeneous mixture composed of two or more substances. In such a mixture, a solute is dissolved in another substance, known as a solvent. Gases may dissolve in liquids, for example, carbon dioxide or oxygen in water. Liquids may dissolve in other liquids. Gases can...
when high concentrations of
macromoleculeA macromolecule is a very large molecule most often created by some form of polymerization. In the context of biochemistry, the term may be applied to the four conventional biopolymers , as well as non-polymeric molecules with large molecular mass such as macrocycles...
s such as
proteinProteins are organic compounds made of amino acids arranged in a linear chain and folded into a globular form. The amino acids in a polymer chain are joined together by the peptide bonds between the carboxyl and amino groups of adjacent amino acid residues...
s are present. Such conditions occur routinely in living
cellsThe cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of an organism that is classified as living, 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
cytosolThe cytosol or intracellular fluid is the liquid found inside cells. In eukaryotes this liquid is separated by cell membranes from the contents of the organelles suspended in the cytosol, such as the mitochondrial matrix inside the mitochondrion...
of
Escherichia coliEscherichia coli , is a Gram negative bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E...
contains about 300-400 milligrammes per millilitre (mg/ml) of macromolecules. Crowding occurs since these high concentrations of macromolecules reduce the volume of
solventA solvent is a liquid, solid, or gas that dissolves another solid, liquid, or gaseous solute, resulting in a solution.The most common solvent in everyday life is water. Most other commonly-used solvents are organic chemicals. These are called organic solvents...
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
enzymeEnzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process are called substrates, and the enzyme converts them into different molecules, called the products. Almost all processes in a biological cell need enzymes to occur at...
s or processes in
metabolismMetabolism is the set of chemical reactions that occur in living organisms to maintain life. These processes allow organisms to grow and reproduce, maintain their structures, and respond to their environments. Metabolism is usually divided into two categories. Catabolism breaks down organic matter,...
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 , is a Gram negative bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E...
cell is only about 2
micrometreA micrometre or micron is one millionth of a metre,or equivalently one thousandth of a millimetre....
s (μm) long and 0.5 μm in diameter, with a cell volume of 0.6 - 0.7 μm
3. 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
RNARibonucleic acid is a biologically important type of molecule that consists of a long chain of nucleotide units. Each nucleotide consists of a nitrogenous base, a ribose sugar, and a phosphate...
and the cell's
DNADeoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms and some viruses. The main role of DNA molecules is the long-term storage of information...
chromosome, giving a total concentration of macromolecules of between 300 to 400 mg/ml. In
eukaryoteA eukaryote is an organism whose cells contain complex structures enclosed within membranes. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear envelope, within which the genetic material is carried...
s the cell's interior is further crowded by the
protein filamentIn biology, a filament is a long chain of protein subunits, 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
cytoskeletonThe cytoskeleton is a cellular "scaffolding" or "skeleton" contained within the cytoplasm. The cytoskeleton is present in all cells; it was once thought this structure was unique to eukaryotes, but recent research has identified the prokaryotic cytoskeleton...
, this meshwork divides the cytosol into a network of narrow pores.
These high concentrations of macromolecules occupy a large proportion of the volume of the cell, which reduces the volume of solvent that is available for other macromolecules. This
excluded volumeThe 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 activityIn chemical thermodynamics activity is a measure of the “effective concentration” of a species in a mixture. By convention, it is a dimensionless quantity. The activity of pure substances in condensed phases is normally taken as unity. Activity depends on temperature, pressure and composition of...
), which in turn alters the
ratesThe reaction rate or rate of reaction for a reactant or product in a particular reaction is intuitively defined as how fast a reaction takes place...
and
equilibrium constantFor a general chemical equilibriumthe equilibrium constant can be defined bywhere {A} is the activity of the chemical species A, etc. . It is conventional to put the activities of the products in the numerator and those of the reactants in the denominator...
s of their reactions. In particular this effect alters
dissociation constantIn 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 complexA multiprotein complex is a group of two or more proteins. Protein complexes are a form of quaternary structure. Proteins in a protein complex are linked by non-covalent protein-protein interactions, and different protein complexes have different degrees of stability over time...
es, or when
DNA-binding proteinDNA-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
genomeIn modern molecular biology the genome refers to all of its hereditary information encoded in DNA .The genome includes both the genes and the non-coding sequences of the DNA. The term was adapted in 1920 by Hans Winkler, Professor of Botany at the University of Hamburg, Germany...
. 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 massThe molecular mass of a substance, frequently referred to by the older term molecular weight and abbreviated as MW, is the mass of one molecule of that substance, relative to the 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 acidAmino acids are molecules containing an amine group, a carboxylic acid group and one of the twenty R-groups. These molecules are particularly important in biochemistry, where this term refers to alpha-amino acids with the general formula H
2NCHRCOOH, where R is an organic substituent...
s or
simple sugarsMonosaccharides are the most basic unit of 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 and ribose...
. 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 is the study of the chemical processes in living organisms. It deals with the structure and function of cellular components such as proteins, carbohydrates, lipids, nucleic acids and other biomolecules....
and
cell biologyCell biology is an academic 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
transcriptionTranscription, or RNA synthesis, is the process of creating an equivalent 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 in the presence of the correct enzymes...
and
DNA replicationDNA replication, the basis for biological inheritance, is a fundamental process occurring in all living organisms to copy their DNA. This process is "semiconservative" in that each strand of the original double-stranded DNA molecule serves as template for the reproduction of the complementary strand...
. Crowding has also been suggested to be involved in processes as diverse as the aggregation of
hemoglobinHemoglobin is the iron-containing oxygen-transport metalloprotein in the red blood cells of vertebrates, and the tissues of some invertebrates....
in
sickle-cell diseaseSickle-cell disease, or sickle-cell anaemia , is a life-long blood disorder characterized by red blood cells that assume an abnormal, rigid, sickle shape. Sickling decreases the cells' flexibility and results in a risk of various complications. The sickling occurs because of a mutation in the...
, and the responses of cells to changes in their volume.
The importance of crowding in
protein foldingProtein folding 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
biophysicsBiophysics is an interdisciplinary science that employs and develops theories and methods of the physical sciences for the investigation of biological systems . Studies included under the branches of biophysics span all levels of biological organization, from the molecular scale to whole organisms...
. 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 aggregationProtein 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
GroELGroEL 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.
A particularly striking example of the importance of crowding effects involves the
crystallinIn biology, a crystallin is a water-soluble structural protein found in the lens of the eye, accounting for the transparency of the structure and also in cornea, where it can play a similar role...
s that fill the interior of the
lensThe 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 sharp real...
. These proteins have to remain stable and in solution for the lens to be transparent;
precipitationPrecipitation 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
aggregationParticle aggregation in materials science is direct mutual attraction between particles via van der Waals forces or chemical bonding....
of crystallins causes
cataractA 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
denaturationDenaturation is a process in which proteins or nucleic acids lose their tertiary 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. If proteins in a living cell are denatured, this...
. 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 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 measurementsBiophysical techniques are methods used for gaining information about biological systems on an atomic or molecular level. They overlap with methods from other branches of science.Main biophysical techniques are:...
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 glycolPoly , also known as poly , polyoxyethylene and under the tradename Carbowax is the most commercially important type of polyether. PEG, PEO or POE refers to an oligomer or polymer of ethylene oxide...
or
ficollFicoll 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.