Protein

Proteins are large biomolecules and macromolecules that comprise one or more long chains of
amino acid residues. Proteins perform a vast array of functions within organisms, including
catalysing metabolic reactions, DNA replication, responding to stimuli, providing structure to cells
and organisms, and transporting molecules from one location to another. Proteins differ from one
another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of
their genes, and which usually results in protein folding into a specific 3D structure that determines
its activity.

A representation of the 3D structure of the

protein myoglobin showing turquoise α-
helices. This protein was the first to have
its structure solved by X-ray
crystallography. Toward the right-center
among the coils, a prosthetic group called
a heme group (shown in gray) with a bound
oxygen molecule (red).

A linear chain of amino acid residues is called a polypeptide. A protein contains at least one long
polypeptide. Short polypeptides, containing less than 20–30 residues, are rarely considered to be
proteins and are commonly called peptides. The individual amino acid residues are bonded together
by peptide bonds and adjacent amino acid residues. The sequence of amino acid residues in a
protein is defined by the sequence of a gene, which is encoded in the genetic code. In general, the
genetic code specifies 20 standard amino acids; but in certain organisms the genetic code can
include selenocysteine and—in certain archaea—pyrrolysine. Shortly after or even during synthesis,
the residues in a protein are often chemically modified by post-translational modification, which
alters the physical and chemical properties, folding, stability, activity, and ultimately, the function of
the proteins. Some proteins have non-peptide groups attached, which can be called prosthetic
groups or cofactors. Proteins can work together to achieve a particular function, and they often
associate to form stable protein complexes.

Once formed, proteins only exist for a certain period and are then degraded and recycled by the
cell's machinery through the process of protein turnover. A protein's lifespan is measured in terms of
its half-life and covers a wide range. They can exist for minutes or years with an average lifespan of
1–2 days in mammalian cells. Abnormal or misfolded proteins are degraded more rapidly either due
to being targeted for destruction or due to being unstable.

Like other biological macromolecules such as polysaccharides and nucleic acids, proteins are
essential parts of organisms and participate in virtually every process within cells. Many proteins
are enzymes that catalyse biochemical reactions and are vital to metabolism. Some proteins have
structural or mechanical functions, such as actin and myosin in muscle, and the cytoskeleton's
scaffolding proteins that maintain cell shape. Other proteins are important in cell signaling, immune
responses, cell adhesion, and the cell cycle. In animals, proteins are needed in the diet to provide the
essential amino acids that cannot be synthesized. Digestion breaks the proteins down for metabolic
use.

History and etymology

Discovery and early studies

Proteins have been studied and recognized since the 1700s by Antoine Fourcroy and others,[1][2]
who often collectively called them "albumins", or "albuminous materials" (Eiweisskörper, in
German).[2] Gluten, for example, was first separated from wheat in published research around 1747,
and later determined to exist in many plants.[1] In 1789, Antoine Fourcroy recognized three distinct
varieties of animal proteins: albumin, fibrin, and gelatin.[3] Vegetable (plant) proteins studied in the
late 1700s and early 1800s included gluten, plant albumin, gliadin, and legumin.[1]

Proteins were first described by the Dutch chemist Gerardus Johannes Mulder and named by the
Swedish chemist Jöns Jacob Berzelius in 1838.[4][5] Mulder carried out elemental analysis of
common proteins and found that nearly all proteins had the same empirical formula,
C400H620N100O120P1S1.[6] He came to the erroneous conclusion that they might be composed of a
single type of (very large) molecule. The term "protein" to describe these molecules was proposed
by Mulder's associate Berzelius; protein is derived from the Greek word πρώτειος (proteios),
meaning "primary",[7] "in the lead", or "standing in front",[2] + -in. Mulder went on to identify the
products of protein degradation such as the amino acid leucine for which he found a (nearly correct)
molecular weight of 131 Da.[6]
Early nutritional scientists such as the German Carl von Voit believed that protein was the most
important nutrient for maintaining the structure of the body, because it was generally believed that
"flesh makes flesh".[8] Around 1862, Karl Heinrich Ritthausen isolated the amino acid glutamic
acid.[9] Thomas Burr Osborne compiled a detailed review of the vegetable proteins at the
Connecticut Agricultural Experiment Station. Osborne, alongside Lafayette Mendel, established
several nutritionally essential amino acids in feeding experiments with laboratory rats.[10] Diets
lacking an essential amino acid stunts the rats' growth, consistent with Liebig's law of the
minimum.[11] The final essential amino acid to be discovered, threonine, was identified by William
Cumming Rose.[12]

The difficulty in purifying proteins impeded work by early protein biochemists. Proteins could be
obtained in large quantities from blood, egg whites, and keratin, but individual proteins were
unavailable. In the 1950s, the Armour Hot Dog Company purified 1 kg of bovine pancreatic
ribonuclease A and made it freely available to scientists. This gesture helped ribonuclease A
become a major target for biochemical study for the following decades.[6]

Polypeptides

polypeptide

The understanding of proteins as polypeptides, or chains of amino acids, came through the work of
Franz Hofmeister and Hermann Emil Fischer in 1902.[13][14] The central role of proteins as enzymes
in living organisms that catalyzed reactions was not fully appreciated until 1926, when James B.
Sumner showed that the enzyme urease was in fact a protein.[15]

Linus Pauling is credited with the successful prediction of regular protein secondary structures
based on hydrogen bonding, an idea first put forth by William Astbury in 1933.[16] Later work by
Walter Kauzmann on denaturation,[17][18] based partly on previous studies by Kaj Linderstrøm-
Lang,[19] contributed an understanding of protein folding and structure mediated by hydrophobic
interactions.[20]
The first protein to have its amino acid chain sequenced was insulin, by Frederick Sanger, in 1949.
Sanger correctly determined the amino acid sequence of insulin, thus conclusively demonstrating
that proteins consisted of linear polymers of amino acids rather than branched chains, colloids, or
cyclols.[21] He won the Nobel Prize for this achievement in 1958.[22] Christian Anfinsen's studies of
the oxidative folding process of ribonuclease A, for which he won the nobel prize in 1972, solidified
the thermodynamic hypothesis of protein folding, according to which the folded form of a protein
represents its free energy minimum.[23][24]

Structure

John Kendrew with model of myoglobin in

progress

With the development of X-ray crystallography, it became possible to determine protein structures
as well as their sequences.[25] The first protein structures to be solved were hemoglobin by Max
Perutz and myoglobin by John Kendrew, in 1958.[26][27] The use of computers and increasing
computing power has supported the sequencing of complex proteins. In 1999, Roger Kornberg
sequenced the highly complex structure of RNA polymerase using high intensity X-rays from
synchrotrons.[25]

Since then, cryo-electron microscopy (cryo-EM) of large macromolecular assemblies[28] has been
developed. Cryo-EM uses protein samples that are frozen rather than crystals, and beams of
electrons rather than X-rays. It causes less damage to the sample, allowing scientists to obtain
more information and analyze larger structures.[25] Computational protein structure prediction of
small protein structural domains[29] has helped researchers to approach atomic-level resolution of
protein structures. As of April 2024, the Protein Data Bank contains 181,018 X-ray, 19,809 EM and
12,697 NMR protein structures.[30]
Classification

Proteins are primarily classified by sequence and structure, although other classifications are
commonly used. Especially for enzymes the EC number system provides a functional classification
scheme.[31] Similarly, gene ontology classifies both genes and proteins by their biological and
biochemical function, and by their intracellular location.[32]

Sequence similarity is used to classify proteins both in terms of evolutionary and functional
similarity. This may use either whole proteins or protein domains, especially in multi-domain
proteins. Protein domains allow protein classification by a combination of sequence, structure and
function, and they can be combined in many ways. In an early study of 170,000 proteins, about two-
thirds were assigned at least one domain, with larger proteins containing more domains (e.g.
proteins larger than 600 amino acids having an average of more than 5 domains).[33]

Biochemistry

Chemical structure of the peptide bond (bottom) and the

three-dimensional structure of a peptide bond between an
alanine and an adjacent amino acid (top/inset). The bond
itself is made of the CHON elements.

Resonance structures of the peptide bond that links

individual amino acids to form a protein polymer
Most proteins consist of linear polymers built from series of up to 20 L-α-amino acids. All
proteinogenic amino acids have a common structure where an α-carbon is bonded to an amino
group, a carboxyl group, and a variable side chain. Only proline differs from this basic structure as
its side chain is cyclical, bonding to the amino group, limiting protein chain flexibility.[34] The side
chains of the standard amino acids have a variety of chemical structures and properties, and it is
the combined effect of all amino acids that determines its three-dimensional structure and chemical
reactivity.[35]

The amino acids in a polypeptide chain are linked by peptide bonds between amino and carboxyl
group. An individual amino acid in a chain is called a residue, and the linked series of carbon,
nitrogen, and oxygen atoms are known as the main chain or protein backbone.[36]: 19 The peptide
bond has two resonance forms that confer some double-bond character to the backbone. The alpha
carbons are roughly coplanar with the nitrogen and the carbonyl (C=O) group. The other two dihedral
angles in the peptide bond determine the local shape assumed by the protein backbone. One
consequence of the N-C(O) double bond character is that proteins are somewhat rigid.[36]: 31 A
polypeptide chain ends with a free amino group, known as the N-terminus or amino terminus, and a
free carboxyl group, known as the C-terminus or carboxy terminus.[37] By convention, peptide
sequences are written N-terminus to C-terminus, correlating with the order in which proteins are
synthesized by ribosomes.[37][38]

The words protein, polypeptide, and peptide are a little ambiguous and can overlap in meaning.
Protein is generally used to refer to the complete biological molecule in a stable conformation,
whereas peptide is generally reserved for a short amino acid oligomers often lacking a stable 3D
structure. But the boundary between the two is not well defined and usually lies near 20–30
residues.[39]

Proteins can interact with many types of molecules and ions, including with other proteins, with
lipids, with carbohydrates, and with DNA.[40][41][42]

Abundance in cells

A typical bacterial cell, e.g. E. coli and Staphylococcus aureus, is estimated to contain about 2 million
proteins. Smaller bacteria, such as Mycoplasma or spirochetes contain fewer molecules, on the
order of 50,000 to 1 million. By contrast, eukaryotic cells are larger and thus contain much more
protein. For instance, yeast cells have been estimated to contain about 50 million proteins and
human cells on the order of 1 to 3 billion.[43] The concentration of individual protein copies ranges
from a few molecules per cell up to 20 million.[44] Not all genes coding proteins are expressed in
most cells and their number depends on, for example, cell type and external stimuli. For instance, of
the 20,000 or so proteins encoded by the human genome, only 6,000 are detected in lymphoblastoid
cells.[45] The most abundant protein in nature is thought to be RuBisCO, an enzyme that catalyzes
the incorporation of carbon dioxide into organic matter in photosynthesis. Plants can consist of as
much as 1% by weight of this enzyme.[46]

Synthesis

Biosynthesis

A ribosome produces a protein using

mRNA as template

The DNA sequence of a gene encodes the

amino acid sequence of a protein

Proteins are assembled from amino acids using information encoded in genes. Each protein has its
own unique amino acid sequence that is specified by the nucleotide sequence of the gene encoding
this protein. The genetic code is a set of three-nucleotide sets called codons and each three-
nucleotide combination designates an amino acid, for example AUG (adenine–uracil–guanine) is
the code for methionine. Because DNA contains four nucleotides, the total number of possible
codons is 64; hence, there is some redundancy in the genetic code, with some amino acids
specified by more than one codon.[42]: 1002–42 Genes encoded in DNA are first transcribed into pre-
messenger RNA (mRNA) by proteins such as RNA polymerase. Most organisms then process the
pre-mRNA (a primary transcript) using various forms of post-transcriptional modification to form the
mature mRNA, which is then used as a template for protein synthesis by the ribosome. In
prokaryotes the mRNA may either be used as soon as it is produced, or be bound by a ribosome
after having moved away from the nucleoid. In contrast, eukaryotes make mRNA in the cell nucleus
and then translocate it across the nuclear membrane into the cytoplasm, where protein synthesis
then takes place. The rate of protein synthesis is higher in prokaryotes than eukaryotes and can
reach up to 20 amino acids per second.[47]

The process of synthesizing a protein from an mRNA template is known as translation. The mRNA
is loaded onto the ribosome and is read three nucleotides at a time by matching each codon to its
base pairing anticodon located on a transfer RNA molecule, which carries the amino acid
corresponding to the codon it recognizes. The enzyme aminoacyl tRNA synthetase "charges" the
tRNA molecules with the correct amino acids. The growing polypeptide is often termed the nascent
chain. Proteins are always biosynthesized from N-terminus to C-terminus.[42]: 1002–42

The size of a synthesized protein can be measured by the number of amino acids it contains and by
its total molecular mass, which is normally reported with the unit dalton (Da), or its derivative unit
kilodalton (kDa). The average size of a protein increases from Archaea to Bacteria to Eukaryote
(283, 311, 438 residues and 31, 34, 49 kDa respectively) due to a bigger number of protein domains
constituting proteins in higher organisms.[48] For instance, yeast proteins are on average 466 amino
acids long and 53 kDa in mass.[39] The largest known proteins are the titins, a component of the
muscle sarcomere, with a molecular mass of almost 3000 kDa and a total length of almost 27 000
amino acids.[49]

Chemical synthesis

Peptide Synthesis

Short proteins can be synthesized chemically by a family of peptide synthesis methods. These rely
on organic synthesis techniques such as chemical ligation to produce peptides in high yield.[50]
Chemical synthesis allows for the introduction of non-natural amino acids into polypeptide chains,
such as attachment of fluorescent probes to amino acid side chains.[51] These methods are useful
in laboratory biochemistry and cell biology, though generally not for commercial applications.
Chemical synthesis is inefficient for polypeptides longer than about 300 amino acids, and the
synthesized proteins may not readily assume their native tertiary structure. Most chemical
synthesis methods proceed from C-terminus to N-terminus, opposite the biological reaction.[52]
Structure

The crystal structure of the chaperonin, a huge protein

complex. A single protein subunit is highlighted.
Chaperonins assist protein folding.

Three possible representations of the three-dimensional

structure of the protein triose phosphate isomerase. Left:
All-atom representation colored by atom type. Middle:
Simplified representation illustrating the backbone
conformation, colored by secondary structure. Right:
Solvent-accessible surface representation colored by
residue type (acidic residues red, basic residues blue, polar
residues green, nonpolar residues white).

Most proteins fold into unique 3D structures. The shape into which a protein naturally folds is
known as its native conformation.[36]: 36 Although many proteins can fold unassisted, simply through
the chemical properties of their amino acids, others require the aid of molecular chaperones to fold
into their native states.[36]: 37 Biochemists often refer to four distinct aspects of a protein's
structure:[36]: 30–34

Primary structure: the amino acid sequence. A protein is a polyamide.

Secondary structure: regularly repeating local structures stabilized by hydrogen bonds. The most
common examples are the α-helix, β-sheet and turns. Because secondary structures are local,
many regions of distinct secondary structure can be present in the same protein molecule.

Tertiary structure: the overall shape of a single protein molecule; the spatial relationship of the
secondary structures to one another. Tertiary structure is generally stabilized by nonlocal
interactions, most commonly the formation of a hydrophobic core, but also through salt bridges,
hydrogen bonds, disulfide bonds, and even post-translational modifications. The term "tertiary
 structure" is often used as synonymous with the term fold. The tertiary structure is what controls
the basic function of the protein.

Quaternary structure: the structure formed by several protein molecules (polypeptide chains),
usually called protein subunits in this context, which function as a single protein complex.

Quinary structure: the signatures of protein surface that organize the crowded cellular interior.
Quinary structure is dependent on transient, yet essential, macromolecular interactions that occur
inside living cells.

Proteins are not entirely rigid molecules. In addition to these levels of structure, proteins may shift
between several related structures while they perform their functions. In the context of these
functional rearrangements, these tertiary or quaternary structures are usually referred to as
"conformations", and transitions between them are called conformational changes. Such changes
are often induced by the binding of a substrate molecule to an enzyme's active site, or the physical
region of the protein that participates in chemical catalysis. In solution, protein structures vary
because of thermal vibration and collisions with other molecules.[42]: 368–75

Molecular surface of several proteins showing their

comparative sizes. From left to right are: immunoglobulin
G (IgG, an antibody), hemoglobin, insulin (a hormone),
adenylate kinase (an enzyme), and glutamine synthetase
(an enzyme).

Proteins can be informally divided into three main classes, which correlate with typical tertiary
structures: globular proteins, fibrous proteins, and membrane proteins. Almost all globular proteins
are soluble and many are enzymes. Fibrous proteins are often structural, such as collagen, the
major component of connective tissue, or keratin, the protein component of hair and nails.
Membrane proteins often serve as receptors or provide channels for polar or charged molecules to
pass through the cell membrane.[42]: 165–85

A special case of intramolecular hydrogen bonds within proteins, poorly shielded from water attack
and hence promoting their own dehydration, are called dehydrons.[53]
Protein domains

Many proteins are composed of several protein domains, i.e. segments of a protein that fold into
distinct structural units.[54]: 134 Domains usually have specific functions, such as enzymatic
activities (e.g. kinase) or they serve as binding modules.[54]: 155–156

Protein domains vs. motifs. Protein domains (such as the EVH1 domain) are functional units within proteins that fold
into defined 3D structures. Motifs are usually short sequences with specific functions but without a stable 3D structure.
Many motifs are binding sites for other proteins (such as the red and green bars shown here in the context of a VASP
protein).[55]

Sequence motif

Short amino acid sequences within proteins often act as recognition sites for other proteins.[56] For
instance, SH3 domains typically bind to short PxxP motifs (i.e. 2 prolines [P], separated by two
unspecified amino acids [x], although the surrounding amino acids may determine the exact binding
specificity). Many such motifs has been collected in the Eukaryotic Linear Motif (ELM) database.[57]

Cellular functions

Proteins are the chief actors within the cell, said to be carrying out the duties specified by the
information encoded in genes.[39] With the exception of certain types of RNA, most other biological
molecules are relatively inert elements upon which proteins act. Proteins make up half the dry
weight of an Escherichia coli cell, whereas other macromolecules such as DNA and RNA make up
only 3% and 20%, respectively.[58] The set of proteins expressed in a particular cell or cell type is
known as its proteome.[54]: 120
 The enzyme hexokinase is shown as a
conventional ball-and-stick molecular
model. To scale in the top right-hand
corner are two of its substrates, ATP and
glucose.

The chief characteristic of proteins that allows their diverse set of functions is their ability to bind
other molecules specifically and tightly. The region of the protein responsible for binding another
molecule is known as the binding site and is often a depression or "pocket" on the molecular
surface. This binding ability is mediated by the tertiary structure of the protein, which defines the
binding site pocket, and by the chemical properties of the surrounding amino acids' side chains.
Protein binding can be extraordinarily tight and specific; for example, the ribonuclease inhibitor
protein binds to human angiogenin with a sub-femtomolar dissociation constant (< 10−15 M) but
does not bind at all to its amphibian homolog onconase (> 1 M). Extremely minor chemical changes
such as the addition of a single methyl group to a binding partner can sometimes suffice to nearly
eliminate binding; for example, the aminoacyl tRNA synthetase specific to the amino acid valine
discriminates against the very similar side chain of the amino acid isoleucine.[59]

Proteins can bind to other proteins as well as to small-molecule substrates. When proteins bind
specifically to other copies of the same molecule, they can oligomerize to form fibrils; this process
occurs often in structural proteins that consist of globular monomers that self-associate to form
rigid fibers. Protein–protein interactions regulate enzymatic activity, control progression through the
cell cycle, and allow the assembly of large protein complexes that carry out many closely related
reactions with a common biological function. Proteins can bind to, or be integrated into, cell
membranes. The ability of binding partners to induce conformational changes in proteins allows the
construction of enormously complex signaling networks.[42]: 830–49 As interactions between proteins
are reversible and depend heavily on the availability of different groups of partner proteins to form
aggregates that are capable to carry out discrete sets of function, study of the interactions between
specific proteins is a key to understand important aspects of cellular function, and ultimately the
properties that distinguish particular cell types.[60][61]
Enzymes

The best-known role of proteins in the cell is as enzymes, which catalyse chemical reactions.
Enzymes are usually highly specific and accelerate only one or a few chemical reactions. Enzymes
carry out most of the reactions involved in metabolism, as well as manipulating DNA in processes
such as DNA replication, DNA repair, and transcription. Some enzymes act on other proteins to add
or remove chemical groups in a process known as posttranslational modification. About 4,000
reactions are known to be catalysed by enzymes.[62] The rate acceleration conferred by enzymatic
catalysis is often enormous—as much as 1017-fold increase in rate over the uncatalysed reaction in
the case of orotate decarboxylase (78 million years without the enzyme, 18 milliseconds with the
enzyme).[63]

The molecules bound and acted upon by enzymes are called substrates. Although enzymes can
consist of hundreds of amino acids, it is usually only a small fraction of the residues that come in
contact with the substrate, and an even smaller fraction—three to four residues on average—that are
directly involved in catalysis.[64] The region of the enzyme that binds the substrate and contains the
catalytic residues is known as the active site.[54]: 389

Dirigent proteins are members of a class of proteins that dictate the stereochemistry of a
compound synthesized by other enzymes.[65]

Cell signaling and ligand binding

Ribbon diagram of a mouse

antibody against cholera that
binds a carbohydrate antigen

Many proteins are involved in the process of cell signaling and signal transduction. Some proteins,
such as insulin, are extracellular proteins that transmit a signal from the cell in which they were
synthesized to other cells in distant tissues. Others are membrane proteins that act as receptors
whose main function is to bind a signaling molecule and induce a biochemical response in the cell.
Many receptors have a binding site exposed on the cell surface and an effector domain within the
cell, which may have enzymatic activity or may undergo a conformational change detected by other
proteins within the cell.[41]: 251–81

Antibodies are protein components of an adaptive immune system whose main function is to bind
antigens, or foreign substances in the body, and target them for destruction. Antibodies can be
secreted into the extracellular environment or anchored in the membranes of specialized B cells
known as plasma cells. Whereas enzymes are limited in their binding affinity for their substrates by
the necessity of conducting their reaction, antibodies have no such constraints. An antibody's
binding affinity to its target is extraordinarily high.[42]: 275–50

Many ligand transport proteins bind particular small biomolecules and transport them to other
locations in the body of a multicellular organism. These proteins must have a high binding affinity
when their ligand is present in high concentrations, and release the ligand when it is present at low
concentrations in the target tissues. The canonical example of a ligand-binding protein is
haemoglobin, which transports oxygen from the lungs to other organs and tissues in all vertebrates
and has close homologs in every biological kingdom.[42]: 222–29 Lectins are sugar-binding proteins
which are highly specific for their sugar moieties. Lectins typically play a role in biological
recognition phenomena involving cells and proteins.[66] Receptors and hormones are highly specific
binding proteins.

Transmembrane proteins can serve as ligand transport proteins that alter the permeability of the
cell membrane to small molecules and ions. The membrane alone has a hydrophobic core through
which polar or charged molecules cannot diffuse. Membrane proteins contain internal channels that
allow such molecules to enter and exit the cell. Many ion channel proteins are specialized to select
for only a particular ion; for example, potassium and sodium channels often discriminate for only
one of the two ions.[41]: 232–34
Structural proteins

Protein Structure

Structural proteins confer stiffness and rigidity to otherwise-fluid biological components. Most
structural proteins are fibrous proteins; for example, collagen and elastin are critical components of
connective tissue such as cartilage, and keratin is found in hard or filamentous structures such as
hair, nails, feathers, hooves, and some animal shells.[42]: 178–81 Some globular proteins can play
structural functions, for example, actin and tubulin are globular and soluble as monomers, but
polymerize to form long, stiff fibers that make up the cytoskeleton, which allows the cell to maintain
its shape and size.[54]: 490

Other proteins that serve structural functions are motor proteins such as myosin, kinesin, and
dynein, which are capable of generating mechanical forces. These proteins are crucial for cellular
motility of single celled organisms and the sperm of many multicellular organisms which reproduce
sexually. They generate the forces exerted by contracting muscles[42]: 258–64, 272 and play essential
roles in intracellular transport.[54]: 481, 490

Methods of study

Methods commonly used to study protein structure and function include immunohistochemistry,
site-directed mutagenesis, X-ray crystallography, nuclear magnetic resonance and mass
spectrometry. The activities and structures of proteins may be examined in vitro, in vivo, and in silico.
In vitro studies of purified proteins in controlled environments are useful for learning how a protein
carries out its function:[67] for example, enzyme kinetics studies explore the chemical mechanism of
an enzyme's catalytic activity and its relative affinity for various possible substrate molecules.[68] By
contrast, in vivo experiments can provide information about the physiological role of a protein in the
context of a cell or even a whole organism, and can often provide more information about protein
behavior in different contexts.[69] In silico studies use computational methods to study proteins.[70]
Protein purification

Proteins may be purified from other cellular components using a variety of techniques such as
ultracentrifugation, precipitation, electrophoresis, and chromatography;[36]: 21–24 the advent of
genetic engineering has made possible a number of methods to facilitate purification.[71]

To perform in vitro analysis, a protein must be purified away from other cellular components. This
process usually begins with cell lysis, in which a cell's membrane is disrupted and its internal
contents released into a solution known as a crude lysate. The resulting mixture can be purified
using ultracentrifugation, which fractionates the various cellular components into fractions
containing soluble proteins; membrane lipids and proteins; cellular organelles, and nucleic acids.
Precipitation by a method known as salting out can concentrate the proteins from this lysate.
Various types of chromatography are then used to isolate the protein or proteins of interest based
on properties such as molecular weight, net charge and binding affinity.[36]: 21–24 The level of
purification can be monitored using various types of gel electrophoresis if the desired protein's
molecular weight and isoelectric point are known, by spectroscopy if the protein has distinguishable
spectroscopic features, or by enzyme assays if the protein has enzymatic activity. Additionally,
proteins can be isolated according to their charge using electrofocusing.[72]

For natural proteins, a series of purification steps may be necessary to obtain protein sufficiently
pure for laboratory applications. To simplify this process, genetic engineering is often used to add
chemical features to proteins that make them easier to purify without affecting their structure or
activity. Here, a "tag" consisting of a specific amino acid sequence, often a series of histidine
residues (a "His-tag"), is attached to one terminus of the protein. As a result, when the lysate is
passed over a chromatography column containing nickel, the histidine residues ligate the nickel and
attach to the column while the untagged components of the lysate pass unimpeded. A number of
tags have been developed to help researchers purify specific proteins from complex mixtures.[71]
Cellular localization

Proteins in various cellular compartments and structures

tagged with green fluorescent protein (here, white)

The study of proteins in vivo is often concerned with the synthesis and localization of the protein
within the cell. Although many intracellular proteins are synthesized in the cytoplasm and
membrane-bound or secreted proteins in the endoplasmic reticulum, the specifics of how proteins
are targeted to specific organelles or cellular structures is often unclear. A useful technique for
assessing cellular localization uses genetic engineering to express in a cell a fusion protein or
chimera consisting of the natural protein of interest linked to a "reporter" such as green fluorescent
protein (GFP).[73] The fused protein's position within the cell can then be cleanly and efficiently
visualized using microscopy.[74]

Other methods for elucidating the cellular location of proteins requires the use of known
compartmental markers for regions such as the ER, the Golgi, lysosomes or vacuoles, mitochondria,
chloroplasts, plasma membrane, etc. With the use of fluorescently tagged versions of these
markers or of antibodies to known markers, it becomes much simpler to identify the localization of
a protein of interest. For example, indirect immunofluorescence will allow for fluorescence
colocalization and demonstration of location. Fluorescent dyes are used to label cellular
compartments for a similar purpose.[75]
Other possibilities exist, as well. For example, immunohistochemistry usually uses an antibody to
one or more proteins of interest that are conjugated to enzymes yielding either luminescent or
chromogenic signals that can be compared between samples, allowing for localization
information.[76] Another applicable technique is cofractionation in sucrose (or other material)
gradients using isopycnic centrifugation.[77] While this technique does not prove colocalization of a
compartment of known density and the protein of interest, it indicates an increased likelihood.[77]

Finally, the gold-standard method of cellular localization is immunoelectron microscopy. This

technique uses an antibody to the protein of interest, along with classical electron microscopy
techniques. The sample is prepared for normal electron microscopic examination, and then treated
with an antibody to the protein of interest that is conjugated to an extremely electro-dense material,
usually gold. This allows for the localization of both ultrastructural details as well as the protein of
interest.[78]

Through another genetic engineering application known as site-directed mutagenesis, researchers

can alter the protein sequence and hence its structure, cellular localization, and susceptibility to
regulation. This technique even allows the incorporation of unnatural amino acids into proteins,
using modified tRNAs,[79] and may allow the rational design of new proteins with novel
properties.[80]

Proteomics

The total complement of proteins present at a time in a cell or cell type is known as its proteome,
and the study of such large-scale data sets defines the field of proteomics, named by analogy to the
related field of genomics. Key experimental techniques in proteomics include 2D electrophoresis,[81]
which allows the separation of many proteins, mass spectrometry,[82] which allows rapid high-
throughput identification of proteins and sequencing of peptides (most often after in-gel digestion),
protein microarrays, which allow the detection of the relative levels of the various proteins present in
a cell, and two-hybrid screening, which allows the systematic exploration of protein–protein
interactions.[83] The total complement of biologically possible such interactions is known as the
interactome.[84] A systematic attempt to determine the structures of proteins representing every
possible fold is known as structural genomics.[85]

Structure determination

Discovering the tertiary structure of a protein, or the quaternary structure of its complexes, can
provide important clues about how the protein performs its function and how it can be affected, i.e.
in drug design. As proteins are too small to be seen under a light microscope, other methods have
to be employed to determine their structure. Common experimental methods include X-ray
crystallography and NMR spectroscopy, both of which can produce structural information at atomic
resolution. However, NMR experiments are able to provide information from which a subset of
distances between pairs of atoms can be estimated, and the final possible conformations for a
protein are determined by solving a distance geometry problem. Dual polarisation interferometry is
a quantitative analytical method for measuring the overall protein conformation and conformational
changes due to interactions or other stimulus. Circular dichroism is another laboratory technique for
determining internal β-sheet / α-helical composition of proteins. Cryoelectron microscopy is used to
produce lower-resolution structural information about very large protein complexes, including
assembled viruses;[41]: 340–41 a variant known as electron crystallography can produce high-
resolution information in some cases, especially for two-dimensional crystals of membrane
proteins.[86] Solved structures are usually deposited in the Protein Data Bank (PDB), a freely
available resource from which structural data about thousands of proteins can be obtained in the
form of Cartesian coordinates for each atom in the protein.[87]

Many more gene sequences are known than protein structures. Further, the set of solved structures
is biased toward proteins that can be easily subjected to the conditions required in X-ray
crystallography, one of the major structure determination methods. In particular, globular proteins
are comparatively easy to crystallize in preparation for X-ray crystallography. Membrane proteins
and large protein complexes, by contrast, are difficult to crystallize and are underrepresented in the
PDB.[88] Structural genomics initiatives have attempted to remedy these deficiencies by
systematically solving representative structures of major fold classes. Protein structure prediction
methods attempt to provide a means of generating a plausible structure for proteins whose
structures have not been experimentally determined.[89]
Structure prediction

Constituent amino-acids can be analyzed to predict secondary,

tertiary and quaternary protein structure, in this case hemoglobin
containing heme units

Complementary to the field of structural genomics, protein structure prediction develops efficient
mathematical models of proteins to computationally predict the molecular formations in theory,
instead of detecting structures with laboratory observation.[90] The most successful type of
structure prediction, known as homology modeling, relies on the existence of a "template" structure
with sequence similarity to the protein being modeled; structural genomics' goal is to provide
sufficient representation in solved structures to model most of those that remain.[91] Although
producing accurate models remains a challenge when only distantly related template structures are
available, it has been suggested that sequence alignment is the bottleneck in this process, as quite
accurate models can be produced if a "perfect" sequence alignment is known.[92] Many structure
prediction methods have served to inform the emerging field of protein engineering, in which novel
protein folds have already been designed.[93] Many proteins (in eukaryotes ~33%) contain large
unstructured but biologically functional segments and can be classified as intrinsically disordered
proteins. Predicting and analysing protein disorder is an important part of protein structure
characterisation.[94]
In silico simulation of dynamical processes

A more complex computational problem is the prediction of intermolecular interactions, such as in

molecular docking,[95] protein folding, protein–protein interaction and chemical reactivity.
Mathematical models to simulate these dynamical processes involve molecular mechanics, in
particular, molecular dynamics. In this regard, in silico simulations discovered the folding of small α-
helical protein domains such as the villin headpiece,[96] the HIV accessory protein[97] and hybrid
methods combining standard molecular dynamics with quantum mechanical mathematics have
explored the electronic states of rhodopsins.[98]

Beyond classical molecular dynamics, quantum dynamics methods allow the simulation of proteins
in atomistic detail with an accurate description of quantum mechanical effects. Examples include
the multi-layer multi-configuration time-dependent Hartree method and the hierarchical equations of
motion approach, which have been applied to plant cryptochromes[99] and bacteria light-harvesting
complexes,[100] respectively. Both quantum and classical mechanical simulations of biological-scale
systems are extremely computationally demanding, so distributed computing initiatives such as the
Folding@home project facilitate the molecular modeling by exploiting advances in GPU parallel
processing and Monte Carlo techniques.[101][102]

Chemical analysis

The total nitrogen content of organic matter is mainly formed by the amino groups in proteins. The
total Kjeldahl nitrogen (TKN) is a measure of nitrogen widely used in the analysis of (waste) water,
soil, food, feed and organic matter in general. As the name suggests, the Kjeldahl method is applied.
More sensitive methods are available.[103][104]

Digestion

Hydrolysis of protein. X = HCl and heat

for industrial proteolysis. X = protease
for biological proteolysis
In the absence of catalysts, proteins are slow to hydrolyze.[105] The breakdown of proteins to small
peptides and amino acids (proteolysis) is a step in digestion; these breakdown products are then
absorbed in the small intestine.[106] The hydrolysis of proteins relies on enzymes called proteases or
peptidases. Proteases, which are themselves proteins, come in several types according to the
particular peptide bonds that they cleave as well as their tendency to cleave peptide bonds at the
terminus of a protein (exopeptidases) vs peptide bonds at the interior of the protein
(endopeptidases).[107] Pepsin is an endopeptidase in the stomach. Subsequent to the stomach, the
pancreas secretes other proteases to complete the hydrolysis, these include trypsin and
chymotrypsin.[108]

Protein hydrolysis is employed commercially as a means of producing amino acids from bulk
sources of protein, such as blood meal, feathers, keratin. Such materials are treated with hot
hydrochloric acid, which effects the hydrolysis of the peptide bonds.[109]

Mechanical properties

The mechanical properties of proteins are highly diverse and are often central to their biological
function, as in the case of proteins like keratin and collagen.[110] For instance, the ability of muscle
tissue to continually expand and contract is directly tied to the elastic properties of their underlying
protein makeup.[111][112] Beyond fibrous proteins, the conformational dynamics of enzymes[113] and
the structure of biological membranes, among other biological functions, are governed by the
mechanical properties of the proteins. Outside of their biological context, the unique mechanical
properties of many proteins, along with their relative sustainability when compared to synthetic
polymers, have made them desirable targets for next-generation materials design.[114][115]

Young's modulus, E, is calculated as the axial stress σ over the resulting strain ε. It is a measure of
the relative stiffness of a material. In the context of proteins, this stiffness often directly correlates
to biological function. For example, collagen, found in connective tissue, bones, and cartilage, and
keratin, found in nails, claws, and hair, have observed stiffnesses that are several orders of
magnitude higher than that of elastin,[116] which is thought to give elasticity to structures such as
blood vessels, pulmonary tissue, and bladder tissue, among others.[117][118] In comparison to this,
globular proteins, such as Bovine Serum Albumin, which float relatively freely in the cytosol and
often function as enzymes (and thus undergoing frequent conformational changes) have
comparably much lower Young's moduli.[119][120]

The Young's modulus of a single protein can be found through molecular dynamics simulation.
Using either atomistic force-fields, such as CHARMM or GROMOS, or coarse-grained forcefields like
Martini,[121] a single protein molecule can be stretched by a uniaxial force while the resulting
extension is recorded in order to calculate the strain.[122][123] Experimentally, methods such as
atomic force microscopy can be used to obtain similar data.[124] The internal dynamics of proteins
involve subtle elastic and plastic deformations induced by viscoelastic forces, which can be probed
by nano-rheology techniques.[125]

At the macroscopic level, the Young's modulus of cross-linked protein networks can be obtained
through more traditional mechanical testing. Experimentally observed values for a few proteins can
be seen below.

Elasticity of various proteins

Protein Protein class Young's modulus

keratin (cross-linked) fibrous 1.5–10 GPa[126]

elastin (cross-linked) fibrous 1 MPa[116]

fibrin (cross-linked) fibrous 1–10 MPa[116]

collagen (cross-linked) fibrous 5–7.5 GPa[116][127]

resilin (cross-linked) fibrous 1–2 MPa[116]

bovine serum albumin (cross-linked) globular 2.5–15 kPa[119]

β-barrel outer membrane proteins membrane 20–45 GPa[128]

See also

Deproteination – Technique in research of live materials

DNA-binding protein – Proteins that bind with DNA

Index of protein-related articles

Intein

List of proteins

Macromolecule – Very large molecule

Protein evolution – Study of changes in DNA and RNA over time

Protein sequence space – Representation of possible genetic sequences

Protein superfamily – Grouping of proteins

Protein toxicity – Buildup of metabolic waste due to kidney dysfunction

Proteopathy – Diseases caused by abnormal protein structure

Proteopedia – 3D encyclopedia of proteins and other molecules

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Further reading

Textbooks

Branden C, Tooze J (1999). Introduction to Protein Structure. New York: Garland Pub. ISBN 978-0-
8153-2305-1.

Murray RF, Harper HW, Granner DK, Mayes PA, Rodwell VW (2006). Harper's Illustrated
Biochemistry. New York: Lange Medical Books/McGraw-Hill. ISBN 978-0-07-146197-9.

Van Holde KE, Mathews CK (1996). Biochemistry ([Link]

h) . Menlo Park, California: Benjamin/Cummings Pub. Co., Inc. ISBN 978-0-8053-3931-4.

History

Tanford C, Reynolds JA (2001). Nature's Robots: A History of Proteins. Oxford New York: Oxford
University Press, USA. ISBN 978-0-19-850466-5.

External links

Databases and projects

NCBI Entrez Protein database ([Link]

NCBI Protein Structure database ([Link]

[Link]/sites/entrez?db=Structure)
 Human Protein Reference Database ([Link]
[Link]/)

Human Proteinpedia ([Link]

[Link]/)

Folding@Home (Stanford University) ([Link] Archived ([Link]

[Link]/web/20120908075542/[Link] 2012-09-08 at the
Wayback Machine

Protein Databank in Europe ([Link] (see also PDBeQuips ([Link]

20130727184433/[Link] , short articles and tutorials on interesting PDB
structures)

Research Collaboratory for Structural Bioinformatics ([Link] (see also Molecule

of the Month ([Link]
h/[Link]) Archived ([Link]
db/[Link]?p=education_discussion%2Fmolecule_of_the_month%[Link]) 2020-07-24 at
the Wayback Machine, presenting short accounts on selected proteins from the PDB)

Proteopedia – Life in 3D ([Link] : rotatable, zoomable 3D model with wiki

annotations for every known protein molecular structure.

UniProt the Universal Protein Resource ([Link]

[Link]/)

Tutorials and educational websites

"An Introduction to Proteins" ([Link]

uction-to-proteins/) from HOPES (Huntington's Disease Outreach Project for Education at
Stanford)

Proteins: Biogenesis to Degradation – The Virtual Library of Biochemistry and Cell Biology (http
s://[Link]/web/20050219090405/[Link]

Portals: Biology Technology Medicine Chemistry Food Ecology

Environment Science Evolutionary biology