A representation of the 3D structure of the proteinmyoglobin showing turquoiseα-helices. This protein was the first to have its structure solved byX-ray crystallography. Toward the right-center among the coils, aprosthetic group called aheme group (shown in gray) with a bound oxygen molecule (red).
A linear chain of amino acid residues is called apolypeptide. 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 calledpeptides. The individual amino acid residues are bonded together bypeptide bonds and adjacent amino acid residues. Thesequence of amino acid residues in a protein is defined by thesequence of a gene, which is encoded in thegenetic code. In general, the genetic code specifies 20 standard amino acids; but in certain organisms the genetic code can includeselenocysteine and—in certainarchaea—pyrrolysine. Shortly after or even during synthesis, the residues in a protein are often chemically modified bypost-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 calledprosthetic groups orcofactors. Proteins can work together to achieve a particular function, and they often associate to form stableprotein complexes.
Once formed, proteins only exist for a certain period and are thendegraded and recycled by the cell's machinery through the process ofprotein turnover. A protein's lifespan is measured in terms of itshalf-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 aspolysaccharides andnucleic acids, proteins are essential parts of organisms and participate in virtually every process withincells. Many proteins areenzymes thatcatalyse biochemical reactions and are vital tometabolism. Some proteins have structural or mechanical functions, such asactin andmyosin in muscle, and thecytoskeleton's scaffolding proteins that maintain cell shape. Other proteins are important in cell signaling,immune responses,cell adhesion, and thecell cycle. In animals, proteins are needed in thediet to provide theessential amino acids that cannot besynthesized.Digestion breaks the proteins down for metabolic use.
Proteins have been studied and recognized since the 1700s byAntoine 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, andgelatin.[3] Vegetable (plant) proteins studied in the late 1700s and early 1800s includedgluten,plant albumin,gliadin, andlegumin.[1]
Proteins were first described by the Dutch chemistGerardus Johannes Mulder and named by the Swedish chemistJöns Jacob Berzelius in 1838.[4][5] Mulder carried outelemental analysis of common proteins and found that nearly all proteins had the sameempirical 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 theGreek wordπρώτειος (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 theamino acidleucine for which he found a (nearly correct) molecular weight of 131Da.[6]
The difficulty in purifying proteins impeded work by early protein biochemists. Proteins could be obtained in large quantities from blood, egg whites, andkeratin, but individual proteins were unavailable. In the 1950s, theArmour Hot Dog Company purified 1 kg of bovine pancreaticribonuclease 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]
The first protein to have its amino acid chainsequenced wasinsulin, byFrederick 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, orcyclols.[21] He won the Nobel Prize for this achievement in 1958.[22]Christian Anfinsen's studies of theoxidative folding process of ribonuclease A, for which he won the nobel prize in 1972, solidified thethermodynamic hypothesis of protein folding, according to which the folded form of a protein represents itsfree energy minimum.[23][24]
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 orprotein domains, especially inmulti-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 600amino 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 analanine and an adjacent amino acid (top/inset). The bond itself is made of theCHON elements.Resonance structures of thepeptide bond that links individual amino acids to form a proteinpolymer
Most proteins consist of linearpolymers built from series of up to 20L-α-amino acids. Allproteinogenic amino acids have a common structure where anα-carbon isbonded to anamino group, acarboxyl group, and a variableside chain. Onlyproline 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 thestandard 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 bypeptide bonds between amino and carboxyl group. An individual amino acid in a chain is called aresidue, and the linked series of carbon, nitrogen, and oxygen atoms are known as themain chain orprotein backbone.[36]: 19 The peptide bond has tworesonance forms that confer somedouble-bond character to the backbone. The alpha carbons are roughlycoplanar with the nitrogen and the carbonyl (C=O) group. The other twodihedral angles in the peptide bond determine the local shape assumed by the protein backbone. One conseqence 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 theN-terminus oramino terminus, and a free carboxyl group, known as theC-terminus orcarboxy terminus.[37] By convention, peptide sequences are written N-terminus to C-terminus, correlating with the order in which proteins aresynthesized by ribosomes.[37][38]
The wordsprotein,polypeptide, andpeptide are a little ambiguous and can overlap in meaning.Protein is generally used to refer to the complete biological molecule in a stableconformation, whereaspeptide 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]
A typicalbacterial cell, e.g.E. coli andStaphylococcus aureus, is estimated to contain about 2 million proteins. Smaller bacteria, such asMycoplasma orspirochetes 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 andhuman 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 inlymphoblastoid cells.[45] The most abundant protein in nature is thought to beRuBisCO, an enzyme that catalyzes the incorporation ofcarbon dioxide into organic matter inphotosynthesis. Plants can consist of as much as 1% by weight of this enzyme,[46]
Synthesis
Biosynthesis
A ribosome produces a protein using mRNA as templateTheDNA sequence of a geneencodes 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 thenucleotide sequence of the gene encoding this protein. Thegenetic code is a set of three-nucleotide sets calledcodons and each three-nucleotide combination designates an amino acid, for example AUG (adenine–uracil–guanine) is the code formethionine. BecauseDNA 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 firsttranscribed into pre-messenger RNA (mRNA) by proteins such asRNA polymerase. Most organisms then process the pre-mRNA (aprimary transcript) using various forms ofpost-transcriptional modification to form the mature mRNA, which is then used as a template for protein synthesis by theribosome. Inprokaryotes the mRNA may either be used as soon as it is produced, or be bound by a ribosome after having moved away from thenucleoid. In contrast,eukaryotes make mRNA in thecell nucleus and thentranslocate it across thenuclear membrane into thecytoplasm, whereprotein 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 astranslation. The mRNA is loaded onto the ribosome and is read three nucleotides at a time by matching each codon to itsbase pairinganticodon located on atransfer RNA molecule, which carries the amino acid corresponding to the codon it recognizes. The enzymeaminoacyl tRNA synthetase "charges" the tRNA molecules with the correct amino acids. The growing polypeptide is often termed thenascent chain. Proteins are always biosynthesized fromN-terminus toC-terminus.[42]: 1002–42
The size of a synthesized protein can be measured by the number of amino acids it contains and by its totalmolecular mass, which is normally reported in units ofdaltons (synonymous withatomic mass units), or the 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 ofprotein 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 thetitins, a component of themusclesarcomere, with a molecular mass of almost 3,000 kDa and a total length of almost 27,000 amino acids.[49]
Short proteins can be synthesized chemically by a family ofpeptide synthesis methods. These rely onorganic synthesis techniques such aschemical 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 offluorescent probes to amino acid side chains.[51] These methods are useful in laboratorybiochemistry andcell 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 nativetertiary structure. Most chemical synthesis methods proceed from C-terminus to N-terminus, opposite the biological reaction.[52]
Structure
The crystal structure of thechaperonin, a huge protein complex. A single protein subunit is highlighted. Chaperonins assist protein folding.Three possible representations of the three-dimensional structure of the proteintriose 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 proteinsfold into unique 3D structures. The shape into which a protein naturally folds is known as itsnative conformation.[36]: 36 Although many proteins can fold unassisted, simply through the chemical properties of their amino acids, others require the aid of molecularchaperones to fold into their native states.[36]: 37 Biochemists often refer to four distinct aspects of a protein's structure:[36]: 30–34
Secondary structure: regularly repeating local structures stabilized byhydrogen bonds. The most common examples are theα-helix,β-sheet andturns. 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 ahydrophobic core, but also throughsalt bridges, hydrogen bonds,disulfide bonds, and evenpost-translational modifications. The term "tertiary structure" is often used as synonymous with the termfold. The tertiary structure is what controls the basic function of the protein.
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 calledconformational changes. Such changes are often induced by the binding of asubstrate molecule to an enzyme'sactive 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
Proteins can be informally divided into three main classes, which correlate with typical tertiary structures:globular proteins,fibrous proteins, andmembrane proteins. Almost all globular proteins aresoluble and many are enzymes. Fibrous proteins are often structural, such ascollagen, the major component of connective tissue, orkeratin, the protein component of hair and nails. Membrane proteins often serve asreceptors or provide channels for polar or charged molecules to pass through thecell membrane.[42]: 165–85
A special case of intramolecular hydrogen bonds within proteins, poorly shielded from water attack and hence promoting their owndehydration, are calleddehydrons.[53]
Many proteins are composed of severalprotein domains, i.e. segments of a protein that fold into distinct structural units.[54]: 134 Domains usually have specific functions, such asenzymatic activities (e.g.kinase) or they serve as binding modules.[54]: 155–156
Protein domains vs. motifs. Protein domains (such as theEVH1 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 aVASP 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. 2prolines [P], separated by two unspecifiedamino acids [x], although the surrounding amino acids may determine the exact binding specificity). Many such motifs has been collected in theEukaryotic 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 ofRNA, most other biological molecules are relatively inert elements upon which proteins act. Proteins make up half the dry weight of anEscherichia 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 itsproteome.[54]: 120
The enzymehexokinase is shown as a conventional ball-and-stick molecular model. To scale in the top right-hand corner are two of its substrates,ATP andglucose.
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 thebinding 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, theribonuclease inhibitor protein binds to humanangiogenin with a sub-femtomolardissociation constant (<10−15 M) but does not bind at all to its amphibian homologonconase (> 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, theaminoacyl tRNA synthetase specific to the amino acidvaline discriminates against the very similar side chain of the amino acidisoleucine.[59]
Proteins can bind to other proteins as well as tosmall-molecule substrates. When proteins bind specifically to other copies of the same molecule, they canoligomerize 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 thecell cycle, and allow the assembly of largeprotein 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 complexsignaling 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]
The best-known role of proteins in the cell is asenzymes, whichcatalyse chemical reactions. Enzymes are usually highly specific and accelerate only one or a few chemical reactions. Enzymes carry out most of the reactions involved inmetabolism, as well as manipulating DNA in processes such asDNA replication,DNA repair, andtranscription. 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 oforotate decarboxylase (78 million years without the enzyme, 18 milliseconds with the enzyme).[63]
The molecules bound and acted upon by enzymes are calledsubstrates. 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 theactive site.[54]: 389
Many proteins are involved in the process ofcell signaling andsignal transduction. Some proteins, such asinsulin, are extracellular proteins that transmit a signal from the cell in which they were synthesized to other cells in distanttissues. Others aremembrane proteins that act asreceptors 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 aconformational change detected by other proteins within the cell.[41]: 251–81
Antibodies are protein components of anadaptive immune system whose main function is to bindantigens, or foreign substances in the body, and target them for destruction. Antibodies can besecreted into the extracellular environment or anchored in the membranes of specializedB cells known asplasma 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 particularsmall biomolecules and transport them to other locations in the body of a multicellular organism. These proteins must have a high binding affinity when theirligand 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 ishaemoglobin, which transportsoxygen from thelungs to other organs and tissues in allvertebrates and has close homologs in every biologicalkingdom.[42]: 222–29 Lectins aresugar-binding proteins which are highly specific for their sugar moieties.Lectins typically play a role in biologicalrecognition phenomena involving cells and proteins.[66]Receptors andhormones are highly specific binding proteins.
Transmembrane proteins can serve as ligand transport proteins that alter thepermeability of the cell membrane tosmall molecules and ions. The membrane alone has ahydrophobic core through whichpolar or charged molecules cannotdiffuse. Membrane proteins contain internal channels that allow such molecules to enter and exit the cell. Manyion channel proteins are specialized to select for only a particular ion; for example,potassium andsodium channels often discriminate for only one of the two ions.[41]: 232–34
Other proteins that serve structural functions aremotor proteins such asmyosin,kinesin, anddynein, which are capable of generating mechanical forces. These proteins are crucial for cellularmotility of single celled organisms and thesperm of many multicellular organisms which reproducesexually. They generate the forces exerted by contractingmuscles[42]: 258–64, 272 and play essential roles in intracellular transport.[54]: 481, 490
Methods commonly used to study protein structure and function includeimmunohistochemistry,site-directed mutagenesis,X-ray crystallography,nuclear magnetic resonance andmass spectrometry. The activities and structures of proteins may be examinedin vitro,in vivo, andin 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 thechemical 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 acell or even a wholeorganism, and can often provide more information about protein behavior in different contexts.[69]In silico studies use computational methods to study proteins.[70]
To performin vitro analysis, a protein must be purified away from other cellular components. This process usually begins withcell lysis, in which a cell's membrane is disrupted and its internal contents released into a solution known as acrude lysate. The resulting mixture can be purified usingultracentrifugation, which fractionates the various cellular components into fractions containing soluble proteins; membranelipids and proteins; cellularorganelles, andnucleic acids.Precipitation by a method known assalting out can concentrate the proteins from this lysate. Various types ofchromatography 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 ofgel electrophoresis if the desired protein's molecular weight andisoelectric point are known, byspectroscopy if the protein has distinguishable spectroscopic features, or byenzyme assays if the protein has enzymatic activity. Additionally, proteins can be isolated according to their charge usingelectrofocusing.[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 ofhistidine residues (a "His-tag"), is attached to one terminus of the protein. As a result, when the lysate is passed over a chromatography column containingnickel, 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]
The study of proteinsin vivo is often concerned with the synthesis and localization of the protein within the cell. Although many intracellular proteins are synthesized in thecytoplasm and membrane-bound or secreted proteins in theendoplasmic reticulum, the specifics of how proteins aretargeted to specific organelles or cellular structures is often unclear. A useful technique for assessing cellular localization uses genetic engineering to express in a cell afusion protein orchimera consisting of the natural protein of interest linked to a "reporter" such asgreen fluorescent protein (GFP).[73] The fused protein's position within the cell can then be cleanly and efficiently visualized usingmicroscopy.[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 usingisopycnic 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 isimmunoelectron 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 assite-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 rationaldesign of new proteins with novel properties.[80]
The total complement of proteins present at a time in a cell or cell type is known as itsproteome, and the study of such large-scale data sets defines the field ofproteomics, named by analogy to the related field ofgenomics. Key experimental techniques in proteomics include2D 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 afterin-gel digestion),protein microarrays, which allow the detection of the relative levels of the various proteins present in a cell, andtwo-hybrid screening, which allows the systematic exploration ofprotein–protein interactions.[83] The total complement of biologically possible such interactions is known as theinteractome.[84] A systematic attempt to determine the structures of proteins representing every possible fold is known asstructural 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. indrug design. As proteins aretoo small to be seen under alight microscope, other methods have to be employed to determine their structure. Common experimental methods includeX-ray crystallography andNMR spectroscopy, both of which can produce structural information atatomic 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 adistance geometry problem.Dual polarisation interferometry is a quantitative analytical method for measuring the overallprotein conformation andconformational 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 assembledviruses;[41]: 340–41 a variant known aselectron crystallography can produce high-resolution information in some cases, especially for two-dimensional crystals of membrane proteins.[86] Solved structures are usually deposited in theProtein Data Bank (PDB), a freely available resource from which structural data about thousands of proteins can be obtained in the form ofCartesian 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 inX-ray crystallography, one of the major structure determination methods. In particular, globular proteins are comparatively easy tocrystallize 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 containingheme units
Complementary to the field of structural genomics,protein structure prediction develops efficientmathematical 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 ashomology 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 thatsequence 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 ofprotein 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 asintrinsically disordered proteins. Predicting and analysing protein disorder is an important part of protein structure characterisation.[94]
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-layermulti-configuration time-dependent Hartree method and thehierarchical 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, sodistributed computing initiatives such as theFolding@home project facilitate themolecular modeling by exploiting advances inGPU parallel processing andMonte 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, theKjeldahl method is applied. More sensitive methods are available.[103][104]
Hydrolysis of protein. X = HCl and heat for industrial proteolysis. X = protease for biological proteolysis
In the absence of catalysts, proteins are slow tohydrolyze.[105] The breakdown of proteins to small peptides and amino acids (proteolysis) is a step indigestion; these breakdown products are then absorbed in the small intestine.[106] The hydrolysis of proteins relies on enzymes calledproteases or peptidases. Proteases, which are themselves proteins, come in several types according to the particularpeptide 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 includetrypsin andchymotrypsin.[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 hothydrochloric acid, which effects the hydrolysis of the peptide bonds.[109]
Mechanical properties
Themechanical properties of proteins are highly diverse and are often central to their biological function, as in the case of proteins likekeratin andcollagen.[110] For instance, the ability ofmuscle 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 ofenzymes[113] and the structure ofbiological 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 tosynthetic 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 relativestiffness of a material. In the context of proteins, this stiffness often directly correlates to biological function. For example,collagen, found inconnective tissue,bones, andcartilage, andkeratin, found in nails, claws, and hair, have observed stiffnesses that are several orders of magnitude higher than that ofelastin,[116] which is though to give elasticity to structures such asblood vessels,pulmonary tissue, andbladder tissue, among others.[117][118] In comparison to this,globular proteins, such asBovine Serum Albumin, which float relatively freely in thecytosol 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 throughmolecular dynamics simulation. Using either atomistic force-fields, such asCHARMM orGROMOS, 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 asatomic force microscopy can be used to obtain similar data.[124]
At the macroscopic level, the Young's modulus of cross-linked protein networks can be obtained through more traditionalmechanical testing. Experimentally observed values for a few proteins can be seen below.
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