Wikipedia

Protein

This article is about a class of molecules. For protein as a nutrient, seeProtein (nutrient).For other uses, seeProtein (disambiguation).

Proteinsare largebiomoleculesandmacromoleculesthat comprise one or more long chains ofamino acidresidues.Proteins perform a vast array of functions within organisms, includingcatalysing metabolic reactions,DNA replication,responding to stimuli,providingstructure to cellsandorganisms,andtransporting moleculesfrom one location to another. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by thenucleotide sequenceof theirgenes,and which usually results inprotein foldinginto a specific3D structurethat determines its activity.

A representation of the 3D structure of the proteinmyoglobinshowing turquoiseα-helices.This protein was the first to have its structure solved byX-ray crystallography.Toward the right-center among the coils, aprosthetic groupcalled 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 bondsand adjacent amino acid residues. Thesequenceof amino acid residues in a protein is defined by thesequenceof 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 includeselenocysteineand—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 groupsorcofactors.Proteins can also 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 thendegradedand recycled by the cell's machinery through the process ofprotein turnover.A protein's lifespan is measured in terms of itshalf-lifeand 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 aspolysaccharidesandnucleic acids,proteins are essential parts of organisms and participate in virtually every process withincells.Many proteins areenzymesthatcatalysebiochemical reactions and are vital tometabolism.Proteins also have structural or mechanical functions, such asactinandmyosinin muscle and the proteins in thecytoskeleton,which form a system ofscaffoldingthat maintains cell shape. Other proteins are important in cell signaling,immune responses,cell adhesion,and thecell cycle.In animals, proteins are needed in thedietto provide theessential amino acidsthat cannot besynthesized.Digestionbreaks the proteins down for metabolic use.

History and etymology

Further information:History of molecular biology

Discovery and early studies

Proteins have been studied and recognized since the 1700s byAntoine Fourcroyand 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]n 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 Mulderand named by the Swedish chemistJöns Jacob Berzeliusin 1838.[4][5][better source needed]Mulder carried outelemental analysisof 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 theGreekwordπρώτειος(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 acidleucinefor which he found a (nearly correct) molecular weight of 131Da.[6]

Early nutritional scientists such as the GermanCarl von Voitbelieved 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 Ritthausenisolated the amino acidglutamic acid.[9]Thomas Burr Osbornecompiled a detailed review of the vegetable proteins at theConnecticut Agricultural Experiment Station.Then, working with[clarification needed]Lafayette Mendeland applyingLiebig's law of the minimum,which states that growth is limited by the scarcest resource, to the feeding of laboratory rats, thenutritionally essential amino acidswere established. The work was continued and communicated byWilliam Cumming Rose.

The difficulty in purifying proteins in large quantities made them very difficult for early protein biochemists to study. Hence, early studies focused on proteins that could be purified in large quantities, including those of blood, egg whites, and various toxins, as well as digestive and metabolic enzymes obtained from slaughterhouses.[clarification needed]In the 1950s, theArmour Hot Dog Companypurified 1 kg of pure bovine pancreaticribonuclease Aand made it freely available to scientists; this gesture helped ribonuclease A become a major target for biochemical study for the following decades.[6]

Polypeptides

The understanding of proteins aspolypeptides,or chains of amino acids, came through the work ofFranz HofmeisterandHermann Emil Fischerin 1902.[10][11]The central role of proteins asenzymesin living organisms that catalyzed reactions was not fully appreciated until 1926, whenJames B. Sumnershowed that the enzymeureasewas in fact a protein.[12]

Linus Paulingis credited with the successful prediction of regular proteinsecondary structuresbased onhydrogen bonding,an idea first put forth byWilliam Astburyin 1933.[13]Later work byWalter Kauzmannondenaturation,[14][15]based partly on previous studies byKaj Linderstrøm-Lang,[16]contributed an understanding ofprotein foldingand structure mediated byhydrophobic interactions.

The first protein to have its amino acid chainsequencedwasinsulin,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.[17]He won the Nobel Prize for this achievement in 1958.[18]Christian Anfinsen's studies of theoxidative foldingprocess of ribonuclease A, for which he won the nobel prize in 1972, solidified thethermodynamic hypothesisof protein folding, according to which the folded form of a protein represents itsfree energyminimum.[19][20]

Structure

John Kendrewwith model of myoglobin in progress

With the development ofX-ray crystallography,it became possible to determine protein structures as well as their sequences.[21]The firstprotein structuresto be solved werehemoglobinbyMax PerutzandmyoglobinbyJohn Kendrew,in 1958.[22][23]The use of computers and increasing computing power also supported the sequencing of complex proteins. In 1999,Roger Kornbergsucceeded in sequencing the highly complex structure ofRNA polymeraseusing high intensity X-rays fromsynchrotrons.[21]

Since then,cryo-electron microscopy(cryo-EM) of largemacromolecular assemblies[24]has been developed. Cryo-EM uses protein samples that are frozen rather than crystals, andbeams of electronsrather than X-rays. It causes less damage to the sample, allowing scientists to obtain more information and analyze larger structures.[21]Computationalprotein structure predictionof small proteinstructural domains[25]has also helped researchers to approach atomic-level resolution of protein structures. As of April 2024,theProtein Data Bankcontains 181,018 X-ray, 19,809EMand 12,697NMRprotein structures.[26]

Classification

Main articles:Protein family,Gene Ontology,andEnzyme Commission number

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. Similarly, thegene ontologyclassifies both genes and proteins by their biological and biochemical function, but also by their intracellular location.

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 different 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 acidshaving an average of more than 5 domains).[27]

Biochemistry

Chemical structure of the peptide bond (bottom) and the three-dimensional structure of a peptide bond between analanineand an adjacent amino acid (top/inset). The bond itself is made of theCHONelements.
Resonancestructures of thepeptide bondthat links individual amino acids to form a proteinpolymer
Main articles:Biochemistry,Amino acid,andPeptide bond

Most proteins consist of linearpolymersbuilt from series of up to 20 differentL-α-amino acids. Allproteinogenic amino acidspossess common structural features, including anα-carbonto which anaminogroup, acarboxylgroup, and a variableside chainarebonded.Onlyprolinediffers from this basic structure as it contains an unusual ring to the N-end amine group, which forces the CO–NH amide moiety into a fixed conformation.[28]The side chains of the standard amino acids, detailed in thelist of standard amino acids,have a great variety of chemical structures and properties; it is the combined effect of all of the amino acid side chains in a protein that ultimately determines its three-dimensional structure and its chemical reactivity.[29] The amino acids in a polypeptide chain are linked bypeptide bonds.Once linked in the protein chain, an individual amino acid is called aresidue,and the linked series of carbon, nitrogen, and oxygen atoms are known as themain chainorprotein backbone.[30]: 19 

The peptide bond has tworesonanceforms that contribute somedouble-bondcharacter and inhibit rotation around its axis, so that the alpha carbons are roughlycoplanar.The other twodihedral anglesin the peptide bond determine the local shape assumed by the protein backbone.[30]: 31 The end with a free amino group is known as theN-terminusor amino terminus, whereas the end of the protein with a free carboxyl group is known as theC-terminusor carboxy terminus (the sequence of the protein is written from N-terminus to C-terminus, from left to right).

The wordsprotein,polypeptide,andpeptideare a little ambiguous and can overlap in meaning.Proteinis generally used to refer to the complete biological molecule in a stableconformation,whereaspeptideis 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.[31]Polypeptidecan refer to any single linear chain of amino acids, usually regardless of length, but often implies an absence of a definedconformation.

Interactions

Proteins can interact with many types of molecules, includingwith other proteins,with lipids,with carbohydrates,andwith DNA.[32][33][30][34]

Abundance in cells

It has been estimated that average-sizedbacteriacontain about 2 million proteins per cell (e.g.E. coliandStaphylococcus aureus). Smaller bacteria, such asMycoplasmaorspirochetescontain fewer molecules, on the order of 50,000 to 1 million. By contrast,eukaryoticcells are larger and thus contain much more protein. For instance,yeastcells have been estimated to contain about 50 million proteins andhumancells on the order of 1 to 3 billion.[35]The concentration of individual protein copies ranges from a few molecules per cell up to 20 million.[36]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 inlymphoblastoidcells.[37]

Synthesis

Biosynthesis

A ribosome produces a protein using mRNA as template
TheDNAsequence of a geneencodesthe amino acid sequence of a protein
Main article:Protein biosynthesis

Proteins are assembled from amino acids using information encoded in genes. Each protein has its own unique amino acid sequence that is specified by thenucleotidesequence of the gene encoding this protein. Thegenetic codeis a set of three-nucleotide sets calledcodonsand each three-nucleotide combination designates an amino acid, for example AUG (adenine–uracil–guanine) is the code formethionine.BecauseDNAcontains 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.[34]: 1002–42 Genes encoded in DNA are firsttranscribedinto pre-messenger RNA(mRNA) by proteins such asRNA polymerase.Most organisms then process the pre-mRNA (also known as aprimary transcript) using various forms ofpost-transcriptional modificationto form the mature mRNA, which is then used as a template for protein synthesis by theribosome.Inprokaryotesthe 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,eukaryotesmake mRNA in thecell nucleusand thentranslocateit across thenuclear membraneinto thecytoplasm,whereprotein synthesisthen takes place. The rate of protein synthesis is higher in prokaryotes than eukaryotes and can reach up to 20 amino acids per second.[38]

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 pairinganticodonlocated on atransfer RNAmolecule, 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-terminustoC-terminus.[34]: 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 domainsconstituting proteins in higher organisms.[39]For instance,yeastproteins are on average 466 amino acids long and 53 kDa in mass.[31]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.[40]

Chemical synthesis

Main article:Peptide synthesis

Short proteins can also be synthesized chemically by a family of methods known aspeptide synthesis,which rely onorganic synthesistechniques such aschemical ligationto produce peptides in high yield.[41]Chemical synthesis allows for the introduction of non-natural amino acids into polypeptide chains, such as attachment offluorescentprobes to amino acid side chains.[42]These methods are useful in laboratorybiochemistryandcell 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.[43]

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).
Main article:Protein structure
Further information:Protein structure prediction

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

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 asubstratemolecule to an enzyme'sactive site,or the physical region of the protein that participates in chemical catalysis. In solution, proteins also undergo variation in structure through thermal vibration and the collision with other molecules.[34]: 368–75 

Molecular surface of several proteins showing their comparative sizes. From left to right are:immunoglobulin G(IgG, anantibody),hemoglobin,insulin(a hormone),adenylate kinase(an enzyme), andglutamine synthetase(an enzyme).

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 aresolubleand 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 asreceptorsor provide channels for polar or charged molecules to pass through thecell membrane.[34]: 165–85 

A special case of intramolecular hydrogen bonds within proteins, poorly shielded from water attack and hence promoting their owndehydration,are calleddehydrons.[44]

Protein domains

Main article:Protein domain

Many proteins are composed of severalprotein domains,i.e. segments of a protein that fold into distinct structural units. Domains usually also have specific functions, such asenzymaticactivities (e.g.kinase) or they serve as binding modules (e.g. theSH3 domainbinds to proline-rich sequences in other proteins).

Sequence motif

Short amino acid sequences within proteins often act as recognition sites for other proteins.[45]For instance,SH3 domainstypically 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.

Protein topology

Topology of a protein describes the entanglement of the backbone and the arrangement of contacts within the folded chain.[46]Two theoretical frameworks ofknot theoryandCircuit topologyhave been applied to characterise protein topology. Being able to describe protein topology opens up new pathways for protein engineering and pharmaceutical development, and adds to our understanding of protein misfolding diseases such as neuromuscular disorders and cancer.

Cellular functions

Proteins are the chief actors within the cell, said to be carrying out the duties specified by the information encoded in genes.[31]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 colicell, whereas other macromolecules such as DNA and RNA make up only 3% and 20%, respectively.[47]The set of proteins expressed in a particular cell or cell type is known as itsproteome.

The enzymehexokinaseis shown as a conventional ball-and-stick molecular model. To scale in the top right-hand corner are two of its substrates,ATPandglucose.

The chief characteristic of proteins that also 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 siteand 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 inhibitorprotein binds to humanangiogeninwith a sub-femtomolardissociation constant(<10−15M) 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 synthetasespecific to the amino acidvalinediscriminates against the very similar side chain of the amino acidisoleucine.[48]

Proteins can bind to other proteins as well as tosmall-moleculesubstrates. When proteins bind specifically to other copies of the same molecule, they canoligomerizeto form fibrils; this process occurs often in structural proteins that consist of globular monomers that self-associate to form rigid fibers.Protein–protein interactionsalso regulate enzymatic activity, control progression through thecell cycle,and allow the assembly of largeprotein complexesthat carry out many closely related reactions with a common biological function. Proteins can also bind to, or even be integrated into, cell membranes. The ability of binding partners to induce conformational changes in proteins allows the construction of enormously complexsignalingnetworks.[34]: 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.[49][50]

Enzymes

Main article:Enzyme

The best-known role of proteins in the cell is asenzymes,whichcatalysechemical 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.[51]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).[52]

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.[53]The region of the enzyme that binds the substrate and contains the catalytic residues is known as theactive site.

Dirigent proteinsare members of a class of proteins that dictate thestereochemistryof a compound synthesized by other enzymes.[54]

Cell signaling and ligand binding

See also:Glycan-protein interactions
Ribbon diagramof a mouse antibody againstcholerathat binds acarbohydrateantigen

Many proteins are involved in the process ofcell signalingandsignal 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 proteinsthat act asreceptorswhose 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 changedetected by other proteins within the cell.[33]: 251–81 

Antibodiesare protein components of anadaptive immune systemwhose main function is to bindantigens,or foreign substances in the body, and target them for destruction. Antibodies can besecretedinto the extracellular environment or anchored in the membranes of specializedB cellsknown 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.[34]: 275–50 

Many ligand transport proteins bind particularsmall biomoleculesand transport them to other locations in the body of a multicellular organism. These proteins must have a high binding affinity when theirligandis present in high concentrations, but must also release the ligand when it is present at low concentrations in the target tissues. The canonical example of a ligand-binding protein ishaemoglobin,which transportsoxygenfrom thelungsto other organs and tissues in allvertebratesand has close homologs in every biologicalkingdom.[34]: 222–29 Lectinsaresugar-binding proteinswhich are highly specific for their sugar moieties.Lectinstypically play a role in biologicalrecognitionphenomena involving cells and proteins.[55]Receptorsandhormonesare highly specific binding proteins.

Transmembrane proteinscan also serve as ligand transport proteins that alter thepermeabilityof the cell membrane tosmall moleculesand ions. The membrane alone has ahydrophobiccore through whichpolaror charged molecules cannotdiffuse.Membrane proteins contain internal channels that allow such molecules to enter and exit the cell. Manyion channelproteins are specialized to select for only a particular ion; for example,potassiumandsodiumchannels often discriminate for only one of the two ions.[33]: 232–34 

Structural proteins

Structural proteins confer stiffness and rigidity to otherwise-fluid biological components. Most structural proteins arefibrous proteins;for example,collagenandelastinare critical components ofconnective tissuesuch ascartilage,andkeratinis found in hard or filamentous structures such ashair,nails,feathers,hooves,and someanimal shells.[34]: 178–81 Someglobular proteinscan also play structural functions, for example,actinandtubulinare globular and soluble as monomers, butpolymerizeto form long, stiff fibers that make up thecytoskeleton,which allows the cell to maintain its shape and size.

Other proteins that serve structural functions aremotor proteinssuch asmyosin,kinesin,anddynein,which are capable of generating mechanical forces. These proteins are crucial for cellularmotilityof single celled organisms and thespermof many multicellular organisms which reproducesexually.They also generate the forces exerted by contractingmuscles[34]: 258–64, 272 and play essential roles in intracellular transport.

Protein evolution

Main article:Molecular evolution

A key question in molecular biology is how proteins evolve, i.e. how canmutations(or rather changes inamino acidsequence) lead to new structures and functions? Most amino acids in a protein can be changed without disrupting activity or function, as can be seen from numeroushomologousproteins across species (as collected in specialized databases forprotein families,e.g.PFAM).[56]In order to prevent dramatic consequences of mutations, agene may be duplicatedbefore it can mutate freely. However, this can also lead to complete loss of gene function and thuspseudo-genes.[57]More commonly, single amino acid changes have limited consequences although some can change protein function substantially, especially inenzymes.For instance, many enzymes can change theirsubstrate specificityby one or a few mutations.[58]Changes in substrate specificity are facilitated bysubstrate promiscuity,i.e. the ability of many enzymes to bind and process multiplesubstrates.When mutations occur, the specificity of an enzyme can increase (or decrease) and thus its enzymatic activity.[58]Thus, bacteria (or other organisms) can adapt to different food sources, including unnatural substrates such as plastic.[59]

Methods of study

Main article:Protein methods

Methods commonly used to study protein structure and function includeimmunohistochemistry,site-directed mutagenesis,X-ray crystallography,nuclear magnetic resonanceandmass spectrometry.

The activities and structures of proteins may be examinedin vitro,in vivo,andin silico.In vitrostudies of purified proteins in controlled environments are useful for learning how a protein carries out its function: for example,enzyme kineticsstudies explore thechemical mechanismof an enzyme's catalytic activity and its relative affinity for various possible substrate molecules. By contrast,in vivoexperiments can provide information about the physiological role of a protein in the context of acellor even a wholeorganism.In silicostudies use computational methods to study proteins.

Protein purification

Main article:Protein purification

Proteins may bepurifiedfrom other cellular components using a variety of techniques such asultracentrifugation,precipitation,electrophoresis,andchromatography;the advent ofgenetic engineeringhas made possible a number of methods to facilitate purification.

To performin vitroanalysis, 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; membranelipidsand proteins; cellularorganelles,andnucleic acids.Precipitationby a method known assalting outcan concentrate the proteins from this lysate. Various types ofchromatographyare then used to isolate the protein or proteins of interest based on properties such as molecular weight, net charge and binding affinity.[30]: 21–24 The level of purification can be monitored using various types ofgel electrophoresisif the desired protein's molecular weight andisoelectric pointare known, byspectroscopyif the protein has distinguishable spectroscopic features, or byenzyme assaysif the protein has enzymatic activity. Additionally, proteins can be isolated according to their charge usingelectrofocusing.[60]

For natural proteins, a series of purification steps may be necessary to obtain protein sufficiently pure for laboratory applications. To simplify this process,genetic engineeringis 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 ofhistidineresidues (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 different tags have been developed to help researchers purify specific proteins from complex mixtures.[61]

Cellular localization

Proteins in differentcellular compartmentsand structures tagged withgreen fluorescent protein(here, white)

The study of proteinsin vivois often concerned with the synthesis and localization of the protein within the cell. Although many intracellular proteins are synthesized in thecytoplasmand membrane-bound or secreted proteins in theendoplasmic reticulum,the specifics of how proteins aretargetedto specific organelles or cellular structures is often unclear. A useful technique for assessing cellular localization uses genetic engineering to express in a cell afusion proteinorchimeraconsisting of the natural protein of interest linked to a "reporter"such asgreen fluorescent protein(GFP).[62]The fused protein's position within the cell can then be cleanly and efficiently visualized usingmicroscopy,[63]as shown in the figure opposite.

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 immunofluorescencewill allow for fluorescence colocalization and demonstration of location. Fluorescent dyes are used to label cellular compartments for a similar purpose.[64]

Other possibilities exist, as well. For example,immunohistochemistryusually 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. Another applicable technique is cofractionation in sucrose (or other material) gradients usingisopycnic centrifugation.[65]While this technique does not prove colocalization of a compartment of known density and the protein of interest, it does increase the likelihood, and is more amenable to large-scale studies.

Finally, the gold-standard method of cellular localization isimmunoelectron microscopy.This technique also 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.[66]

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,[67]and may allow the rationaldesignof new proteins with novel properties.[68]

Proteomics

Main article:Proteomics

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,[69]which allows the separation of many proteins,mass spectrometry,[70]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.[71]The total complement of biologically possible such interactions is known as theinteractome.[72]A systematic attempt to determine the structures of proteins representing every possible fold is known asstructural genomics.[73]

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 seenunder alight microscope,other methods have to be employed to determine their structure. Common experimental methods includeX-ray crystallographyandNMR spectroscopy,both of which can produce structural information atatomicresolution. 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 geometryproblem.Dual polarisation interferometryis a quantitative analytical method for measuring the overallprotein conformationandconformational changesdue to interactions or other stimulus.Circular dichroismis another laboratory technique for determining internal β-sheet / α-helical composition of proteins.Cryoelectron microscopyis used to produce lower-resolution structural information about very large protein complexes, including assembledviruses;[33]: 340–41 a variant known aselectron crystallographycan also produce high-resolution information in some cases, especially for two-dimensional crystals of membrane proteins.[74]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 coordinatesfor each atom in the protein.[75]

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 tocrystallizein preparation for X-ray crystallography. Membrane proteins and large protein complexes, by contrast, are difficult to crystallize and are underrepresented in the PDB.[76]Structural genomicsinitiatives have attempted to remedy these deficiencies by systematically solving representative structures of major fold classes.Protein structure predictionmethods attempt to provide a means of generating a plausible structure for proteins whose structures have not been experimentally determined.[77]

Structure prediction

Constituent amino-acids can be analyzed to predict secondary, tertiary and quaternary protein structure, in this case hemoglobin containinghemeunits
Main articles:Protein structure predictionandList of protein structure prediction software

Complementary to the field of structural genomics,protein structure predictiondevelops efficientmathematical modelsof proteins to computationally predict the molecular formations in theory, instead of detecting structures with laboratory observation.[78]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.[79]Although producing accurate models remains a challenge when only distantly related template structures are available, it has been suggested thatsequence alignmentis the bottleneck in this process, as quite accurate models can be produced if a "perfect" sequence alignment is known.[80]Many structure prediction methods have served to inform the emerging field ofprotein engineering,in which novel protein folds have already been designed.[81]Also proteins (in eukaryotes ~33%) contain large unstructured but biologically functional segments and can be classified asintrinsically disordered proteins.[82]Predicting and analysing protein disorder is, therefore, an important part of protein structure characterisation.[83]

Bioinformatics

Main article:Bioinformatics

A vast array of computational methods have been developed to analyze the structure, function and evolution of proteins. The development of such tools has been driven by the large amount of genomic and proteomic data available for a variety of organisms, including thehuman genome.It is simply impossible to study all proteins experimentally, hence only a few are subjected to laboratory experiments while computational tools are used to extrapolate to similar proteins. Suchhomologous proteinscan be efficiently identified in distantly related organisms bysequence alignment.Genome and gene sequences can be searched by a variety of tools for certain properties.Sequence profiling toolscan findrestriction enzymesites,open reading framesinnucleotidesequences, and predictsecondary structures.Phylogenetic treescan be constructed andevolutionaryhypotheses developed using special software likeClustalWregarding the ancestry of modern organisms and the genes they express. The field ofbioinformaticsis now indispensable for the analysis of genes and proteins.

In silico simulation of dynamical processes

A more complex computational problem is the prediction of intermolecular interactions, such as inmolecular docking,[84]protein folding,protein–protein interactionand chemical reactivity. Mathematical models to simulate these dynamical processes involvemolecular mechanics,in particular,molecular dynamics.In this regard,in silicosimulations discovered the folding of small α-helicalprotein domainssuch as thevillinheadpiece,[85]theHIVaccessory protein[86]and hybrid methods combining standard molecular dynamics withquantum mechanicalmathematics have explored the electronic states ofrhodopsins.[87]

Beyond classical molecular dynamics,quantum dynamicsmethods 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(MCTDH) method and thehierarchical equations of motion(HEOM) approach, which have been applied to plant cryptochromes[88]and bacteria light-harvesting complexes,[89]respectively. Both quantum and classical mechanical simulations of biological-scale systems are extremely computationally demanding, sodistributed computinginitiatives (for example, theFolding@homeproject[90]) facilitate themolecular modelingby exploiting advances inGPUparallel processing andMonte Carlotechniques.

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 methodis applied. More sensitive methods are available.[91][92]

Nutrition

Further information:Protein (nutrient)andProtein quality

Mostmicroorganismsand plants can biosynthesize all 20 standardamino acids,while animals (including humans) must obtain some of the amino acids from thediet.[47]The amino acids that an organism cannot synthesize on its own are referred to asessential amino acids.Key enzymes that synthesize certain amino acids are not present in animals—such asaspartokinase,which catalyses the first step in the synthesis oflysine,methionine,andthreoninefromaspartate.If amino acids are present in the environment, microorganisms can conserve energy by taking up the amino acids from their surroundings anddownregulatingtheir biosynthetic pathways.

In animals, amino acids are obtained through the consumption of foods containing protein. Ingested proteins are then broken down into amino acids throughdigestion,which typically involvesdenaturationof the protein through exposure toacidandhydrolysisby enzymes calledproteases.Some ingested amino acids are used for protein biosynthesis, while others are converted toglucosethroughgluconeogenesis,or fed into thecitric acid cycle.This use of protein as a fuel is particularly important understarvationconditions as it allows the body's own proteins to be used to support life, particularly those found inmuscle.[93]

In animals such asdogsandcats,protein maintains the health and quality of the skin by promoting hair follicle growth and keratinization, and thus reducing the likelihood of skin problems producing malodours.[94]Poor-quality proteins also have a role regarding gastrointestinal health, increasing the potential for flatulence and odorous compounds in dogs because when proteins reach the colon in an undigested state, they are fermented producing hydrogen sulfide gas, indole, and skatole.[95]Dogs and cats digest animal proteins better than those from plants, but products of low-quality animal origin are poorly digested, including skin, feathers, and connective tissue.[95]

Mechanical Properties

Themechanical propertiesof proteins are highly diverse and are often central to their biological function, as in the case of proteins likekeratinandcollagen.[96]For instance, the ability ofmuscle tissueto continually expand and contract is directly tied to the elastic properties of their underlying protein makeup.[97][98]Beyond fibrous proteins, the conformational dynamics ofenzymes[99]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.[100][101]

Young's Modulus

Young's modulus,E,is calculated as the axial stress σ over the resulting strain ε. It is a measure of the relativestiffnessof 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,[102]which is though to give elasticity to structures such asblood vessels,pulmonary tissue,andbladder tissue,among others.[103][104]In comparison to this,globular proteins,such asBovine Serum Albumin,which float relatively freely in thecytosoland often function as enzymes (and thus undergoing frequent conformational changes) have comparably much lower Young's moduli.[105][106]

The Young's modulus of a single protein can be found throughmolecular dynamicssimulation. Using either atomistic force-fields, such asCHARMMorGROMOS,or coarse-grained forcefields like Martini,[107]a single protein molecule can be stretched by a uniaxial force while the resulting extension is recorded in order to calculate the strain.[108][109]Experimentally, methods such asatomic force microscopycan be used to obtain similar data.[110]

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.

Elasticity of Various Proteins
Protein Protein Class Young's modulus
Keratin (Cross-Linked) Fibrous 1.5-10 GPa[111]
Elastin (Cross-Linked) Fibrous 1 MPa[102]
Fibrin (Cross-linked) Fibrous 1-10 MPa[102]
Collagen (Cross-linked) Fibrous 5-7.5 GPa[102][112]
Resilin (Cross-Linked) Fibrous 1-2 MPa[102]
Bovine Serum Albumin (Cross-Linked) Globular 2.5-15 KPa[105]
β-Barrel Outer Membrane Proteins Membrane 20-45 GPa[113]

Viscosity

In addition to serving as enzymes within the cell,globular proteinsoften act as key transport molecules. For instance,Serum Albumins,a key component ofblood,are necessary for the transport of a multitude of small molecules throughout the body.[114]Because of this, the concentration dependent behavior of these proteins in solution is directly tied to the function of thecirculatory system.On way of quantifying this behavior is through theviscosityof the solution.

Viscosity, η, is generally given is a measure of a fluid's resistance to deformation. It can be calculated as the ratio between the applied stress and the rate of change of the resulting shear strain, that is, the rate of deformation. Viscosity of complex liquid mixtures, such as blood, often depends strongly on temperature and solute concentration.[115]For serum albumin, specificallybovine serum albumin,the following relation between viscosity andtemperatureandconcentrationcan be used.[116]

Wherecis the concentration,Tis the temperature,Ris thegas constant,and α, β,B,D,and ΔEare all material-based property constants. This equation has the form of anArrhenius equation,assigning viscosity an exponential dependence on temperature and concentration.

See also

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Further reading

Textbooks
  • Branden C, Tooze J (1999).Introduction to Protein Structure.New York: Garland Pub.ISBN978-0-8153-2305-1.
  • Murray RF, Harper HW, Granner DK, Mayes PA, Rodwell VW (2006).Harper's Illustrated Biochemistry.New York: Lange Medical Books/McGraw-Hill.ISBN978-0-07-146197-9.
  • Van Holde KE, Mathews CK (1996).Biochemistry.Menlo Park, California: Benjamin/Cummings Pub. Co., Inc.ISBN978-0-8053-3931-4.

Databases and projects

Tutorials and educational websites

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