protien structure
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Primary structure
The sequence of the different amino acids is called the primary structure of the peptide or protein. Counting of residues always starts at the N-terminal end (NH2-
group), which is the end where the amino group is not involved in a peptide bond. The primary structure of a protein is determined by the gene corresponding to
the protein. A specific sequence of nucleotides in DNA is transcribed into mRNA, which is read by the ribosome in a process called translation. The sequence of a
protein is unique to that protein, and defines the structure and function of the protein. The sequence of a protein can be determined by methods such as Edman
degradation or tandem mass spectrometry. Often however, it is read directly from the sequence of the gene using the genetic code. Post-transcriptional
modifications such as disulfide formation, phosphorylations and glycosylations are usually also considered a part of the primary structure, and cannot be read from
the gene.
[edit]Secondary structure
By building models of peptides using known information about bond lengths and angles, the first elements of secondary structure, the alpha helix and the beta
sheet, were suggested in 1951 by Linus Pauling and coworkers.<ref name = pauling51>PAULING L, COREY RB, BRANSON HR. Proc Natl Acad Sci U S A. 1951
Apr;37(4):205-11. The structure of proteins; two hydrogen-bonded helical configurations of the polypeptide chain. PMID 14816373</ref> Both the alpha helix and
the beta-sheet represent a way of saturating all the hydrogen bond donors and acceptors in the peptide backbone. These secondary structure elements only
depend on properties that all the residues have in common, explaining why they occur frequently in most proteins. Since then other elements of secondary
structure have been discovered such as various loops and other forms of helices. The part of the backbone that is not in a regular secondary structure is said to
be random coil. Each of these two secondary structure elements have a regular geometry, meaning they are constrained to specific values of the dihedral angles
ψ and φ. Thus they can be found in a specific region of the Ramachandran plot.
Image :Helices.png
The left panel shows the hydrogen bonding in an actual α-helix backbone. Note that the nth residue O (Lys 143) bonds to the (n+4)th following residue's N (Arg 147). The actual values of some
displayed H-bond distances give you some idea about the variations to expect within a helix. The center panel includes the side chains which were omitted in the left panel for clarity. You see
the side chains pointing towards the N-terminal of the chain (lower residue numbers) and thus it is usually possible to determine the direction of the helix quite well during initial model building.
A 0.2 nm electron density is shown in the right panel
Here are some more representation of the same helix.
Image:Alpha-h2.png
Backbone
Image:Alpha-h3.png
Secondary structure cartoon ("ribbon" or "linguini diagram")
Turns, loops and a few other secondary structure elements such as a 3-10 helix complete the picture. We have now enough pieces to assemble a complete
protein, displaying its typical tertiary structure.
[edit]Tertiary structure
The elements of secondary structure are usually folded into a compact shape using a variety of loops and turns. The formation of tertiary structure is usually driven
by the burial of hydrophobic residues, but other interactions such as hydrogen bonding, ionic interactions and disulfide bonds can also stabilize the tertiary
structure. The tertiary structure encompasses all the noncovalent interactions that are not considered secondary structure, and is what defines the overall fold of
the protein, and is usually indispensable for the function of the protein.
[edit]Quaternary structure
The quaternary structure is the interaction between several chains of peptide bonds. The individual chains are called subunits. The individual subunits are not
necessarily covalently connected, but might be connected by a disulfide bond. Not all proteins have quaternary structure, since they might be functional as
monomers. The quaternary structure is stabilized by the same range of interactions as the tertiary structure. Complexes of two or more polypeptides (i.e. multiple
subunits) are called multimers. Specifically it would be called a dimer if it contains two subunits, a trimer if it contains three subunits, and a tetramer if it contains
four subunits. Multimers made up of identical subunits may be referred to with a prefix of "homo-" (e.g. a homotetramer) and those made up of different subunits
may be referred to with a prefix of "hetero-" (e.g. a heterodimer). Tertiary structures vary greatly from one protein to another. They are held together by glycosydic
and covalent bonds.
Primary structure
Main article: Protein primary structure
The primary structure refers to amino acid sequence of the polypeptide chain. The primary structure is held together by covalentor peptide bonds, which are made
during the process of protein biosynthesis or translation. The two ends of the polypeptide chain are referred to as the carboxyl terminus (C-terminus) and the
amino terminus (N-terminus) based on the nature of the free group on each extremity. Counting of residues always starts at the N-terminal end (NH2-group), which
is the end where the amino group is not involved in a peptide bond. The primary structure of a protein is determined by the gene corresponding to the protein. A
specific sequence of nucleotides in DNA is transcribed into mRNA, which is read by the ribosome in a process called translation. The sequence of a protein is
unique to that protein, and defines the structure and function of the protein. The sequence of a protein can be determined by methods such as Edman
degradation or tandem mass spectrometry. Often however, it is read directly from the sequence of the gene using thegenetic code. Post-translational modifications
such as disulfide formation, phosphorylations and glycosylations are usually also considered a part of the primary structure, and cannot be read from the gene.
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[edit]Amino acid residues
Main article: Amino acid
Main article: Proteinogenic amino acid
Each α-amino acid consists of a backbone part that is present in all the amino acid types, and a side chain that is unique to each type of residue. An exception
from this rule is proline. Because the carbon atom is bound to four different groups it is chiral, however only one of the isomers occur in biological proteins. Glycine
however, is not chiral since its side chain is a hydrogen atom. A simple mnemonic for correct L-form is "CORN": when the Cα atom is viewed with the H in front, theresidues read "CO-R-N" in a c lockwise direction.
[edit]Secondary structure
An alpha-helix with hydrogen bonds (yellow dots)
Main article: Protein secondary structure
Secondary structure refers to highly regular local sub-structures. Two main types of secondary structure, the alpha helix and the beta strand, were suggested in1951 by Linus Pauling and coworkers.[4] These secondary structures are defined by patterns of hydrogen bonds between the main-chain peptide groups. They
have a regular geometry, being constrained to specific values of the dihedral angles ψ and φ on the Ramachandran plot . Both the alpha helix and the beta-sheet
represent a way of saturating all the hydrogen bond donors and acceptors in the peptide backbone. Some parts of the protein are ordered but do not form any
regular structures. They should not be confused with random coil, an unfolded polypeptide chain lacking any fixed three-dimensional structure. Several sequential
secondary structures may form a "supersecondary unit ".[5]
[edit]Tertiary structure
Main article: Protein tertiary structure
Tertiary structure refers to three-dimensional structure of a single protein molecule. The alpha-helices and beta-sheets are folded into a compact globule. The
folding is driven by the non-specific hydrophobic interactions (the burial of hydrophobic residues from water), but the structure is stable only when the parts of a
protein domain are locked into place by specific tertiary interactions, such assalt bridges, hydrogen bonds, and the tight packing of side chains and disulfide bonds.
The disulfide bonds are extremely rare in cytosolic proteins, since the cytosol is generally a reducing environment.
[edit]Quaternary structure
Main article: Protein quaternary structure
Quaternary structure is a larger assembly of several protein molecules or polypeptide chains, usually called subunits in this context. The quaternary structure is
stabilized by the same non-covalent interactions and disulfide bonds as the tertiary structure. Complexes of two or more polypeptides (i.e. multiple subunits) are
called multimers. Specifically it would be called a dimer if it contains two subunits, a trimer if it contains three subunits, and a tetramer if it contains four subunits.
The subunits are frequently related to one another by symmetry operations, such as a 2-fold axis in a dimer. Multimers made up of identical subunits are referred
to with a prefix of "homo-" (e.g. a homotetramer) and those made up of different subunits are referred to with a prefix of "hetero-" (e.g. a heterotetramer, such as
the two alpha and two beta chains of hemoglobin).
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Proteins are polymers of amino acids covalently linked through peptide bonds into achain. Within and outside of cells, proteins serve a myriad of functions, including structuralroles (cytoskeleton), as catalysts (enzymes), transporter to ferry ions and molecules acrossmembranes, and hormones to name just a few.
With few exceptions, biotechnology is about understanding, modifying and ultimately
exploiting proteins for new and useful purposes. To accomplish these goals, one would like tohave a firm grasp of protein structure and how structure relates to function. This goal is, of course, much easier to articulate than to realize! The objective of this brief review is tosummarize only the fundamental concepts of protein structure.
Amino Acids
Proteins are polymers of amino acids joined together by peptidebonds. There are 20 different amino acids that make up essentially all proteinson earth. Each of these amino acids has a fundamental design composed of acentral carbon (also called the alpha carbon) bonded to:
• a hydrogen
• a carboxyl group
• an amino group
• a unique side chain or R-group
Thus, the characteristic that distinguishes one amino acid from another is its unique sidechain, and it is the side chain that dictates an amino acids chemical properties.Examples of three amino acids are shown below, and structures of all 20 are available. Note that the aminoacids are shown with the amino and carboxyl groups ionized, as they are at physiologic pH.
Except for glycine, which has a hydrogen as its R-group, there is asymmetry about the alphacarbon in all amino acids. Because of this, all amino acids except glycine can exist in either of
two mirror-image forms. The two forms - called stereoisomers - are referred to as D and L aminoacids. With rare exceptions, all of the amino acids in proteins are L amino acids.
The unique side chains confer unique chemical properties on amino acids, and dictate howeach amino acid interacts with the others in a protein. Amino acids can thus be classified as being hydrophobic versus hydrophilic, and uncharged versus positively-charged versusnegatively-charged . Ultimately, the three dimensional conformation of a protein - and its activity- is determined by complex interactions among side chains. Some aspects of protein structure can be deduced by examining the properties of clusters of amino acids. For example, a computer program that plots the hydrophobicity profile is often used to predict membrane-spanningregions of a protein or regions that are likely to be immunogenic.
Peptides and Proteins
Amino acids are covalently bonded together in chains by peptide bonds. If the chain length isshort (say less than 30 amino acids) it is called a peptide; longer chains are called polypeptides or proteins. Peptide bonds are formed between the carboxyl group of one amino acid and theamino group of the next amino acid. Peptide bond formation occurs in a condensation reactioninvolving loss of a molecule of water.
The head-to-tail arrangment of amino acids in a protein means that there is a amino group on oneend (called the amino-terminus or N-terminus) and a carboxyl group on the other end (carboxyl-
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terminus or C-terminus). The carboxy-terminal amino acid corresponds to the last one addedto the chain during translation of the messenger RNA.
Levels of Protein Structure
Structural features of proteins are usually described at four levels of complexity:
• Primary structure: the linear arrangment of amino acids in a protein and
the location of covalent linkages such as disulfide bonds between aminoacids.
• Secondary structure: areas of folding or coiling within a protein; examplesinclude alpha helices and pleated sheets, which are stabilized by hydrogenbonding.
• Tertiary structure: the final three-dimensional structure of a protein, whichresults from a large number of non-covalent interactions between aminoacids.
• Quaternary structure: non-covalent interactions that bind multiple
polypeptides into a single, larger protein. Hemoglobin has quaternarystructure due to association of two alpha globin and two beta globinpolyproteins.
• The primary structure of a protein can readily be deduced from the nucleotidesequence of the corresponding messenger RNA. Based on primary structure,many features of secondary structure can be predicted with the aid of computer
programs. However, predicting protein tertiary structure remains a very tough problem, although some progress has been made in this important area.
Structure of protein
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A Ramachandran plot generated from the protein PCNA, a human DNA clamp protein that is composed of both beta sheets and alpha helices(PDB ID 1AXC). Points that lie on the axes
indicateN - and C-terminal residues for each subunit. The green regions show possible angle formations that include glycine, while the blue areas are for formations that don't include glycine.
[edit]Primary structure of protein
Ramachandran diagram (φ,ψ plot), with data points for α-helical residues forming a dense diagonal cluster below and left of center, around the global energy minimum for backbone
conformation.[14]
The proposal that proteins were linear chains of α-amino acids was made nearly simultaneously by two scientists at the same conference in 1902, the 74th
meeting of the Society of German Scientists and Physicians, held in Karlsbad. Franz Hofmeister madethe proposal in the morning, based on his observations of
the biuret reaction in proteins. Hofmeister was followed a few hours later by Emil Fischer , who had amassed a wealth of chemical details supporting the peptide-
bond model. For completeness, the proposal that proteins contained amide linkages was made as early as 1882 by the French chemist E. Grimaux[15].
Despite these data and later evidence that proteolytically digested proteins yielded only oligopeptides, the idea that proteins were linear, unbranched polymers of
amino acids was not accepted immediately. Some well-respected scientists such as William Astbury doubted that covalent bonds were strong enough to hold such
long molecules together; they feared that thermal agitations would shake such long molecules asunder. Hermann Staudinger faced similar prejudices in the 1920s
when he argued that rubber was composed of macromolecules. Thus, several alternative hypotheses arose. The colloidal protein hypothesis stated that
proteins were colloidal assemblies of smaller molecules. This hypothesis was disproved in the 1920s by ultracentrifugation measurements by Theodor Svedberg
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that showed that proteins had a well-defined, reproducible molecular weight and by electrophoretic measurements by Arne Tiselius that indicated that proteins
were single molecules.
A second hypothesis, the cyclol hypothesis advanced by Dorothy Wrinch, proposed that the linear polypeptide underwent a chemical cyclol rearrangement C=O
+ HN C(OH)-N that crosslinked its backbone amide groups, forming a two-dimensional fabric. Other primary structures of proteins were proposed by various
researchers, such as the diketopiperazine model of Emil Abderhalden and the pyrrol/piperidine model of Troensegaard in 1942. Although never given much
credence, these alternative models were finally disproved when Frederick Sanger successfully sequenced insulin and by the crystallographic determination of
myoglobin and hemoglobin by Max Perutz and John Kendrew.
The primary structure of peptides and proteins refers to the linear sequence of its amino acid structural units. The term "primary structure" was first coined by
Linderstrøm-Lang in 1951. By convention, the primary structure of a protein is reported starting from the amino-terminal (N) end to the carboxyl-terminal (C) end.
The post-translational modifications of protein such as disulfide formation, phosphorylations and glycosylations are usually also considered a part of the primary
structure, and cannot be read from the gene.
[edit]Secondary structure of protein
The Hemoglobin molecule has four heme-binding subunits, each largely made of alpha helices.
Secondary structure refers to highly regular local sub-structures. Two main types of secondary structure, the alpha helix and the beta strand, were suggested in
1951 by Linus Pauling'and coworkers.[16]. These secondary structures are defined by patterns of hydrogen bonds between the main-chain peptide groups. They
have a regular geometry, being constrained to specific values of the dihedral angles ψ and φ on the Ramachandran plot . Both the alpha helix and the beta-sheetrepresent a way of saturating all the hydrogen bond donors and acceptors in the peptide backbone. Some parts of the protein are ordered but do not form any
regular structures. They should not be confused with random coil, an unfolded polypeptide chain lacking any fixed three-dimensional structure. Several sequential
secondary structures may form a "supersecondary unit ".[17]
Beta-meander motif
Portion of outer surface Protein A o f Borrelia burgdorfe ri complexed with a murine monoclonal antibody.
Amino acids vary in their ability to form the various secondary structure elements. Proline and glycine are sometimes known as "helix breakers" because they
disrupt the regularity of the α helical backbone conformation; however, both have unusual conformational abilities and are commonly found in turns. Amino acids
that prefer to adopt helical conformations in proteins include methionine, alanine, leucine, glutamate and lysine ( "MALEK" in amino-acid 1-letter codes); by
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contrast, the large aromatic residues (tryptophan, tyrosine and phenylalanine) and Cβ-branched amino acids (isoleucine, valine, and threonine) prefer to adopt β-
strand conformations. However, these preferences are not strong enough to produce a reliable method of predicting secondary structure from sequence
alone.Secondary structure in proteins consists of local inter-residue interactions mediated by hydrogen bonds, or not. The most common secondary structures are
alpha helices and beta sheets. Other helices, such as the 310 helix and π helix, are calculated to have energetically favorable hydrogen-bonding patterns but are
rarely if ever observed in natural proteins except at the ends of α helices due to unfavorable backbone packing in the center of the helix.
A Ramachandran plot (also known as a Ramachandran map or a Ramachandran diagram or a [φ,ψ] plot), developed by Gopalasamudram Narayana Ramachandran and
Viswanathan Sasisekharan is a way to visualize dihedral angles ψ against φ of amino acid residues in protein structure. [18]. It shows the possible conformations of ψ and φ
angles for a polypeptide. Mathematically, the Ramachandran plot is the visualization of a function . The domain
of this function is the torus. Hence, the conventional Ramachandran plot is a projection of the torus on the plane, resulting in a distorted view and the presence of discontinuities.
One would expect that larger side chains would result in more restrictions and consequently a smaller allowable region in the Ramachandran plot. In practice this does not
appear to be the case; only the methylene group at the α position has an influence. Glycine has a hydrogen atom, with a smaller van der Waals radius, instead of a methyl
group at the α position. Hence it is least restricted and this is apparent in the Ramachandran plot for glycine for which the allowable area is considerably larger. In contrast, the
Ramachandran plot for proline shows only a very limited number of possible combinations of ψ and φ. The Ramachandran plot was calculated just before the first protein
structures at atomic resolution were determined. Forty years later there were tens of thousands of high-resolution protein structures determined by X-ray crystallography and
deposited in the Protein Data Bank (PDB). From one thousand different protein chains, Ramachandran plots of over 200 000 amino acids were plotted, showing some
significant differences, especially for glycine (Hovmöller et al. 2002). The upper left region was found to be split into two; one to the left containing amino acids in beta sheets
and one to the right containing the amino acids in random coil of this conformation. One can also plot the dihedral angles in polysaccharides and other polymers in this fashion.
For the first two protein side-chain dihedral angles a similar plot is the Janin Plot.
α helix
The amino acids in an α helix are arranged in a right-handed helical structure where each amino acid residue corresponds to a 100° turn in the helix (i.e., the helix
has 3.6 residues per turn), and a translation of 1.5 Å (0.15 nm) along the helical axis. (Short pieces of left-handed helix sometimes occur with a large content of
achiral glycine amino acids, but are unfavorable for the other normal, biological L-amino acids.) The pitch of the alpha-helix (the vertical distance between one
consecutive turn of the helix) is 5.4 Å (0.54 nm) which is the product of 1.5 and 3.6. What is most important is that the N-H group of an amino acid forms a
hydrogen bond with the C=O group of the amino acid four residues earlier; this repeated hydrogen bonding is the most prominent characteristic of an α-helix.
Official international nomenclature specifies two ways of defining α-helices, rule 6.2 in terms of repeating φ,ψ torsion angles and rule 6.3 in terms of the combined
pattern of pitch and hydrogen bonding. Different amino-acid sequences have different propensities for forming α-helical structure. Methionine, alanine, leucine,
uncharged glutamate, and lysine ("MALEK" in the amino-acid 1-letter codes) all have especially high helix-forming propensities, whereas proline and glycine have
poor helix-forming propensities. Proline either breaks or kinks a helix, both because it cannot donate an amide hydrogen bond (having no amide hydrogen), and
also because its sidechain interferes sterically with the backbone of the preceding turn - inside a helix, this forces a bend of about 30° in the helix axis.[9] However,
proline is often seen as the first residue of a helix, presumably due to its structural rigidity. At the other extreme, glycine also tends to disrupt helices because its
high conformational flexibility makes it entropically expensive to adopt the relatively constrained α-helical structure[19].
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Representation of abeta hairpin
Greek-key motif in protein structure.
β sheet The first β sheet structure was proposed by William Astbury in the 1930s. He proposed the idea of hydrogen bonding between the peptide bonds of
parallel or antiparallel extended β strands. However, Astbury did not have the necessary data on the bond geometry of the amino acids in order to build accurate
models, especially since he did not then know that the peptide bond was planar. A refined version was proposed by Linus Pauling and Robert Corey in 1951.
The β sheet (also β-pleated sheet) is the second form of regular secondary structure in proteins, only somewhat less common than alpha helix. Beta sheets
consist of beta strands connected laterally by at least two or three backbone hydrogen bonds, forming a generally twisted, pleated sheet. A beta s trand (also β
strand) is a stretch of polypeptide chain typically 3 to 10 amino acids long with backbone in an almost fully extended conformation.
A very simple structural motif involving β sheets is the β hairpin, in which two antiparallel strands are linked by a short loop of two to five residues, of which one is
frequently a glycine or a proline, both of which can assume the unusual dihedral-angle conformations required for a tight turn. However, individual strands can also
be linked in more elaborate ways with long loops that may contain alpha helices or even entire protein domains.
Greek key motif The Greek key motif consists of four adjacent antiparallel strands and their linking loops. It consists of three antiparallel strands connected by
hairpins, while the fourth is adjacent to the first and linked to the third by a longer loop. This type of structure forms easily during the protein folding process.[20][21] It
was named after a pattern common to Greek ornamental artwork (see meander (art)).
The β-α-β motif Due to the chirality of their component amino acids, all strands exhibit a "right-handed" twist evident in most higher-order β sheet structures. In
particular, the linking loop between two parallel strands almost always has a right-handed crossover chirality, which is strongly favored by the inherent twist of the
sheet. This linking loop frequently contains a helical region, in which case it is called a β-α-β motif. A closely related motif called a β-α-β-α motif forms the basic
component of the most commonly observed protein tertiary structure, the TIM barrel.
β-meander motif A simple supersecondary protein topology composed of 2 or more consecutive antiparallel β-strands linked together by hairpin loops.[22][23] This
motif is common in β-sheets and can be found in several structural architectures including β-barrels and β-propellers.
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Psi-loop motif
Portion of Carboxypept idase A.
Psi-loop motif The psi-loop, Ψ-loop, motif consists of two antiparallel strands with one strand in between that is connected to both by hydrogen bonds.[24] There are
four possible strand topologies for single Ψ-loops as cited by Hutchinson et al. (1990). This motif is rare as the process resulting in its formation seems unlikely to
occur during protein folding. The Ψ-loop was first identified in the aspartic protease family.[25]
Coiled coils
The possibility of coiled coils for α-keratin was proposed by Francis Crick in 1952 as well as mathematical methods for determining their structure. Remarkably,
this was soon after the structure of the alpha helix was suggested in 1951 by Linus Pauling and coworkers.
Coiled coils usually contain a repeated pattern, hxxhcxc , of hydrophobic (h) and charged (c) amino-acid residues, referred to as a heptad repeat. The positions in
the heptad repeat are usually labeled abcdefg, where a and d are the hydrophobic positions, often being occupied by isoleucine, leucine or valine. Folding a
sequence with this repeating pattern into an alpha-helical secondary structure causes the hydrophobic residues to be presented as a 'stripe' that coils gentlyaround the helix in left-handed fashion, forming an amphipathic structure. The most favorable way for two such helices to arrange themselves in the water-filled
environment of the cytoplasm is to wrap the hydrophobic strands against each other sandwiched between the hydrophilic amino acids. It is thus the burial of
hydrophobic surfaces, that provides the thermodynamic driving force for the oligomerization. The packing in a coiled-coil interface is exceptionally tight, with almost
complete van der Waals contact between the side chains of the a and d residues. This tight packing was originally predicted by Francis Crick in 1952 and is
referred to as Knobs into holes packing. The α-helices may be parallel or anti-parallel, and usually adopt a left-handed super-coil. Although disfavored, a few right-
handed coiled coils have also been observed in nature and in designed proteins[26].
Structural features of the three major forms of protein helices[27]
Geometry attribute α-helix 310 helix π-helix
Residues per turn 3.6 3.0 4.4
Translation per residue 1.5Å 2.0Å 1.1Å
Radius of helix 2.3Å 1.9Å 2.8Å
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Pitch 5.4Å 6.0Å 4.8Å
The four levels of protein structure, from top to bottom: primary structure, secondary structure (β-sheet left, right α-helix), tertiary and quaternary structure.
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[edit]Tertiary structure of protein
Tertiary structure is considered to be largely determined by the protein's primary structure - the sequence of amino acids of which it is composed. Efforts to predict
tertiary structure from the primary structure are known generally as protein structure prediction. However, the environment in which a protein is synthesized and
allowed to fold are significant determinants of its final shape and are usually not directly taken into account by current prediction methods.
In globular proteins, tertiary interactions are frequently stabilized by the sequestration of hydrophobic amino acid residues in the protein core, from which water is
excluded, and by the consequent enrichment of charged or hydrophilic residues on the protein's water-exposed surface. In secreted proteins that do not spend
time in the cytoplasm, disulfide bonds between cysteine residues help to maintain the protein's tertiary structure. A variety of common and s table tertiary structures
appear in a large number of proteins that are unrelated in both function and evolution - for example, many proteins are shaped like a TIM barrel, named for theenzyme triosephosphateisomerase. Another common structure is a highly stable dimeric coiled coil structure composed of 2-7 alpha helices.
The majority of protein structures known to date have been solved with the experimental technique of X-ray crystallography, which typically provides data of high
resolution but provides no time-dependent information on the protein's conformational flexibility. A second common way of solving protein structures uses NMR,
which provides somewhat lower-resolution data in general and is limited to relatively small proteins, but can provide time-dependent information about the motion
of a protein in solution. Dual polarisation interferometry is a time resolved analytical method for determining the overall conformation and conformational changes
in surface captured proteins providing complementary information to these high resolution methods. More is known about the tertiary structural features of soluble
globular proteins than about membrane proteins because the latter class is extremely difficult to study using these methods[28].
[edit]Quaternary structure of proteins
Several proteins are actually assemblies of more than one polypeptide chain, which in the context of the larger assemblage are known as protein subunits. In
addition to the tertiary structure of the subunits, multiple-subunit proteins possess a quaternary structure, which is the arrangement into which the subunits
assemble. Enzymes composed of subunits with diverse functions are sometimes called holoenzymes, in which some parts may be known as regulatory subunits
and the functional core is known as the catalytic subunit. Examples of proteins with quaternary structure include hemoglobin, DNA polymerase, and ion channels.
Other assemblies referred to instead as multiprotein complexes also possess quaternary structure. Examples include nucleosomes and microtubules.
Changes in quaternary structure can occur through conformational changes within individual subunits or through reorientation of the subunits relative to each
other. It is through such changes, which underlie cooperativity and allostery in "multimeric" enzymes, that many proteins undergo regulation and perform their
physiological function. The above definition follows a classical approach to biochemistry, established at times when the distinction between a protein and a
functional, proteinaceous unit was difficult to elucidate. More recently, people refer to protein-protein interaction when discussing quaternary structure of proteins
and consider all assemblies of proteins as protein complexes[29].
[edit]Protein structure determination
Ribbon diagram of the structure of myoglobin, showing colored alpha helices. Such proteins are long, linear molecules with thousands of atoms; yet the relative position of each atom has been
determined with sub-atomic resolution by X-ray crystallography. Since it is difficult to visualize all the atoms at once, the ribbon shows the rough path of the protein polymer from its N-terminus
(blue) to its C-terminus (red).
Around 90% of the protein structures available in the Protein Data Bank have been determined by X-ray crystallography. This method allows one to measure the
3D density distribution of electrons in the protein (in the crystallized state) and thereby infer the 3D coordinates of all the atoms to be determined to a certain
resolution. Roughly 9% of the known protein structures have been obtained by Nuclear Magnetic Resonance techniques. The secondary structure composition
can be determined via circular dichroism or dual polarisation interferometry. Cryo-electron microscopy has recently become a means of determining protein
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structures to high resolution (less than 5 angstroms or 0.5 nanometer) and is anticipated to increase in power as a tool for high resolution work in the next decade.
This technique is still a valuable resource for researchers working with very large protein complexes such as virus coat proteins and amyloid fibers.
[edit]X-ray crystallography
X-ray crystallography of biological molecules took off with Dorothy Crowfoot Hodgkin, who solved the structures of cholesterol (1937), vitamin B12 (1945)
and penicillin (1954), for which she was awarded the Nobel Prize in Chemistry in 1964. In 1969, she succeeded in solving the structure of insulin, on which she
worked for over thirty years.[30]
X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and diffracts into many
specific directions. Crystal structures of proteins (which are irregular and hundreds of times larger than cholesterol) began to be solved in the late 1950s, beginning
with the structure of sperm whale myoglobin by Max Perutzand Sir John Cowdery Kendrew, for which they were awarded the Nobel Prize in Chemistry in 1962.[31] Since that success, over 61840 X-ray crystal structures of proteins, nucleic acids and other biological molecules have been determined.[32] For comparison, the
nearest competing method in terms of structures analyzed isnuclear magnetic resonance (NMR) spectroscopy, which has resolved 8759 chemical structures.[33] Moreover, crystallography can solve structures of arbitrarily large molecules, whereas solution-state NMR is restricted to relatively small ones (less than 70
kDa). X-ray crystallography is now used routinely by scientists to determine how a pharmaceutical drug interacts with its protein target and what changes might
improve it.[34] However, intrinsic membrane proteins remain challenging to crystallize because they require detergents or other means to solubilize them in
isolation, and such detergents often interfere with crystallization. Such membrane proteins are a large component of the genome and include many proteins of
great physiological importance, such as ion channels and receptors.[35][36]
[edit]Nuclear magnetic resonance spectroscopy or NMR
Protein nuclear magnetic resonance spectroscopy (usually abbreviated protein NMR) is a field of structural biology in which NMR spectroscopy is used to obtain
information about the structure and dynamics of proteins. The field was pioneered by Richard R. Ernst and Kurt Wüthrich[1], among others. Protein NMR
techniques are continually being used and improved in both academia and the biotech industry. Structure determination by NMR spectroscopy usually consists of
several following phases, each using a separate set of highly specialized techniques. The sample is prepared, resonances are assigned, restraints are generatedand a structure is calculated and validated
[edit]How to sequence a protein?
Protein sequencing is a technique to determine the amino acid sequence of a protein, as well as which conformation the protein adopts and the extent to which it
is complexed with any non-peptide molecules. Discovering the structures and functions of proteins in living organisms is an important tool for understanding
cellular processes, and allows drugs that target specific metabolic pathways to be invented more easily. The two major direct methods of protein sequencing
are mass spectrometry and the Edman degradation reaction. It is also possible to generate an amino acid sequence from the DNA or mRNA sequence
encoding the protein, if this is known. However, there are a number of other reactions which can be used to gain more limited information about protein sequences
and can be used as preliminaries to the aforementioned methods of sequencing or to overcome specific inadequacies within them[37].
[edit]Edman degradation
The Edman degradation is a very important reaction for protein sequencing, because it allows the ordered amino acid composition of a protein to be discovered.
Automated Edman sequencers are now in widespread use, and are able to sequence peptides up to approximately 50 amino acids long. A reaction scheme for
sequencing a protein by the Edman degradation follows - some of the steps are elaborated on subsequently. Break any disulfide bridges in the protein with an
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oxidising agent likeperformic acid or reducing agent like 2-mercaptoethanol. A protecting group such as iodoacetic acid may be necessary to prevent the bonds
from re-forming. Separate and purify the individual chains of the protein complex, if there are more than one.
Determine the amino acid composition of each chain.
Determine the terminal amino acids of each chain.
Break each chain into fragments under 50 amino acids long.
Separate and purify the fragments.Determine the sequence of each fragment.
Repeat with a different pattern of cleavage.
Construct the sequence of the overall protein.
Digestion into peptide fragments Peptides longer than about 50-70 amino acids long cannot be sequenced reliably by the Edman degradation. Because of this,
long protein chains need to be broken up into small fragments which can then be sequenced individually. Digestion is done either by endopeptidases such as
trypsin or pepsin or by chemical reagents such as cyanogen bromide. Different enzymes give different cleavage patterns, and the overlap between fragments can
be used to construct an overall sequence.
Phenylisothiocyanate is reacted with an uncharged terminal amino group, under mildly alkaline conditions, to form a cyclical phenylthiocarbamoyl derivative. Then,
under acidic conditions, this derivative of the terminal amino acid is cleaved as a thiazolinone derivative. The thiazolinone amino acid is then selectively extracted
into an organic solvent and treated with acid to form the more stable phenylthiohydantoin (PTH)- amino acid derivative that can be identified by
using chromatography or electrophoresis. This procedure can then be repeated again to identify the next amino acid. A major drawback to this technique is that
the peptides being sequenced in this manner cannot have more than 50 to 60 residues (and in practice, under 30). The peptide length is limited due to the cyclical
derivitization not always going to completion. The derivitization problem can be resolved by cleaving large peptides into smaller peptides before proceeding withthe reaction. It is able to accurately sequence up to 30 amino acids with modern machines capable of over 99% efficiency per amino acid. An advantage of the
Edman degradation is that it only uses 10 - 100 picomoles of peptide for the sequencing process. Edman degradation reaction is automated to speed up the
process.[38] [39]
[edit]N-terminal amino acid analysis
Determining which amino acid forms the N-terminus of a peptide chain is useful for two reasons: to aid the ordering of individual peptide fragments' sequences into
a whole chain, and because the first round of Edman degradation is often contaminated by impurities and therefore does not give an accurate determination of the
N-terminal amino acid. A generalised method for N-terminal amino acid analysis follows: React the peptide with a reagent which will selectively label the terminal
amino acid. Hydrolyse the protein. Determine the amino acid by chromatography and comparison with standards. There are many different reagents which can be
used to label terminal amino acids. They all react with amine groups and will therefore also bind to amine groups in the side chains of amino acids such as lysine -
for this reason it is necessary to be careful in interpreting chromatograms to ensure that the right spot is chosen. Two of the more common reagents are Sanger's
reagent (1-fluoro-2,4-dinitrobenzene) and dansyl derivatives such as dansyl chloride. Phenylisothiocyanate, the reagent for the Edman degradation, can also be
used. The same questions apply here as in the determination of amino acid composition, with the exception that no stain is needed, as the reagents produce
coloured derivatives and only qualitative analysis is required, so the amino acid does not have to be eluted from the chromatography column, just compared with a
standard. Another consideration to take into account is that, since any amine groups will have reacted with the labelling reagent, ion exchange chromatography
cannot be used, and thin layer chromatography or high pressure liquid chromatography should be used instead[40]
.
[edit]C-terminal amino acid analysis
The number of methods available for C-terminal amino acid analysis is much smaller than the number of available methods of N-terminal analysis. The most
common method is to add carboxypeptidases to a solution of the protein, take samples at regular intervals, and determine the terminal amino acid by analysing a
plot of amino acid concentrations against time
[edit]Mass spectrometry
Present day researchers are using Mass spectrometry an important tool for the characterization of proteins. Protein mass spectrometry refers to the
application of mass spectrometry to the study of proteins. The two primary methods for ionization of whole proteins are electrospray ionization (ESI) and
matrix-assisted laser desorption/ionization (MALDI). In keeping with the performance and mass range of available mass spectrometers, two approaches are used
for characterizing proteins. In the first, intact proteins are ionized by either of the two techniques described above, and then introduced to a mass analyzer. This
approach is referred to as "top-down" strategy of protein analysis. In the second, proteins are enzymatically digested into smaller peptides using a protease such
as trypsin. Subsequently these peptides are introduced into the mass spectrometer and identified by peptide mass fingerprinting or tandem mass spectrometry.
Hence, this latter approach (also called "bottom-up" proteomics) uses identification at the peptide level to infer the existence of proteins.
Whole protein mass analysis is primarily conducted using either time-of-flight (TOF) MS, or Fourier transform ion cyclotron resonance(FT-ICR). These two
types of instrument are preferable here because of their wide mass range, and in the case of FT-ICR, its high mass accuracy. Mass analysis of proteolytic
peptides is a much more popular method of protein characterization, as cheaper instrument designs can be used for characterization. Additionally, sample
preparation is easier once whole proteins have been digested into smaller peptide fragments. The most widely used instrument for peptide mass analysis are the
MALDI time-of-flight instruments as they permit the acquisition of peptide mass fingerprints (PMFs) at high pace (1 PMF can be analyzed in approx. 10 sec).
Multiple stage quadrupole-time-of-flight and the quadrupole ion trap also find use in this application.
[edit]Types of protein
[edit]Conjugated protein
A conjugated protein is a protein that functions in interaction with other chemical groups attached by covalent bonds or by weak interactions. Many proteins
contain only amino acids and no other chemical groups, and they are called simple proteins. However, other kind of proteins yield, on hydrolysis, some other
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chemical component in addition to amino acids and they are called conjugated proteins. The nonamino part of a conjugated protein is usually called its prosthetic
group. Most prosthetic groups are formed from vitamins. Conjugated proteins are classified on the basis of the chemical nature of their prosthetic groups. Some
examples of conjugated proteins are
[edit]Lipoproteins
A lipoprotein is a biochemical assembly that contains both proteins and lipids water-bound to the proteins. Many enzymes, transporters, structural proteins,
antigens, adhesins and toxins are lipoproteins. Examples include the high density (HDL) and low density (LDL) lipoproteins which enable fats to be carried in the
blood stream, the transmembrane proteins of the mitochondrion and the chloroplast, and bacterial lipoproteins.
[edit]Glycoproteins
Glycoproteins are proteins that contain oligosaccharide chains (glycans) covalently attached to polypeptide side-chains. The carbohydrate is attached to the
protein in a cotranslational or posttranslational modification. This process is known as glycosylation. In proteins that have segments extending extracellularly, the
extracellular segments are often glycosylated. Glycoproteins are often important integral membrane proteins, where they play a role in cell-cell interactions.
Glycoproteins also occur in the cytosol, but their functions and the pathways producing these modifications in this compartment are less well-
understood.Glycoproteins are generally the largest and most abundant group of conjugated proteins . They range from glycoproteins in cell surface
membranes that constitute the glycocalyx, to important antibodies produced by leukocytes.
[edit]phosphoproteins
Phosphoproteins are proteins which are chemically bonded to a substance containing phosphoric acid (see phosphorylation for more). The category of organic
molecules that includes Fc receptors, Ulks, Calcineurins, K chips, and urocortins.
[edit]Metalloprotein
A protein that contains a metal ion ass cofactor known as Metalloprotein. Metalloproteins have many different functions in cells, such as enzymes, transport andstorage proteins, and signal transduction proteins. Indeed, about one quarter to one third of all proteins require metals to carry out their functions. The metal ion is
usually coordinated by nitrogen, oxygen or sulfur atoms belonging to amino acids in the polypeptide chain and/or a macrocyclic ligand incorporated into the
protein. The presence of the metal ion allows metalloenzymes to perform functions such as redox reactions that cannot easily be performed by the limited set of
functional groups found in amino acids.
Computer-generated 3-D representation of the zinc finger motif of proteins, consisting of an α hel ix and an antiparallel β sheet. The zinc ion (green) is coordinated by two histidine residues and
two cysteine residues.
Metal IonExamples of enzymes containing this
ion
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Magnesium
Glucose 6-phosphatase
Hexokinase
DNA polymerase
Vanadium vanabins
Manganese Arginase
Iron
Catalase
Hydrogenase
IRE-BP
Aconitase
Nickel[41]Urease
Hydrogenase
Copper Cytochrome oxidase
Laccase
Zinc
Alcohol dehydrogenase
Carboxypeptidase
Aminopeptidase
Beta amyloid
Molybdenum Nitrate reductase
Selenium Glutathione peroxidase
variousMetallothionein
Phosphatase
[edit]hemoproteins
A hemeprotein (or hemoprotein or haemoprotein), or heme protein, is a metalloprotein containing a heme prosthetic group, either covalently or noncovalently
bound to the protein itself. The iron in the heme is capable of undergoing oxidation and reduction (usually to +2 and +3, though stabilized Fe+4 and even Fe+5
species are well known in the peroxidases). Hemoproteins probably evolved from a primordial strategy allowing to incorporate the iron (Fe) atom contained within
the protoporphyrin IX ring of heme into proteins. This strategy has been maintained throughout evolution as it makes hemoproteins responsive to molecules thatcan bind divalent iron (Fe). These molecules included, but are probably not restricted to, gaseous molecules, such as oxygen (O2) nitric oxide (NO), carbon
monoxide (CO) and hydrogen sulfide (H2S). Once bound to the prosthetic heme groups of hemoproteins these gaseous molecules can modulate the
activity/function of those hemoproteins in a way that is said to afford signal transduction. Therefore, when produced in biologic systems (cells), these gaseous
molecules are referred to as gasotransmitters.Haemoglobin contains the prosthetic group containing iron , which is the haem. It is with in the haem group that
carries the oxygen molecule through the binding of the oxygen molecule to the iron ion (Fe2+) found in the haem group[42].
Hemoglobin Hemoglobin (also spelled haemoglobin and abbreviated Hb or Hgb) is the iron-containing oxygen-transport metalloprotein in the red blood cells of all
vertebrates[1] (except the fish family Channichthyidae ) and the tissues of some invertebrates. Hemoglobin in the blood is what transports oxygen from the lungs
or gills to the rest of the body (i.e. the tissues) where it releases the oxygen for cell use, and collects carbon dioxide to bring it back to the lungs. In mammals the
protein makes up about 97% of the red blood cells' dry content, and around 35% of the total content (including water)[citation needed]. Hemoglobin has an oxygen
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binding capacity of 1.34 ml O2 per gram of hemoglobin, which increases the total blood oxygen capacity seventyfold. Hemoglobin is involved in the transport of
other gases: it carries some of the body's respiratory carbon dioxide (about 10% of the total) as carbaminohemoglobin, in which CO2 is bound to the globin
protein. The molecule also carries the important regulatory molecule nitric oxide bound to a globin protein thiol group, releasing it at the same time as oxygen.
Hemoglobin is also found outside red blood cells and their progenitor lines. Other cells that contain hemoglobin include the A9 dopaminergic neurons in the
substantia nigra, macrophages, alveolar cells, and mesangial cells in the kidney. In these tissues, hemoglobin has a non-oxygen-carrying function as an
antioxidant and a regulator of iron metabolism. Hemoglobin and hemoglobin-like molecules are also found in many invertebrates, fungi, and plants. In these
organisms, hemoglobins may carry oxygen, or they may act to transport and regulate other things such as carbon dioxide, nitric oxide, hydrogen sulfide and
sulfide. A variant of the molecule, called leghemoglobin, is used to scavenge oxygen, to keep it from poisoning anaerobic systems, such as nitrogen-fixing nodules
of leguminous plants. phytochromes,
Cytochromes
Cytochrome c with heme c .
Cytochromes are, in general, membrane-bound hemoproteins that contain heme groups and carry out electron transport. They are found either as monomeric
proteins (e.g., cytochrome c) or as subunits of bigger enzymatic complexes that catalyze redox reactions. They are found in the mitochondrial inner membrane andendoplasmic reticulum of eukaryotes, in the chloroplasts of plants, in photosynthetic microorganisms, and in bacteria.
Cytochromes Combination
a and a3
Cytochrome c oxidase ("Complex IV") with electrons delivered to complex by soluble cytochrome c (hence the
name)
b and c 1 Coenzyme Q - cytochrome c reductase ("Complex III")
b6 and f Plastoquinol—plastocyanin reductase
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3-dimensional structure of bovine rhodopsin. The seven transmembrane domains are shown in varying colors. The chromophore is shown in red.
Type prosthetic group
Cytochrome a heme a
Cytochrome b heme b
Cytochrome d tetrapyrrolic chelate of iron
[edit]Opsins
Opsins are a group of light-sensitive 35-55 kDa membrane-bound G protein-coupled receptors of the retinylidene protein family found in photoreceptor cells of theretina. Five classical groups of opsins are involved in vision, mediating the conversion of a photon of light into an electrochemical signal, the first step in the visual
transduction cascade. Another opsin found in the mammalian retina, melanopsin, is involved in circadian rhythms and pupillary reflex but not in image-forming.
[edit]Flavoproteins
Flavoproteins are proteins that contain a nucleic acid derivative of riboflavin: the flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN). Flavoproteins
are involved in a wide array of biological processes, including, but by no means limited to, bioluminescence, removal of radicals contributing to oxidative stress,
photosynthesis, DNA repair, and apoptosis. The spectroscopic properties of the flavin cofactor make it a natural reporter for changes occurring within the active
site; this makes flavoproteins one of the most-studied enzyme families.
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[edit]Simple proteins
The proteins which upon hydrolysis yield only amino acids are known as simple proteins.
[edit]Albumin
Albumin (Latin: albus, white) refers generally to any protein that is water soluble, which is moderately soluble in concentrated salt solutions, and experiences heat
denaturation. They are commonly found in blood plasma, and are unique to other plasma proteins in that they are not glycosylated. Substances containing
albumin, such as egg white, are called albuminoids.
[edit]Globulin
Globulin is one of the three types of serum proteins, the others being albumin and fibrinogen. Some globulins are produced in the liver, while others are made by
the immune system. The term globulin encompasses a heterogeneous group of proteins with typical high molecular weight, and both solubility and electrophoretic
migration rates lower than for albumin.
[edit]Histones
In biology, histones are highly alkaline proteins found in eukaryotic cell nuclei, which package and order the DNA into structural units called nucleosomes. They
are the chief protein components of chromatin, acting as spools around which DNA winds, and play a role in gene regulation.
[edit]Derived protein
[edit]Peptones
Peptones are derived from animal milk or meat digested by proteolytic digestion. In addition to containing small peptides, the resulting spray-dried material
includes fats, metals, salts, vitamins and many other biological compounds. Peptone is used in nutrient media for growing bacteria and fungi
[edit]Proteases
Proteases occur naturally in all organisms. These enzymes are involved in a multitude of physiological reactions from simple digestion of food proteins to highly-
regulated cascades (e.g., the blood-clotting cascade, the complement system, apoptosis pathways, and the invertebrate prophenoloxidase-activating cascade).
Proteases can either break specific peptide bonds (limited proteolysis), depending on the amino acid sequence of a protein, or break down a complete peptide to
amino acids (unlimited proteolysis). The activity can be a destructive change, abolishing a protein's function or digesting it to its principal components; it can be an
activation of a function, or it can be a signal in a signaling pathway.
[edit]Protein data bank or PDB
Like fuel and flame, two forces converged to initiate the Protein data bank (PDB): 1) a small but growing data base of sets of protein structures determined by X-
ray diffraction and 2) the newly available (1968) molecular graphics display, the BRookhaven Raster Display (BRAD), to inspect these structures in 3-D. In 1969,
with the sponsorship of Dr. Walter Hamilton at the Brookhaven National Laboratory, Dr. Edgar Meyer (Texas A&M University) began to write software to store
atomic coordinate files in a common format to make them available for geometric and graphical evaluation. By 1971 program SEARCH was executed remotely to
extract and examine structural data and thereby was instrumental in initiating networking, thus marking the functional beginning of the PDB.
Upon Hamilton's death in 1973, Dr. Tom Koeztle took over direction of the PDB for the subsequent 20 years. In January 1994, Dr. Joel Sussman of
Israel's Weizmann Institute of Science was appointed head of the PDB. In October 1998,[43] the PDB was transferred to the Research Collaboratory for Structural
Bioinformatics (RCSB); the transfer was completed in June 1999. The new director was Dr. Helen M. Berman of Rutgers University (one of the member institutionsof the RCSB).[44] In 2003, with the formation of the wwPDB, the PDB became an international organization. The founding members are PDBe (Europe),
RCSB(USA), and PDBj (Japan). The BMRB joined in 2006. Each of the four members of wwPDB can act as deposition, data processing and distribution centers
for PDB data. The data processing refers to the fact that wwPDB staff review and annotates each submitted entry. The data are then automatically checked for
plausibility (the source code for this validation software has been made available to the public at no charge).
The Protein Data Bank (PDB) is a repository for the 3-D structural data of large biological molecules, such as proteins and nucleic acids. (See also crystallographic
database). The data, typically obtained by X-ray crystallography or NMR spectroscopy and submitted by biologists and biochemists from around the world, are
freely accessible on the Internet via the websites of its member organisations (PDBe, PDBj, and RCSB). The PDB is overseen by an organization called the
Worldwide Protein Data Bank, wwPDB.
[edit]Insulin
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The structure of insulin. The left side is a space-filling model of the insulin monomer, believed to be biologically active. Carbon is green, hydrogenwhite, oxygen red, and nitrogen blue. On the
right side is a ribbon diagram of the insulin hexamer, believed to be the stored form. A monomer unit is highlighted with the A chain in blue and the B chain in cyan. Yellow denotes disulfide
bonds, and magenta spheres are zinc ions.
Insulin hexamers highlighting the threefold symmetry, the zinc ions (center) binding with histidine.
Within vertebrates, the amino acid sequence of insulin is extremely well preserved. Bovine insulin differs from human in only three amino acid residues, and
porcine insulin in one. Even insulin from some species of fish is similar enough to human to be clinically effective in humans. Insulin in some invertebrates is quitesimilar in sequence to human insulin, and has similar physiological effects. The strong homology seen in the insulin sequence of diverse species suggests that it
has been conserved across much of animal evolutionary history. The C-peptide of proinsulin , however, differs much more amongst species; it is also a hormone,
but a secondary one.
Insulin is produced and stored in the body as a hexamer (a unit of six insulin molecules), while the active form is the monomer. The hexamer is an inactive form
with long-term stability, which serves as a way to keep the highly reactive insulin protected, yet readily available. The hexamer-monomer conversion is one of the
central aspects of insulin formulations for injection. The hexamer is far more stable than the monomer, which is desirable for practical reasons, however the
monomer is a much faster reacting drug because diffusion rate is inversely related to particle size. A fast reacting drug means that insulin injections do not have to
precede mealtimes by hours, which in turn gives diabetics more flexibility in their daily schedule. Insulin can aggregate and form fibrillar interdigitated beta-sheets.
This can cause injection amyloidosis, and prevents the storage of insulin for long periods[45].
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In 1869 Paul Langerhans, a medical student in Berlin, was studying the structure of the pancreas under a microscope when he identified some previously un-
noticed tissue clumps scattered throughout the bulk of the pancreas. The function of the "little heaps of cells," later known as the Islets of Langerhans, was
unknown, but Edouard Laguesse later suggested that they might produce secretions that play a regulatory role in digestion. Paul Langerhans' son, Archibald, also
helped to understand this regulatory role. The term insulin origins from insula, the Latin word for islet/island. In 1889, the Polish-German physician Oscar
Minkowski in collaboration with Joseph von Mering removed the pancreas from a healthy dog to test its assumed role in digestion. Several days after the dog's
pancreas was removed, Minkowski's animal keeper noticed a swarm of flies feeding on the dog's urine. On testing the urine they found that there was sugar in the
dog's urine, establishing for the first time a relationship between the pancreas and diabetes. In 1901, another major step was taken by Eugene Opie, when he
clearly established the link between the Islets of Langerhans and diabetes: Diabetes mellitus … is caused by destruction of the islets of Langerhans and occursonly when these bodies are in part or wholly destroyed. Before his work, the link between the pancreas and diabetes was clear, but not the specific role of the
islets.
The Nobel Prize committee in 1923 credited the practical extraction of insulin to a team at the University of Toronto and awarded the Nobel Prize to two men;
Fredericus Bantam and J.J.R. Macleon. They were awarded the Nobel Prize in Physiology or Medicine in 1923 for the discovery of insulin. Bantam, insulted that
Best was not mentioned, shared his prize with Best, and Macleon immediately shared his with James Collip. The patent for insulin was sold to the University of
Toronto for one half-dollar.
The primary structure of insulin was determined by British molecular biologist Frederick Sanger. It was the first protein to have its sequence be determined. He
was awarded the 1958 Nobel Prize in Chemistry for this work. In 1969, after decades of work, Dorothy Crowfoot Hodgkin determined the spatial conformation of
the molecule, the so-called tertiary structure, by means of X-ray diffraction studies. She had been awarded a Nobel Prize in Chemistry in 1964 for the development
of crystallography. Rosalyn Sussman Yalow received the 1977 Nobel Prize in Medicine for the development of the radioimmunoassay for insulin[46].