3/27/23, 9:08 PM
`Levels of Protein Organization
`A 2014 Foundations of Medicine eLAB
`Levels of Protein Organization
`
`Levels of Protein Organization
`
`A protein's primary structure is defined as the amino acid sequence of its polypeptide chain; secondary structure is the local
`spatial arrangement of a polypeptide's backbone (main chain) atoms; tertiary structure refers to the three-dimensional
`structure of an entire polypeptide chain; and quaternary structure is the three-dimensional arrangement of the subunits in a
`multisubunit protein. In this series of pages we examine the different levels of protein organization. We also view structures in
`lots of ways -- Cα backbone, ball-and-stick, CPK, ribbon, spacefilling -- as well color is used to highlight different aspects of
`the amino acids, structure, etc. As you traverse though this module please note these aspects.
`
`This module includes links to KiNG (Kinemage, Next Generation), which displays
`three-dimensional structures in an animated, interactive format. These "kinemages"
`(kinetic images) can be rotated, moved, and zoomed, and parts can be hidden or
`shown. Kinemages were originally implemented under the auspices of the Innovative
`Technology Fund and the Protein Society, and the programming and maintenance
`are done by David C. Richardson and Jane S. Richardson.
`
`Reference: "THE KINEMAGE: A TOOL FOR SCIENTIFIC COMMUNICATION" D.C.
`Richardson and J.S. Richardson (1992) Protein Science 1: 3-9. Also Trends in
`Biochem. Sci. (1994) 19: 135-8.
`
`Text adapted from: Demo5_4a.kin
`
`Primary Structure (1˚)
`
`The primary structure of a peptide or protein is the linear sequence of its amino acids (AAs). By convention, the primary
`structure of a protein is read and written from the amino-terminal (N) to the carboxyl-terminal (C) end. Each amino acid is
`connected to the next by a peptide bond.
`Secondary Structure (2˚) -- Alpha Helices
`While primary structure describes the sequence of amino acids forming a peptide chain, secondary structure refers to the
`local arrangement of the chain in space. Several common secondary structures have been identified in proteins. These will
`be described in the following sections and visualized using the KiNG software mentioned previously.
`
`To load the KiNG Java Applet, just click here. Upon loading this page, the KiNG Java Applet should automatically spawn. If
`you need information on using King, please hover here.
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`https://comis.med.uvm.edu/VIC/coursefiles/MD540/MD540-Protein_Organization_10400_574581210/Protein-org/Protein_Organization_print.html
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`The Alpha Helix
`
`Levels of Protein Organization
`
`An alpha helix is an element of
`secondary structure in which
`the amino acid chain is
`arranged in a spiral. The
`kinemage linked above shows
`an individual alpha helix,
`viewed from the N-terminal
`end to resemble the "helical
`wheel" (see figure below). The
`O and N atoms of the helix
`main chain are shown as red
`and blue balls, respectively.
`The non-integral, 3.6-residue-
`per-turn repeat of the alpha
`helix means that the Cα's of
`successive turns are about
`halfway offset, giving the main
`chain a distinctive 7-pointed
`star appearance in end view.
`Notice that the Cα-Cβ bonds
`do not point out radially from
`the helix axis but "pinwheel"
`along the line of one of the
`adjacent peptides, giving the side chains an asymmetrical start.
`
`The hydrophobic side chains are shown in seagreen, polar ones in skyblue, and charged ones in red. These can be turned on
`by clicking on the checkbox labeled "side ch". Now TURN ON and OFF the various display groups and sets, by clicking in the
`appropriate button box.
`
`When you clicked the different sidechain types on, what did you observe? Did you notice that the helix has one side with
`mainly polar residues, and the other with mainly hydrophobic residues?. This is a typical globular-protein helix; in its native
`configuration, the polar residues would face the solvent while the hydrophobic residues would face the protein interior. In the
`view menu in KiNG, choose View2 or View3 to see more of the structure.
`
`The figure to the left shows a helical wheel
`representation of an amino acid sequence, as if
`looking down the axis of an alpha helix that is
`perpendicular to the page. The amino acid residues
`are numbered from nearest to most distant and are
`arranged as an ideal alpha helix with 3.6 residues per
`complete turn. This figure is a snaphot of a Java
`Applet written by Edward K. O'Neil and Charles M.
`Grisham (University of Virginia in Charlottesville,
`Virginia).
`
`In KiNG, choose View4 for a close-up from the side,
`with the helical hydrogen bonds (H-bonds) in brown.
`Turn on "Hbonds" on the button panel, to see the H-
`bonds in brown. Click on backbone atoms at either
`end of one of the H-bonds, to verify that the alpha-
`helical H-bond pattern does indeed go from a donor
`NH at residue i to an acceptor O at residue i-4 (as
`shown in the figure to the right). Check to see if this
`alpha helix has 3.6 residues per turn. If you were to
`mesure, the rise of a full turn is 5.4 Angstroms (Â).
`
`Alpha helices are nearly all right-handed. To see that this one is righthanded, hold your right hand with the thumb pointing up
`and the fingers loosely curled; trying to match the spiral of the helix, move slowly along the direction your thumb points and
`curl along the line of your fingers, as though tightening a screw. When that motion matches the backbone spiral if done with
`the right hand, then the helix is righthanded.
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`To measure phi,psi angles for the KiNG example helix, turn on "Measure angle &
`dihedral" on the "Tools" pulldown menu. Start by clicking on a carbonyl C atom near
`the top, then the next N, then the Cα, and then a C again; at that point the information
`line will show a dihedral angle that is the phi angle of the central N-Cα bond of those 4
`atoms. For a righthanded alpha-helix, it should be in the range of -50 to -80 degrees.
`Click on the next N and you will get the psi angle, which should be between -25 and
`-60 degrees. Continue down the helix backbone, getting omega (near 180 degrees),
`phi, psi, etc. These helical phi,psi values are in the well-populated area in the lower left
`of the Ramachandran plot (shown on the right).
`
`In summary, the ideal alpha helix has the following properties:
`
`It completes one turn every 3.6 residues;
`It rises approximately 5.4 Â with each turn;
`It is a right-handed helix;
`It is held together by hydrogen bonds between the C=O of residue i and the NH
`of residue i+4;
`It is typically slightly curved.
`
`Some general properties of alpha-helices:
`
`An average alpha-helix is 10 residues long (15 Â in length), although alpha-helices can range between 4 to 40 residues
`in length in a standard globular protein.
`All residues participating in an alpha-helix have similar (phi,psi) angles. These angles, which are approximately -60 and
`-50, are from the bottom left quadrant of the Ramachandran plot.
`Some amino acids are preferred in an alpha-helix. Residues such as Ala, Glu, Leu and Met have a high tendency to
`participate in a helix , while residues such as Pro and Gly have a small such tendency. Of special interest is Proline,
`which cannot fit into a helix, and introduces a kink.
`The helix has an overall dipole moment, which is a vector sum of the aligned dipole moments of the individual peptide
`bonds. The positive pole is at the N-terminus and the negative pole is at the C-terminus. Sometimes this dipole has a
`functional role.
`
`
`
`Some text adapted from: Kinemage Supplement to Branden & Tooze "Introduction to Protein Structure", Chapter 2 -
`MOTIFS OF PROTEIN STRUCTURE by Jane S. and David C. Richardson.
`
`
`Secondary Structure (2˚) -- Beta Strands
`
`A beta strand is an element of secondary structure in which the protein chain is nearly linear. Adjacent beta strands can
`hydrogen bond to form a beta sheet (also referred to as a beta pleated sheet). The participating beta strands are not
`continuous in the primary sequence, and do not even have to be close to each other in the sequence, i.e. the strands forming
`a beta sheet can be separated in primary structure by long sequences of amino acids that are not part of the sheet.
`Approximately a quarter of all residues in a typical protein are in beta strands, though this varies greatly between proteins
`
`To view a beta sheet in the KiNG Java Applet, click here. Kinemage 1 shows the 6-stranded parallel beta sheet from domain
`1 of lactate dehydrogenase (file 1LDM). This doubly-wound parallel beta sheet is the most common folding pattern found in
`known protein structures. This "fold" is also known as the "nucleotide-binding domain", because most examples bind a
`mononucleotide (such as FMN) or a dinucleotide (such as NAD) near the middle of one end of the beta sheet. Lactate
`dehydrogenase is the classic, first-seen example of this type of structure and has the most frequently-observed topology of
`beta connections.
`
`Notice that the H-bonds in this parallel shet are slanted in alternate directions, rather than perpendicular to the strands as we
`will see in antiparallel sheets. Drag right or left to better see that the sheet as a whole twists. This twist is usually described by
`the twist in orientation of the peptide planes (or H-bond plane) as one progresses along the strand; by this definition beta
`sheet twist is always right-handed, although by varying amounts. Click on atoms along a strand to tell its direction from the
`residue numbers, and satisfy yourself that all six strands are indeed parallel. The strand labels show strand sequence order.
`Note that most sequential pairs are next to each other, and that the chain starts in the middle, moves to one edge, skips back
`to the middle and then moves out to the other edge.There are three possible ways to form a beta sheet from beta strands,
`discussed below.
`
`
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`https://comis.med.uvm.edu/VIC/coursefiles/MD540/MD540-Protein_Organization_10400_574581210/Protein-org/Protein_Organization_print.html
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`Levels of Protein Organization
`Types of Beta Sheets Observed in Proteins
`
`1) Parallel beta sheet - All bonded strands have the same N to C direction. As a result they have to be separated by long
`sequence stretches. The hydrogen bonds are equally distanced.
`
`The figure to the left shows a three-stranded parallel beta sheet from the protein
`thioredoxin. The three parallel strands are shown in both cartoon format (left) and
`in stick form containing backbone atoms N, CA, C, and O' (right). Hydrogen bonds
`are identified by arrows connecting the donor nitrogen and acceptor oxygens.
`Strands are numbered according to their relative position in the polypeptide
`sequence.
`
` 2) Antiparallel beta sheet - The beta strands run in alternating directions and
`therefore can be quite close on the primary sequence. The distance between
`successive hydrogen bonds alternates between shorter and longer.
`
`The figure to the right shows a three-
`stranded antiparallel beta sheet from
`thioredoxin. The three antiparallel
`strands are shown in both cartoon format (left) and in stick form containing
`backbone atoms N, CA, C, and O' (right). Hydrogen bonds are identified by
`arrows connecting the donor nitrogen and acceptor oxygens. Strands are
`numbered according to their relative position in the polypeptide sequence.
`
`3) Mixed beta sheet - a mixture of parallel and antiparallel hydrogen bonding.
`About 20% of all beta sheets are mixed.
`
`Hydrogen bond patterns in a
`mixed beta sheet (figure to the
`left). Here a four-stranded beta sheet containing three antiparallel strands
`and one parallel strand is drawn schematically. Hydrogen bonds between
`antiparallel strands are indicated with red lines, those between parallel
`strands with green lines.
`
`Some of the main features of beta sheets include:
`
`The extended conformation in a beta strand is about 3.5 Â per residue,
`and beta strands can be extended as much as 35 Â in length.
`The overall geometry of a sheet is not planar but rather is pleated, with
`alternating Cα carbons above and below the average plane of the sheet.
`Due to the chirality of the amino acids (L amino acids) all beta strands
`have a right-handed twist, whereas a beta sheet has an overall left-
`
`handed twist.
`
`Since the strands do not have to be adjacent on the sequence there are many possible ways to arrange strands in a
`sheet, these arrangements are called topologies and can be quite complicated.
`
`Turn on the side chains in KiNG to examine their arrangment. Along a given strand the sidechains alternate between one side
`of the sheet (gold) and the other (sea or sky). On adjacent strands the alternation is in register, so that the side chains form
`rows that are in quite close contact. On parallel beta sheet, the geometry is such that sidechains with branched beta-carbons
`(Val, Ile, or Thr) make quite favorable contact along a row; since these positions are usually buried and hydrophobic, the
`result is that Val and Ile are the dominant residues found in these positions. The edge strands, or the very ends of a given
`strand, can be exposed to solvent and often have significantly more hydrophilic residues (as, for instance, in row 0 here, or
`the Ser on strand 3).
`
`
`Some text adapted from: "The Protein Tourist: DOUBLY-WOUND PARALLEL ALPHA/BETA PROTEINS, OR NUCLEOTIDE-
`BINDING DOMAINS" by J.S. Richardson and D.C. Richardson.
`
`
`Secondary Structure (2˚) -- Beta Turns and Random Coils
`The Beta Turn
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`https://comis.med.uvm.edu/VIC/coursefiles/MD540/MD540-Protein_Organization_10400_574581210/Protein-org/Protein_Organization_print.html
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`Levels of Protein Organization
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`Turns generally occur when the protein chain needs to change direction in order to connect
`two other elements of secondary structure. The most common is the beta turn, in which the
`change of direction is executed in the space of four residues. Some commonly observed
`features of beta turns are a hydrogen bond between the C=O of residue i and the N-H of
`residue i+3 (i.e, between the first and the fourth residue of the turn) and a strong tendency to
`involve glycine and/or proline. You will sometimes hear the phrase "beta hairpin" which can
`be used to describe a beta turn joining two anti-parallel beta strands together. Beta turns are
`subdivided into numerous types on the basis of the details of their geometry.
`
`Gamma turns are three-residue turns which often incorporate a hydrogen bond between the
`C=O of residue i and the N-H of residue i+2.
`Random Coil
`
`Some regions of the protein chain do not form regular secondary structure and are not
`characterized by any regular hydrogen bonding pattern. These regions are known as random
`coils and are found in two locations in proteins:
`
`Terminal arms - both at the N-terminus and the C-terminus of the protein;
`Loops - Loops are unstructured regions found between regular secondary structure elements.
`
`Random coils can be 4 to 20 residues long, although most loops are not longer than 12 residues. Most loops are exposed to
`the solvent and are have polar or charged side-chains. In some cases loops have a functional role, but in many cases they do
`not. As a result, loop regions are often poorly conserved (i.e. more prone to change) during evolution.
`
`Some text adapted from: "EXERCISE 3. PROTEIN SECONDARY STRUCTURES" by Kim M. Gernert and Kim M. Kitzler.
`
`Propensity of AAs to Form Secondary Structures
`As we have learned, the order of the AAs is the primary structure and all residues in a polypeptide chain have the same main-
`chain atoms. What vary are the side chains (R groups). Do the specific AAs present dictate the secondary structure? As
`shown in the figure, all amino acids can be found in all secondary structure elements, but some are more or less common in
`certain elements. Pro and Gly, for isntance, aren't good in helices but are favored in beta-turns. If we take this a step further
`and ask whether 2, 3, or 4 amino acids combinations dictate secondary structure we find a stronger correlation, but still not
`strong enough to reliably predict tertiary structure.
`Tertiary (3˚) Structure
`Proteins are abundant in all organisms and are fundamental to life.
`The diversity of protein structure underlies the very large range of
`their functions: enzymes (biological catalysts), storage, transport,
`messengers, antibodies, regulation, and structural proteins.
`
`Proteins are linear heteropolymers of fixed length; i.e. a single type
`of protein always has the same number and composition of AAs, but
`different proteins may have 100 to more than 1000 AAs. There is
`therefore a great diversity of possible protein sequences. The linear
`chains fold into specific three-dimensional conformations, which are
`determined by the sequence of amino acids and therefore are also
`extremely diverse, ranging from completely fibrous to globular.
`Covalent disulfide bonds can be introduced between cysteine
`residues placed in close proximity in 3D space -- this provides rigidity
`for the resulting 3D structure. Ribbon diagrams like the one shown
`here are a common way to visualize proteins.
`
`Protein structures can be determined to an atomic level by X-ray diffraction and neutron-diffraction studies of crystallized
`proteins, and more recently by nuclear magnetic resonance (NMR) spectroscopy of proteins in solution. The structures of
`many proteins, however, remain undetermined.
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`To view an example of tertiary structure in
`KiNG, click here. This is ribonuclease A, an
`enzyme responsible for the degradation of
`RNA. The image depicts all atoms of one
`half of the molecule (cyan for side chains,
`brown for hydrogen atoms) and just main
`chain and side chains for the other half. The
`alternate view shows main-chain atoms and
`H-bonds (purple). Click "Animate" to cycle
`between the views.
`
`Although hydrogens constitute about half
`the atoms in a protein, they are seldom
`shown explicitly because they are hard to
`detect with x-ray crystallography (due to low
`electron density) and they very much
`complicate the picture. This ribonuclease
`image is a joint x-ray/neutron diffraction
`structure, for which hydrogens are always
`included. Even without H atoms, an all-atom
`view is too crowded to be very useful but is
`a good way to appreciate where simplified
`versions start from.
`
`
`
`
`
`
`Some text adapted from: Kinemage Supplement to Branden & Tooze "Introduction to Protein Structure", Chapter 2 -
`MOTIFS OF PROTEIN STRUCTURE, Jane S. and David C. Richardson.
`
`
`Protein Folding
`
`Protein folding is the physical process by which a linear polypeptide folds into its characteristic and functional three-
`dimensional structure. Folding of a polypeptide chain is strongly influenced by the solubility of the AA R-groups in water. Each
`protein exists as an unfolded polypeptide or random coil when translated from a sequence of mRNA to a linear chain of amino
`acids. This polypeptide lacks any stable (long-lasting) three-dimensional structure (the left hand side of the neighboring
`figure). Amino acids interact with each other to produce a well-defined three-dimensional structure, the folded protein (the
`right hand side of the figure), known as the native state. All the information for the native fold appears therefore to be
`contained within the primary structure (Anfinsen received the Nobel Prize for this), and proteins are self-folding (although in
`vivo, polypeptide folding is often assisted additional molecules known as molecular chaperones).
`
`Minimizing the number of hydrophobic side-chains exposed to
`water (the hydrophobic effect) is an important driving force behind
`the folding process. Intramolecular hydrogen bonds also contribute
`to protein stability (think of their importance in secondary
`structures). Ionic interactions (attraction between unlike electric
`charges of ionized R-groups) also contribute to the stability of
`tertiary structures. Disulfide bridges (covalent bonds) between
`neighboring cysteine residues can also stabilize three-dimensional
`structures. Note that disulfide bonds are rarely observed in
`intracellular proteins because of the reducing intracellular environment.
`
`The correct 3D structure of a protein is essential to its function, although some parts of functional proteins may remain
`unfolded. Failure to fold into native structure generally produces inactive proteins, but in some instances misfolded proteins
`have modified or toxic functionality (think prions & amyloid fibrils). Consistent with their functional importance, three-
`dimensional structures of proteins are more conserved during evolution time than are the primary amino-acid sequences.
`
`For those who want to contribute to science while playing games I suggest you check out FoldIt. Recently players of this
`game were able to correctly predict the structure of a retroviral protease. For those who want their spare CPU cycles to go to
`a good use, I suggest you check out Folding@home.
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`Levels of Protein Organization
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`Quaternary (4˚) Structure
`Quaternary structure in proteins is the most intricate degree of organization still considered a single molecule. To be
`considered to have quaternary structure, a protein must have two or more peptide chains forming subunits. The subunits can
`be different or identical, and in most cases they are arranged symmetrically. In general, a protein with two subunits is called a
`dimer; one with three subunits a trimer; and one with four subunits a tetramer.
`
`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. A good example would be a DNA polymerase (see image)
`and ion channels. Subunits are held together by the same types of
`interactions that stabilize the tertiary structure of proteins.
`
`There is debate as to whether quaternary structure should be
`defined to include peptides linked by covalent (disulfide) bonds. In
`CMB, we will us quaternary structure to refer only to arrangement of
`subunits that are not covalently linked, although covalent disulfide
`bonds may occur within the individual subunits.
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