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Chapter
4: Protein Structure
Amino
acids are the subunits of proteins.
The
repeating units are amide planes containing peptide bonds.
The amide planes can twist about there connecting carbon atoms to
create the 3D conformations of proteins.
Primary structure-
amino acid sequence.
Secondary structure-
local folding of the protein chain a-helices (Linus Pauling) and b-pleated sheets.
Tertiary structure
is the structure of a complete protein chain.
Quaternary structure
more than one tertiary protein coming together to form one functional
protein (hemoglobin).
Levels of Protein Structure.
The native
conformation is the three-dimensional structure of a protein that is
functional. Each protein has a precise structure needed for proper function.
There are
four levels of protein conformation. Primary structure is the actually order of the amino acids from the N-terminus to the C-terminus. Secondary structure includes the hydrogen-bonded arrangements a-helices and b-pleated sheets (no side chains).
Domains
are independently folding portions of a protein (supersecondary
structure). Tertiary structure is 3D folding due to all atoms including side chains and prosthetic groups (groups other than amino acids).
Quaternary structure
a functional protein consisting of multiple polypeptides called subunits.
Interactions between subunits is mediated by noncovalent
interactions (hydrogen bonds and hydrophobic interactions).
Importance
of primary structure. The amino acid sequence is the basis for structure and function of the protein. Ex: Sickle-cell anemia is due to a single amino acid change (primary structure change) in the protein hemoglobin which has 574 total amino acids.
Results in clumping of hemoglobin and malformation of red blood
cells which leads to poor oxygen binding. It is possible to change one amino acid with another amino acid and it have no effect on function of the protein or it may result in a nonfunctional protein.
Primary
Structure of Proteins.
Determining
the primary sequence of a protein is complex process. Step one: .
Heat a sample of the protein in 6 M HCL at 100-110oC for
12-36 hours.
Analyze types and quantities in an amino acid analyzer. Step two: Identify N and C-terminal residues. Step three and four: Edman degradation cleavage and identification of each individual amino acid (automated).
Most proteins are too long and must be first cleaved into small
peptides and then subjected to Edmund degradation.
Usually two enzyme proteases are used (trypsin and chymotrypsin). »
Trypsin cleaves peptide bonds preferentially at amino acids with
positively charged R groups. »
The amino acid with the charged side chain ends up the C-terminal
amino acid in one of the peptides produced. »
This can help identify the original C-terminal residue if it is an
amino acid without a positively charged side chain. »
Chymotrypsin cleaves preferentially at aromatic amino acids (tyr,
trp, and phe) these residues also end up the C-terminal residue in
peptides produced.
Cyanogen bromide cleaves at internal methionine residues. »
Sulfur of the methionine reacts with the carbon of the cyanogen
bromide to produce a homoserine lactone at the end of the fragment (Fig.
4.3).
Cleavage into peptides will allow you to find overlaps and to
rebuild the proteins primary sequence (Fig. 4.4).
Practice Problem: Treatment of two samples of the same protein with trypsin and
chymotrypsin resulted in the following peptides. Deduce the sequence of the original peptide. Trypsin Leu-Ser-Tyr-Ala-Ile-Arg and Asp-Gly-Met-Phe-Val-Lys. Chymotrypsin Val-Lys-Leu-Ser-Tyr, Ala-Ile-Arg and Asp-Gly-Met-Phe. Asp-Gly-Met-Phe-Val-Lys-Leu-Ser-Tyr-Ala-Ile-Arg
Edman
Method. The sequence of a peptide containing 10-20 residues may then be sequenced by repeated application of the Edman degradation procedure. Uses as little as 10 pm (picomoles) of material. After amino acid sequences of individual peptides are known then the entire peptide may be pieced back together.
Edman reagent phenyl
isothiocyanate reacts with the peptides N-terminal residue, this residue
is then cleaved (anhydrous acid) and detected as a phenylthiohydantoin
derivative of the amino acid.
This is repeated as necessary until the peptide sequence is known
with an apparatus known as a sequencer.
A more commonly used
method is to deduce the amino acid sequence from the DNA sequence.
It is easier to sequence DNA and relatively easy to deduce the
amino acid sequence encoded for in the DNA sequence.
It will not determine all posttranslational modifications however
(Ex: hydroxyproline).
Secondary Structure:
The
hydrogen bonded arrangement of the protein backbone (N-C-C).
Within
each amino acid residue there are two bonds with relatively free rotation. 1) Bond between the a-carbon and the amino group. 2) Bond between the a-carbon and the carboxyl group.
Rigid
planer peptide groups are similar to playing cards that can rotate about
the central a-carbon. Remember we are only interested in the backbone for secondary structure. The angles (F) phi (C-N bond) and psi (y) (C-C bond) are called Ramachandran angles.
Conformation of a
protein backbone can be indicated by giving the values for phi and psi for
each residue.
Two kinds of common
secondary structures are the a-helix
and b-sheets.
Not the only possible secondary structures but the most common and
most important.
In regular secondary structures like the a-helix and b-sheet
the F and y
angles repeat themselves.
The a-helix and b-sheet are periodic structures; their features repeat at regular
intervals. »
The a-helix is rod shaped and only involves one polypeptide chain. »
The
b-pleated
sheet can involve more than one polypeptide chain.
The
a-helix. The a-helix is stabilized by hydrogen bonds parallel to the helical axis within the backbone. The C=O group of each amino acid is hydrogen bonded to the N-H group of the amino acid four residues away. The helical conformation allows for the linear arrangement of the hydrogen bonds.
Gives the bonds and the entire structure added strength. There are 3.6 residues for each turn of the helix and pitch of 5.4 angstroms (1 angstrom = 10-8 cm = 0.54 nm). Proteins have varying amounts of a-helices from a few percent to nearly 100%.
Causes for a-helix
disruption.
Proline has restricted movement about the F disrupting a-helix
formation.
Groups of amino acids with the same charge will also disrupt an a-helix
(electrostatic repulsion).
Bulky side chains (steric repulsion) can also result in a-helix
disruption. »
Especially those that have more than just two hydrogens bonded to
the b-carbon
like: valine, isoleucine and threonine.
The b-pleated sheet.
Very
different than the a-helix. The peptide backbone is almost completely extended. The hydrogen bonds may form different parts of the same protein chain (intrachain) or between different protein chains (interchain).
The hydrogen bonding between the chains or different segments of the same chain give it a repeating zigzag structure.
The hydrogen bonds form perpendicular to the protein chain
not parallel like in the a-helix.
R groups alternate direction pointing up or down.
The a-helix
and b-sheet
in proteins. These two secondary structure forms are used in many different combinations in the formation of proteins. The polypeptide chain twists and turns to form its functional 3D structure.
Glycine is commonly found in reverse turns at which the protein
chain changes direction. Why? »
Prevents steric hindrance.
Proline is also very common in reverse turns because its cyclic
structure gives it the correct geometry to put a kink in the protein
chain.
The most common
feature formed by a combination of these two forms is the bab
unit, in which two parallel b-sheets
are connected by a a-helix. aa unit - two sections of antiparallel a-helices Greek key - a type of antiparallel b-sheet, named for a decorative design in classical Greek pottery The b -barrel - created when b-sheets are extensive enough to fold back on themselves
Two types
of protein conformations: Fibrous and Globular. Difficult to clearly separate secondary and tertiary structure the nature of the side chains (tertiary) can influence the backbone structure (secondary). Fibrous proteins: contain polypeptide chains organized approximately parallel along a single axis.
They consist of long fibers or large sheets.
Tend to be mechanically strong.
Are insoluble in water and dilute salt solutions.
Play important structural roles in nature.
Examples: »
keratin of hair and wool »
collagen of connective tissue of animals including cartilage,
bones, teeth, skin, and blood vessels Globular proteins: proteins which are folded into a more or less spherical shape.
They tend to be soluble in water or in salt solutions.
Most of their polar side chains are on the outside and interact
with the aqueous environment by hydrogen bonding and ion-dipole
interactions.
Most of their nonpolar side chains are buried inside. Why is this
important?
Nearly all have substantial sections of a-helix and b-sheet.
Complex tertiary and quaternary structures.
Examples: »
myoglobin (Figure 4.16) »
hemoglobin (Figure 4.21)
Tertiary Structure:
Three-dimensional
arrangement of the atoms in the molecules. Conformations of the side chains, positions of any prosthetic groups are parts of the tertiary structure and relationships of secondary structures to one another. Secondary tells you much about the tertiary structure of a fibrous protein, except for arrangement of the atoms of the side chains. Globular proteins it is necessary to determine how those secondary structures fold back on each other, in addition to side-chain and prosthetic group positions.
Forces
involved in protein folding. Primary structure depends on the covalent peptide bonds while secondary and tertiary structure relies on noncovalent interactions. Multiple polypeptides that come together as a functional unit is the quaternary structure may depend on covalent or noncovalent bonds.
Can include complexes with a metal ion (electrostatic). A disulfide bond between cysteine side chains is a covalent bond and can play a role in structure. Not all proteins have every structural feature and specialized structures can bring residues that are linearly far apart close together. The interplay of all the amino acids and their side groups give each protein their structure and function. X-ray crystallography is an experimental technique designed to determine tertiary structure.
In order to use this technique highly purified crystals of the
protein must be grown.
The protein crystal is then exposed to a beam of x-rays and a
diffraction pattern is produced on a photographic plate.
The pattern is due to the scattering of x-rays by the electrons of
each atom.
Specific patterns are due to certain protein structures.
Information is then extracted by Fourier series calculations. NMR (nuclear magnetic resonance spectroscopy) can supplement the results of x-ray diffraction.
2-D NMR
uses protein samples in solution.
Uses Fourier series as well for data analysis.
Ex:
Myoglobin. Globular protein that carries oxygen in muscle. First tertiary structure found using x-ray crystallography. Single polypeptide of 153 amino acids including a prosthetic group, the heme group.
Heme group also found in hemoglobin. Compact internal structure with close interior atoms. 75% of all residues in myoglobin are found as part of 8 a-helices (no b-sheets). Residue side-chains are involved in hydrogen bonding, the exterior of the protein is almost exclusively polar and the interior is nearly all nonpolar. Two polar histidine residues are found in the interior and interact with the heme group. The heme group positions itself in a hydrophobic pocket of the protein and drastically affects structure by adding to how tightly folded the protein is (apoprotein). Heme group consists of protoporphyrin IX (organic) and Fe(II) (Fe2+) Fe(II) of heme has 6 coordinates sites; 4 interact with N atoms of porphoryn to give the heme group, the 5th with N of a His side chain, and the 6th with an O2 molecule.
The other His acts as a gate to open and close as oxygen comes in
to bind the heme.
Denaturation-
The unfolding of a protein. Noncovalent interactions are relatively weak and can be relatively easily disrupted. Disruption of first disulfide bonds leaves the protein susceptible to easy denaturation. Under proper conditions a completely denatured protein may regain its structure, renaturation. Ways to denature a protein:
Heat
increasing temp. will denature a protein (increase in vibrations).
pH
extremes changes charge which would interrupt interactions.
Detergents
like (sodium dodecyl sulfate) SDS can interrupt hydrophobic and charged
interactions.
b-Mercaptoethanol is a reducing agent commonly used to reduce disulfide bridges to
sulfhydryl groups. »
Usually used with Urea to interrupt hydrogen bonding as well. »
Important research especially in the field of reactive oxygen
species.
Quaternary structure.
A
functional protein consisting of more than one chain.
Dimers,
trimers and tetramers.
Normally
due to noncovalent interactions. Subtle changes in structure at one site can dramatically change properties at a distant site, proteins that exibit this property are called allosteric (often enzymes).
Ex:
Hemoglobin (Hb) an allosteric tetramer. Hemoglobin consists of 2 a-globin (141) and 2 b-globin (146) chains (heme group is the same). Both a- and b-globin are similar to myoglobin and similar to each other, homologous.
Most amino acid residues are the same and in the same position. 4 molecules of oxygen can bind hemoglobin cooperatively.
Cooperative binding means once one oxygen is bound it becomes easier for the next to bind.
Myoglobin oxygen binding curve hyperbolic while hemoglobin
is sigmoidal. »
Sigmoidal indicates cooperative binding. Myoglobin is for storage of oxygen (muscle) and must hold oxygen more tightly wile hemoglobin is for transport.
Hemoglobin gives up oxygen easily when and where it is needed
(capillaries). Hemoglobin changes structurally depending on whether oxygen is bound or not.
The b-chains are significantly more compact when oxygenated and
results in different crystal structures.
Conformational
changes accompany hemoglobin function. H+ and CO2 can affect the affinity of hemoglobin for oxygen by altering the structure. .
What does this mean physiologically? CO2 is produced by metabolism which in turn forms H2CO3 with a pKa of 6.35 which means HCO3- (releases H+) is the predominant form of dissolved CO2 at physiologic pH.
Therefore increased CO2 production leads to release of
oxygen by hemoglobin.
Allows for fine tuning of pH and oxygen concentration. 2,3-bisphosphoglycerate (BPG) also binds hemoglobin and serves to lower hemoglobins affinity for oxygen.
Hemoglobin with out BPG would not release much oxygen at all.
BPG (- charges) binds electrostatically to Hb (+charges).
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