Peptide Cleavage

Ramachandran plot A Ramachandran plot (also known as a Ramachandran diagram or a [φ,ψ] plot), originally developed in 1963 by G. N. Ramachandran, C. Ramakrishnan, and V. Sasisekharan,[1] is a way to visualize backbone dihedral angles ψ against φ of amino acid residues in protein structure and identify sterically allowed regions for these angles.

The figure at left illustrates the definition of the φ and ψ backbone dihedral angles[2] (called φ and φ' by Ramachandran). The ω angle at the peptide bond is normally 180°, since the partial-double-bond character keeps the peptide planar.[3] The figure at top right shows the allowed φ,ψ backbone conformational regions from the Ramachandran et al. 1963 and 1968 hard-sphere calculations: full radius in solid outline, reduced radius in dashed, and relaxed tau (N-Calpha-C) angle in dotted lines.[4] 

Because dihedral angle values are circular and 0° is the same as 360°, the edges of the Ramachandran plot "wrap" right-to-left and bottom-to-top. For instance, the small strip of allowed values along the lower-left edge of the plot are a continuation of the large, extended-chain region at upper left.

Biuret Testlab procedure:1.mix albumin and 10% NaOH and add 0.5% CuSO4 drop by drop –> dark violet color2.mix pentose and 10% NaOH and add 0.5% CuSO4 drop by drop –> light violet colorexplanation:•reacts with peptide bonds in protein•blue to violet color•Two peptide bonds at leastXanthoproteic reactionlab procedure:1.mix albumin with conc. nitric acid and heat –> yellow solution2.cool the solution and add ammonium hydroxide –> orange solutionexplanation:•Nitric acid gives a color when heated with proteins containing tyrosine (yellow color) or tryptophan (orange color); the color is due to nitration•aromatic phenyl ring is nitrated to give yellow colored nitro-derivatives•at alkaline pH, the color changes to orange due to the ionization of the phenolic group

Millon’s testlab procedure:1.mix albumin and millon’s rgt and heat–> red flocculent pptexplanation:•positive results with proteins containing the phenolic amino acid “tyrosine”

Glyoxylic acid reactionlab procedure:1.mix albumin with hopkins-cole reagent2.incline the test tube and add conc. sulfuric acid –>violet ringexplanation:•Hopkins-Colé reagent (magnesium salt of oxalic acid) gives positive results with proteins containing the essential amino acid “tryptophan” indicating a high nutritive value•specific test for detecting tryptophan

Heller’s Ring testlab procedure:1.mix 5 ml of conc. nitric acid with albumin by inclining the test tube –>white ringexplanation:•Nitric acid causes denaturation of proteins with the formation of a white ppt•used to test the presence of albumin in urine

Reduced Sulfur Testlab procedure:1.mix albumin with 40% NaOH and add 10 drops of lead acetate solution –> black pptexplanation:•Proteins containing sulfur (in Cysteine and cystine) give a black deposit of lead sulfide (PbS) when heated with lead acetate in alkaline medium

Adamkiewicz Reactionlab procedure:1.mix 3 drops of albumin with glacial acetic acid2.add conc. sulfuric acid to the solution–> violet ringexplanation:•detect the presence of the amino acid tryptophan in proteins•red/purple colour

The biuret test is a chemical test used for detecting the presence of peptide bonds. In the presence of peptides, acopper(II) ion forms violet-colored coordination complexes in an alkaline solution.[1] Several variants on the test have been developed, such as the BCA test and the Modified Lowry test.[2]The biuret reaction can be used to assess the concentration of proteins because peptide bonds occur with the same frequency per amino acid in the peptide. The intensity of the color, and hence the absorption at 540 nm, is directly proportional to the protein concentration, according to the Beer-Lambert law.Despite its name, the reagent does not in fact contain biuret ((H2N-CO-)2NH). The test is so named because it also gives a positive reaction to the peptide-like bonds in the biuret molecule.

Procedure[edit]An aqueous sample is treated with an equal volume of 1% strong base (sodium or potassium hydroxide most often) followed by a few drops of aqueous copper(II) sulfate. If the solution turns purple, protein is present. 5–160 mg/mL can be determined. A peptide of a chain length of at least 3 amino acids is necessary for a significant, measurable color shift with these reagents. [3]

Biuret reagent[edit]The Biuret reagent is made of sodium hydroxide (NaOH) and hydrated copper(II) sulfate, together with potassium sodium tartrate.[4] Potassium sodium tartrate[5] is added to complex to stabilize the cupric ions. The reaction of the cupric ions with the nitrogen atoms involved in peptide bonds leads to the displacement of the peptide hydrogen atoms under the alkaline conditions. A tri or tetra dentate chelate of with the peptide nitrogen produces the "buret" color. This is found with dipeptides (Datta,S.P., Leberman,R., and Rabin,B.R., Trans.Farad.Soc. (1959), 55, 2141.)The reagent is commonly used in the biuret protein assay, a colorimetric test used to determine protein concentration by UV/VIS spectroscopy at wavelength 565 nm.

High sensitivity variants of the biuret test[edit]Two major modifications of the biuret test are commonly applied in modern colorimetric analysis of peptides: the bicinchoninic acid (BCA) assay and the Lowry assay. In these tests, the Cu+ formed during the biuret reaction reacts further with other reagents, leading to a deeper color.In the BCA test, Cu+ forms a deep purple complex with bicinchoninic acid (BCA),[6] which absorbs around 562 nm, producing the signature violet color. The water-soluble BCA/copper complex absorbs much more strongly than the peptide/copper complex, increasing the sensitivity of the biuret test by a factor of around 100: the BCA assay allows to detect proteins in the range of 0.0005 to 2 mg/mL). Additionally, the BCA protein assay gives the important benefit of compatibility with substances like up to 5% surfactants in protein samples.In the Lowry protein assay Cu+ is oxidized back to Cu2+ by MoVI in Folin-Ciocalteu's reagent, which forms molybdenum blue (MoIV). Tyrosine residues in the protein also form molybdenum blue under these circumstances. In this way, proteins can be detected in concentrations between 0.005 and 2 mg/mL.[7]Molybdenum blue in turn can bind certain organic dyes such as malachite green and Auramin O, resulting in further amplification of the signal.[8]

Theory:                                                             Amino acids are building blocks of all proteins, and are linked in series by peptide bond (-CONH-) to form the primary structure of a protein. Amino acids possess an amine group, a carboxylic acid group and a varying side chain that differs between different amino acids.There are 20 naturally occurring amino acids, which vary from one another with respect to their side chains. Their melting points are extremely high (usually exceeding 200°C), and at their pI, they exist as zwitterions, rather than as unionized molecules.Amino acids respond to all typical chemical reactions associated with compounds that contain carboxylic acid and amino groups, usually under conditions where the zwitter ions form is present in only small quantities. All amino acids (except glycine) exhibit optical activity due to the presence of an asymmetric α – Carbon atom. Amino acids with an L – configuration are present in all naturally occurring proteins, whereas those with D – forms are found in antibiotics and in bacterial cell walls.

Protein folding

Protein folding is the process by which a protein structure assumes its functional shape or conformation. It is the physical process by which a polypeptide folds into its characteristic and functional three-dimensional structure

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. The resulting three-dimensional structure is determined by the amino acid sequence (Anfinsen's dogma).[2] Experiments [3] beginning in the 1980s indicate the codon for an amino acid can also influence protein structure.

The correct three-dimensional structure is essential to function, although some parts of functional proteins may remain unfolded,[4]

 so that protein dynamics is important. Failure to fold into native structure generally produces inactive proteins, but in some instances misfolded proteins have modified or toxic functionality. 

Relationship between folding and amino acid sequenceThe amino-acid sequence of a protein determines its native conformation. [7] A protein molecule folds spontaneously during or after biosynthesis. While these macromolecules may be regarded as "folding themselves", the process also depends on the solvent (water or lipid bilayer),[8] the concentration of salts, the pH, the temperature, the possible presence of cofactors and of molecular chaperones.

Minimizing the number of hydrophobic side-chains exposed to water is an important driving force behind the folding process. [9]

 Formation of intramolecular hydrogen bonds provides another important contribution to protein stability. [10] The strength of hydrogen bonds depends on their environment, thus H-bonds enveloped in a hydrophobic core contribute more than H-bonds exposed to the aqueous environment to the stability of the native state. [11]

The process of folding often begins co-translationally, so that the N-terminus of the protein begins to fold while the C-terminal portion of the protein is still being synthesized by the ribosome. Specialized proteins called chaperones assist in the folding of other proteins.[12]

Chaperonin

Chaperonins are proteins that provide favourable conditions for the correct folding of other proteins, thus preventing aggregation. Newly made proteins usually must fold from a linear chain of amino acids into a three-dimensional form. Chaperonins belong to a large class of molecules that assist protein folding, calledmolecular chaperones.[1] The energy to fold proteins is supplied by adenosine triphosphate (ATP).

Ensuring Proteins Behave Themselves

Chaperonins are made of two major subunits; in humans these subunits are Hsp60 and Hsp10. The larger subunit, Hsp60, is made of multiple ring-shaped structures that form a cage. The cage has a place (domain) to bind a protein and a domain to bind ATP in order to provide energy. Misfolded proteins become attached to the larger subunit by forming a temporary chemical bonds with the protein binding domain.

The smaller subunit, Hsp10, forms the cap of the chaperonin protein. When the cap binds to chaperonin, the larger subunit changes shape, which forces the misfolded protein trapped inside to fold into the proper shape. Once the protein has been folded properly, the smaller subunit comes off and the protein is released as depicted in Figure 2 above. Some proteins may go through more than one round of refolding before they resume the normal protein shape.

Chaperonins and Disease: Monitoring Protein Folding is Exhausting

Msfolded proteins form aggregates or clumps called amyloid plaques that are difficult for cells to destroy. Amyloid plaques interfere with signaling processes that normally occur in cells simply because they form large indestructible structures in the cell or they take on new functions that damage the cell.

Since misfolded proteins interfere with normal cellular functions, they are associated with diseases such as cystic fibrosis, Alzheimer's, Mad Cow, and Huntington's. The misfolded protein or proteins present in the aggregates will depend on the disease. For example, in Huntington's disease, huntington protein becomes misfolded and forms aggregates. In Alzheimer's disease, amyloid-beta peptide and tau protein form aggregates