DEXPITE UNIVERSITY OF ENTERTAINMENT

BCH 308

Protein Structure

 Factors contributing to the variation in 
protein structure and function:
 The type of amino acids (composition)
 The order in which they occur (sequence)

Classification of Proteins

 There are several levels of classifying 
proteins
 Two levels will be discussed in this course

Level I 
Proteins can be classified according to their 
shape and certain physical properties:
 Fibrous Proteins:  Are those in which the polypeptide chains are either 
extended or coiled to form linear fibres.  They are structural proteins, generally insoluble in an 
aqueous environment because the chain are held 
together in fibres (helices) or sheets by interchain
bonds.  Examples are: hair keratin, silk fibroin and collagen

.Level I Cont’d

 Globular Proteins: Are those in which the polypeptide chain or chains are tightly coiled

 in three dimensions to form approximately spherical shape molecules. 

 They are usually soluble in an aqueous environment and diffuse readily

. Most globular proteins have a dynamic or mobile 

function. 
 They contain a mixture of a-helix, b-pleated sheet and 
random structures.
 Globular proteins include; enzymes, transport 
proteins, hormones and immunoglobulins etc

.Level II:Proteins are also classified based on 
the function: 
 Structural Proteins: These are fibrous proteins. They 
are called structural proteins because they serve as 
supporting elements to biological structures strength 
and protection. Most common example is keratin.
 Dynamic proteins: They are globular proteins. They 
are called dynamic proteins because they are dynamic 
in nature and perform different functions in the cell.

 Polypeptides
 Two amino acids joined by a peptide bond = dipeptide . Three aas 
joined by two peptide bonds = tripeptide . Many aas joined = 
polypeptide . Oligopeptides are short peptide chains up to 20 aas.
 A polypeptide chain is a chain of amino acid residues linked together 
by peptide bonds. The backbone of the polypeptide is given by the 
repeated sequence of three atoms of each residue in the chain: the 
amide N, the alpha Carbon and the Carboxyl Carbon. 
 The existence of an amino group (N-Terminal) at one end of the chain 
and a carboxy group (C-Terminal) at the other end gives a direction 
(polarity) to the chain. 
 Conventionally the beginning of a polypetide is its N-Terminal. Polypeptides and Proteins
 " Polypeptide " refers to the structure of a single chain. Every 
polypeptide has one free amino group (called the "N-terminus") 
and one free carboxyl group (called the "C-terminus"). 
 " Protein " refers to the overall functional assembly, created 
when one or more polypeptides fold up and become functional 
units. Some proteins consist of only a single folded polypeptide 
chain, but many proteins contain multiple polypeptides, and 
frequently inorganic atoms as well, such as Zinc, Iron, 
Magnesium, etc. 
 Subunits are identical and/or nonidentical polypeptide 
chains of multisubunit proteins. Some important reactions of Peptides
 Peptides have characteristic chemical 
reactions:
 The peptide bond holding the amino acid residues 
in a peptide can be hydrolysed (enzyme, acid or 
base hydrolysis)
 The a-amino group of amino terminal residue of a 
peptide react with 1-fluoro-2,4-dinitrobenzene 
(FDNB) to form dinitrophenylpeptide. Therefore 
the amino terminal residue of a polypeptide chain 
can be labelledSome proteins are basically biologically 
active peptides:
 Examples are: Insulin (51 aa), glucagon (29 
aa), corticotropin (39 aa), oxytocin (9 aa), 
bradykinin (9 aa) etc

.Protein Structure
 What is structure? The arrangement of and 
relations between the parts or elements of 
something complex (New Oxford Dictionary).

Why study protein structure?
 Recognise the important aa residues in a 
protein
 The molecular basis of genetic disorders can 
be elucidated
 Can understand the relationship between 
homologous proteins
 Understand the mechanism of action of a 
proteinDifferent Levels of Protein Structure 
Organisation
 Proteins fold in three dimensions. Protein 
structure is organised hierarchically from socalled primary structure to quaternary structure. 
Higher-level structures are motifs and domains

. 
Protein Structure:Types of Protein Structure
 Four main types
 Primary  Structure
 Secondary Structure
 Tertiary Structure
 Quaternary  Structure

Forces holding the different protein 
structures
 To be able to perform their biological function, proteins 
fold into one, or more, specific spatial conformations, 
driven by a number of non-covalent interactions such as 
hydrogen bonding, ionic interactions, Van der Waals' 
forces and hydrophobic packing. 
 Primary structure: peptide bonds (covalent)
 Secondary structure: Mostly hydrogen bonds 
 Tertiary structure: Hydrogen bonds, ionic interations, Van der 
Waals’s forces and hydrophobic interactions.
 Quaternary structures: ionic interations, Van der Waals’s forces 
and hydrophobic interactions.Organisation of Protein StructureAMINO ACIDS (aa)
 Each amino acid has an amino group (NH2), and a carboxyl group

 (COOH) and a side chain (R) joined by a 

single Carbon atom (the alpha carbon, Ca). In exception of 
glycine, the Ca of all amino acids is a chiral centre.All amino acids found in proteins encoded by the genome 
have the L-configuration at this chiral centre. This 
configuration can be remembered as the CORN law. 
Imagine looking along the H-Ca bond with the H atom 
closest to you. When read clockwise, the groups attached 
to the Ca atom, spell the word CORN

The side chains of amino acids
 The R groups on the standard amino acids confer 
specific properties on each. These properties may 
depend on the solution pH. However, amino acids at pH 
7 can be classified into four families on the basis of how 
their R groups interact with water:
 Nonpolar (apolar): Gly, Ala, Val, Leu, Ile, Met, Pro, Phe 
and Trp.
 Polar uncharged: Ser, Thr, Asn, Gln, Tyr and Cys.
 Charged
 Negatively: Asp and Glu
 Positively: Lys, Arg and His.Steps in protein structure determination
1. Prepare a pure sample of protein of interest
2. Determine number of subunits
3. Sequence protein
4. Work out the primary structure
5. Work out the secondary structure
6. Determine tertiary structure and work out 
quaternary structure if possiblePrepare pure sample of protein for 
sequencing
 The protein of interest must be isolated:
 Cell disruption techniques
 Preparation of crude extract
 It must be purified:
 Proteins are purified by fractionation procedures. In a 
series of independent steps, the various 
physicochemical properties of the protein of interest 
are used to progressively separate it from other 
substances.

Some Practical Problems

 The major difficulty is that most proteins occur 
in small amounts: For this reason, the earliest 
proteins to be characterised were abundant e. 
g. haemoglobin. 
 To solve this problem of amount of sample
 Some proteins are studied in micro organisms that 
can be grown in large quantities
 Molecular cloning allows most protein-encoding 
genes to be isolated from the parent organism, 
specifically altered (genetically engineered) if 
desired and expressed at high levels in a micro 
organisms.Cell disruption and preparation of 
crude extract
 The first step in isolation of proteins or other biological 
molecule is to get them out of the cell and into 
solution.
 Many cells require some sort of mechanical disruption to 
release their content.
 Most procedures for lysing cells involve; crushing or grinding 
followed by filtration or centrifugation to remove large 
particles.The extract obtained at this stage is termed the 
crude extract. 
 If the protein of interest is tightly associated with a lipid 
membrane, a detergent or organic solvent may be used to 
solubilise the lipids and recover the protein.Separation techniques
 The idea here is not to minimise the loss of the 
desired protein, but to eliminate selectively the other 
component of the mixture so that only the required 
substance remains.
 Protein purification is considered as much an art as a 
science, with many options available at each step. 
Whilst “trial-and-error” approach can work, knowing 
something about the target protein simplifies the 
selection of separation procedures. Selective solubility
 Factors affecting solubility of proteins are:
 the concentration of dissolved salts 
 the polarity of the solvent
 the pH and temperature 
 By manipulating some or all these variables, 
certain proteins can be precipitated from 
others (selective precipitation).Selective solubility cont’d
 Proteins may be selectively precipitated 
by adding 
 Neutral salts such as ammonium sulphate
 Organic solvents such as ethanol or acetone
 Potent precipitating agents such as trichloroacetic 
acid
 Salting in is the phenomenon whereby the solubility of a 
protein increases at low ion concentration as salts are 
added.
 Salting out is the phenomenon by which the solubility of 
a protein decreases as more salts are added beyond a 
certain critical point.Below is a summary of some of the procedures of 
protein purification and the characteristic they depend 
on:
 Charge
 Polarity
 Size
 Binding affinity
 Ion exchange
 Electrophoresis
 Hydrophobic interaction 
chromatography
 Gel filtration 
chromatography
 SDS-PAGE
 Ultracentrifugation
 Affinity chromatographyDialysis
 This is the process used to separate proteins 
from low-molecular-weight substances 
present in the cell or tissue extract. 
 Proteins are high molecular weight 
compounds are therefore do not pass 
through cellophane.Gel filtration or molecular sieve
 This process sort proteins out on the basis of 
size and shape.
 This process is a form of chromatography. The mixture 
of proteins is passed down a column containing very 
small porous beads of highly hydrated polymer. The 
smaller particles penetrate into the pores in the bead 
and thus are retarded, but larger molecules cannot 
penetrate into the beads and pass down the column 
more rapidly.
 The gel material commonly in use is called dextran, a 
polymer glucose, which is commercially available as 
Sephadex.
 Note: The principle of molecular sieving is different from the everyday 
understanding

Sample question
 In what order would the following proteins 
emerge upon gel filtration of a mixture on 
sephadex-200:
 Myoglobin (Mr = 16,000), catalase (Mr = 500,000), 
cytochrome c (Mr = 12,000), chymotrypsin (Mr 
26,000), and serum albumin (Mr = 65,000)?Ion-exchange chromatography
 This process separates proteins on the basis of 
density and sign of their electric charges at a given 
pH.
 The principle here is that charged molecules bind to 
oppositely charged groups that have been immobilised on 
a matrix. Anions bind to cation group on anion exchanger, 
and cation binds to anion group on cation exchanger.
 The most frequently used anion exchanger is a matrix with 
attached diethylaminoethyl (DEAE) groups, and the most 
frequently used cation exchanger is a matrix bearing 
carboxymethyl (CM) groups.
DEAE: Matrix-CH2-CH2-NH(CH2CH3) 2+
CM: Matrix-CH-COO-

Sample question
 A solution containing egg albumin (pHI = 4.6), 
b-lactoglobulin (pHI = 5.2), and 
chymotrypsinogen (pHI = 9.5) was loaded 
onto a column of diethylaminoethyl cellulose 
(DEAE-cellulose) at pH 5.4. The column was 
then eluted with pH 5.4 buffer, with an 
increasing salt concentration. Predict the 
elution pattern.

END OF LECTURE 1

SOURCE: MR. A. JAMES(2012)