This action might not be possible to undo. Are you sure you want to continue?
Chapter 1 Protein Structure and analysis
Weihong Hou, Ph.D., Associate Prof. Department of Biochemistry
Section 1 Chemical Composition of Protein Amino Acids Section 2 Protein structure: Structure: primary→secondary → tertiary →
Domains, motif Section 3 Protein analysis Purification, sequence, mass, and structure analysis
1. 20 standard amino acids (except for proline) COOH3N+ Cα R 1) α -carbon is chiral (asymmetric) except in glycine (R is H) 2) Amino acids can exit in both D- and L- stereoisomers, but only Lisomers are found in proteins 3) Amino acids are dipolar ions (zwitterions in aqueous solution ) 4) The side chains (R) differ in size, shape, charge and chemical reactivity 5) A few proteins contain nonstandard amino acids that are formed by post-translational modification of the parent amino acids. H
2. Classification of Amino Acids
1) Amino acids with charged side chains (salt bridges, hydrophilic ) “Acidic” amino acids: containing additional carboxyl groups which are usually
aspartic acid (Asp, D)
glutamic acid (Glu, E)
“Basic” amino acids: containing positively charged groups
Lysine (Lys, K): a second amino group attached to the ε -carbon atom
Arginine (arg, R): a guanidine group attached to the δ -carbon atom
Histidine (His, H): a imidazole group has a pKa near neutrality. This group
can be reversibly protonated under physiological conditions, which contribute to the catalytic mechanism of many enzymes.
(2) Amino acids with polar uncharged side chains (hydrophilic) --containing groups that form hydrogen bonds with water Serine (Ser, S) & threonine (Thr, T) have hydroxyl groups.
Asparagine (Asn, N) & Glutamine (Gln, Q) are the amide derivatives of
Asp and Glu
Cysteine (Cys, C) has a thiol group which is often oxidizes to cystine
(3) Amino acids with nonpolar side chains aliphatic side chains Glycine (Gly, G)
Proline (Pro, P): imino acid
Methionine (Met, M): contains a sulfur atom
Alkyl side chains Alanine (Ala, A)
Valine (Val, V)
Leucine (Leu, L)
Isoleucine (Ile, I)
aromatic side chains
--aromatic structure accounts for most of UV absorbance of proteins at 280 nm
Phenylalanine (Phe, F)
Tyrosine (Tyr, Y)
Tryptophan (Trp, W)
Section 2 Protein structure 1. Shapes:
(1) Globular proteins( enzymes)
Complementary fit of a substrate molecule to the catalytic site on an enzyme molecule.
(2) Fibrous proteins: important structural
proteins (silk fibroin, keratin in hair and wools )
Keratin ( 角蛋白 )
Protofibril ( 初原纤维 )
microfibril ( 微管 )
keratin in hair
2. Protein structure (1) Primary structure
Polypeptides contain N- and C- termini and are directional, usually ranging from 100-1500 AAs
Formation of a peptide bond (shaded in gray) in a dipeptide.
N terminus C terminus
Structure of the pentapeptide Ser-Gly-Tyr-Ala-Leu.
(2) Secondary structure
• right-handed • 3.6 aa per turn • hydrogen bond N-H···O=C
A stereo, space-filling representation
Collagen triple helix: three polypeptide intertwined
Model of the α helix. The polypeptide backbone is folded into a spiral that is held in place by hydrogen bonds (black dots) between backbone oxygen atoms and hydrogen atoms. All the hydrogen bonds have the same polarity. The outer surface of the helix is covered by the side-chain R groups.
β -sheet: hydrogen bonding of the pepetide bond
N-H and C=O groups to the complementary groups of another section of the polypeptide chain
Parallel β sheet: sections run in the same direction Antiparallel β sheet: sections run in the opposite direction
A stereo, space-filling representation of the sixstranded antiparallel β sheet.
A two-stranded β sheet
β SHEETS. (a) A simple two-stranded β sheet with antiparallel β strands. A sheet is stabilized by hydrogen bonds (black dots) between the β strands. The planarity of the peptide bond forces a β sheet to be pleated; hence, this structure is also called a β pleated sheet, or simply a pleated sheet. (b) Side view of a β sheet showing how the R groups protrude above and below the plane of the sheet. (c) Model of binding site in class I MHC (major histocompatibility complex) molecules, which are involved in graft rejection. A sheet comprising eight antiparallel β strands (green) forms the bottom of the binding cleft, which is lined by a pair of α helices (blue). A disulfide bond is shown as two connected yellow spheres. The MHC binding cleft is large enough to bind a peptide 8 10 residues long.
(3) Tertiary structure
The different sections of secondary structure and connecting regions fold into a well-defined tertiary structure, with hydrophilic amino acids mostly on the surface and hydrophobic ones in the interior. The structure is stabilized by noncovalent interactions and sometimes disulfide bonds. Denaturation leads to loss of secondary and tertiary structure. Noncovalent interaction between side chains that hold the tertiary structure together: van der Waals forces, hydrogen bonds, electrostatic salt bridges, hydrophobic
Covalent interaction: disulfide bonds
The different sections of α -helix, β -sheet, other minor secondary structure and connecting loops of a polypeptide fold in three dimensions
Noncovalent interaction between side chains that hold the tertiary structure together: van der Waals forces, hydrogen bonds, electrostatic salt bridges, hydrophobic interactions Covalent interaction: disulfide bonds
Denaturation of protein by disruption of its 2o and 3o structure will lead to a random coil conformation
(4) Quaternary structure
Many proteins are composed of two or more polypeptide chains (subunits). These subunits may be identical or different. The same forces which stabilize tertiary structure hold these subunits together. This level of organization called quaternary structure.
The quaternary structure of hemoglobin 。 1-yellow; β 1-light
blue; α 2-green; β 2-dark blue; heme-red back
Domains: structurally independent units (tertiary) of a protein, being connected by sections with limited higher order structure within the same polypeptide. (Figure) They can also have specific function such as substrate binding
of secondary structural elements that frequently occur in globular proteins • Often have functional significance and represent the essential parts of binding or catalytic sites conserved among a protein family
β α β motif
The tertiary structures of the above c-type cytochromes
Cys, Met and His side chains covalently linked to the heme to the protein Back
Section 3 Protein analysis
1. Purification: to obtain enough pure sample for study 2. Sequencing: determine the primary structure of a pure protein sample 3. Mass determination: determine the molecular weight (MW) of an interested protein. 4. Determine the tertiary structure:X-ray crystallography and NMR.
1. Protein purification
• To purify the interested protein from other proteins and nonprotein molecules existing in the cells • An essential experimental step prior to study of any individual protein
The principal properties of proteins used for purification
Size: gel filtration chromatography Charge: ion-exchange chromatography, isoelectric focusing, electrophoresis Affinity: affinity chromatography Recombinant techniques: involving DNA manipulation and making protein purification so easy
(1) Gel filtration chromatography
Bead: the matrix determine the size of the pore
Column + anions
Column + proteins Column + anions
Protein migrate at different position depending on their net charge
(4) Affinity chromatography
• • • Enzyme-substrate binding Receptor-ligand binding Antibody-antigen binding
(5) Recombinant techniques
•Clone the interested protein encoding gene in an expression vector with a purification tag added at the 5’- or 3’ end of the gene •Protein overexpression in a cell •Protein purification with affinity chromatography.
2. Mass Determination
Gel filtration chromatography and SDS-PAGE
•Comparing of the unknown protein with a proper standard •Popular SDS-PAGE: cheap and easy with a 5-10% error •SDS: sodium dodecyl sulfate, makes the proteins negatively charged and the overall charge of a protein is dependent on its mass.
Mass spectrometry: •Molecules are vaporized and ionized, and the degree of deflection (mass-dependent) of the ions in an electromagnetic field is measured •extremely accurate, but expensive •MALDI can measure the mass of proteins smaller than 100 KDa •Helpful to detect post-translational modification •Protein sequencing: relying on the protein data base
3. Protein sequencing : Determine the
primary structure of a protein Specific enzyme/chemical cleavage: •Trypsin cleaves after lusine(K) or arginine (R) •V8 proteease cleaves after glutamic acid (E) • Cyanogen bromide cleaves after methionine (M) Edaman degradation: •Performed in an automated protein sequencer •Determine the sequence of a polypeptide from Nterminal amino acid one by one. •Expensive and laborious
Most protein sequences are deduced from the DNA/cDNA sequence Direct sequencing: determine the N-terminal sequences or some limited internal sequence construction of an oligonulceotide or antibody probe fishing the gene or cDNA
4. X-ray crystallography and NMR Determing the tertiary structure (3-D) of a protein
(1) X-ray crystallography: •Measuring the pattern of diffraction of a beam of X-rays as it pass through a crystal. The first hand data obtained is electron density map, the crystal structure is then deduced. •A very powerful tool in understanding protein tertiary structure •Many proteins have been crystallized and analyzed
(2) NMR •Measuring the relaxation of protons after they have been excited by radio frequencies in a strong magnetic field •Measure protein structure in liquid but not in crystal •Protein measured can not be larger than 30 KDa
This action might not be possible to undo. Are you sure you want to continue?
We've moved you to where you read on your other device.
Get the full title to continue listening from where you left off, or restart the preview.