chapter 2. structures and functions of proteins fei jia department of biochemistry and molecular...
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Chapter 2.
Structures and Functions of Proteins
Fei Jia Department of biochemistry and molecular biology, medical college of Ji Nan university
Functions of Proteins
A. Structural support: providing matrix, giving form to liv
ing organism, serving as structural components of cells.
B. Catalysis: enzymes catalyze chemical reaction
C. Transport: oxygen, iron, lipid, etc.
D. Coordinated motion: myosine and actin in muscle contraction
E. Specific binding to molecules Immune protection: immunoglobulin(Ig), interferon. Regulation of metabolism: insulin, growth hormone Regulation of gene expression: transcription factor
What is protein ?
Proteins are polypeptides, which are linear polymers of amino acids linked together by peptide bonds.
Proteins are synthesized from 20 different amino acids.
Chemical elements composition of proteins
C 50%-55%
H 6%-7%
O 19%-24%
N 13%-19%
S 0-4%
P a little
others a little
chemical elements composition of proteins
CHONSothers
Quantification of proteins
Proteins are the major compounds containing nitrogen, and with the average of 16% of the molecular weight, proteins can be quantified by testing the quantity of the component nitrogen.
1 gram of nitrogen equals to 6.25 gram of protein
Amino Acids: there are 20 standard amino acids that make up almost all naturally existing proteins.
Amino acid structures can be displayed as:
Structure of amino acids
H
glycine
CH3
alanineL-amino acidgeneral structure
R C +NH3
COO-
H
Mirror image structure of amino acids
D : dextrorotation L:laevogyrate
(D) COOH or (L) COOH
H C H2N H2N C H
R R (Only L-amino acids are found in proteins)
20 Standard Amino Acids
Name 3-letter 1-letter Name 3-letter 1-letterAlanine Ala A Leucine Leu LArginine Arg R Lysine Lys KAsparagine Asn N Methionine Met MAspartate Asp D Phenylalanine Phe FCysteine Cys C Proline Pro PGlutamine Gln Q Serine Ser SGlutamic acids Glu E Threonine Thr TGlycine Gly G Tryptophan Trp WHistidine His H Tyrosine Tyr YIsoleucineIle I Valine Val V
Essential Amino Acids
There are 8 amino acids that can not be adequately synthesized by the body and therefore must be obtained from the diet. These amino acids are called essential amino acids. Otherwise called nonessential amino acids. Essential amino acids include:
Lys, Trp, Val, Leu, Ile, Thr, Met, Phe.
Structural characteristics of Amino Acids
1) each has an -amino group, an -carboxyl group, and a side-chain. Amino acids are different in their side-chains (or R-groups).
2) each has two enantiomers due to the asymmetric -carbon atom (except Gly), but in proteins there are only L-amino acids.
3) the groups bounded to the -carbon atom can rotate to give different conformations (except Pro).
Classification of Amino Acids
According to the polarities of their R-groups,
amino acids can be classified as:
1) Neutral non-polar hydrophobic AA : Gly, Ala, Val, Leu, Ile, Phe, Pro.(with non-polar aliphatic or aromatic side chains )
2) Neutral polar AA: Trp, Ser, Tyr, Cys, Met, Asn, Gln,
Thr. (with polar non-ionic side chains )
3) Acidic AA: Asp, Glu. (with ionic side chains )
4) Basic AA: Arg, Lys, His. (with ionic side chains )
甘氨酸 glycine Gly G 5.97
丙氨酸 alanine Ala A 6.00
缬氨酸 valine Val V 5.96
亮氨酸 leucine Leu L 5.98
异亮氨酸 isoleucine Ile I 6.02
苯丙氨酸 phenylalanine Phe F 5.48
脯氨酸 proline Pro P 6.30
1.Neutral non-polar hydrophobic AA. Structure Name Name 3-letter 1-letter pI
色氨酸 tryptophan Try W 5.89
丝氨酸 serine Ser S 5.68
酪氨酸 tyrosine Try Y 5.66
半胱氨酸 cysteine Cys C 5.07
蛋氨酸 methionine Met M 5.74
天冬酰胺 asparagine Asn N 5.41
谷氨酰胺 glutamine Gln Q 5.65
苏氨酸 threonine Thr T 5.60
2. Neutral polar AA
天冬氨酸 aspartic acid Asp D 2.97
谷氨酸 glutamic acid Glu E 3.22
赖氨酸 lysine Lys K 9.74
精氨酸 arginine Arg R 10.76
组氨酸 histidine His H 7.59
3. Acidic AA
4. Basic AA
-OOC-CH-CH2-S+NH3
S-CH2-CH-COO-
+NH3
-OOC-CH-CH2-S+NH3
S-CH2-CH-COO-
+NH3
Cysteine
+
CystineDisulfide bond
-HH
-OOC-CH-CH2-SH
+NH3
-OOC-CH-CH2-SH
+NH3
HS-CH2-CH-COO-
+NH3
HS-CH2-CH-COO-
+NH3
Special amino acids
Proline( imino acid)
CH2
CHCOO-
NH2+
CH2
CH2
CH2
CHCOO-
NH2+
CH2
CH2
Physicochemical Properties of Amino Acids
1) Isoelectric point: at physiological pH, amino acids exist as dipolar ion (zwitterion).
R-CH-COOH
NH2
R-CH-COOH R-CH-COO- R-CH-COO-
NH3+
NH3 + NH2
Cation Zwitterion Anion
+OH- +OH-
+H++H+
Therefore, the isoelectric point (pI) of an amino acid is the pH at which it has no net charge.
pI depends on dissociation constants of α-NH
3+ and α-COOH of an amino acid.
When pH=pI, net charge=0 (zwitterion);
When pH>pI, net charge<0 (negative);
When pH<pI, net charge>0 (positive).
Roughly calculate the pI for an amino acid:
A) If:
K1 K2
+H3N-R-COOH +H3N-R-COO- H2N-R-COO-
then: pI= (pK1+ pK2)/2
2) Ultraviolet light absorption: some amino acids such as Trp(tryptophan ) and Tyr (tyrosine ) have a peak absorption at a wavelength of 280nm. This property is commonly used to determine the concentration of proteins in solution.
3) Ninhydrin reaction: Ninhydrin + amino acid blue-purple compound
This reaction is also used to quantitatively determine amino acids and proteins.
2. Peptide1) Peptide bond: a covalent bond between
the -amino group of one amino acid and the -carboxyl group of another.
H2N-CH-COOH + H2N-CH-COOH
R1 R2
H2O
H2N-CH-CO HN-CH-COOH
R1 R2
NH2-CH-CH OH
NH -O
2 CH-CH OH
glycine
+
-HOH
GlycylglycinePeptide
bond
NH2-CH-C-N-CH-CO
OHHHH
ONH2-CH-C-N-CH-C
O
OHHHH
O
glycine
NH CH COO
- -H OHH
2) Peptide: a linear structure formed by two or more amino acids linked via peptide bonds.
e.g. dipeptide, tripeptide, and octapeptide, are formed from two, three, and eight amino acids, respectively.
Generally, a peptide containing 2-10 amino acids is called oligopeptide; more than 10 amino acids form a longer peptide called polypeptide.
Amino acids in a peptide are called amino acid residues.
3. Molecular structure of proteins
Primary structure
Secondary structure
Tertiary structure
Quaternary structure
HigherStructures
Primary structure of proteins
1) Primary structure: refers to the linear sequence of amino acids in a protein.
2) The major linkage of the primary structure is peptide bond. In some cases disulfide bond is also involved.
3) Spatial structure and function of a protein depend on its primary structure. Notice: primary structure is not the only factor to maintain the spatial structure of a protein. 4) If the primary structure is altered, the function of the protein may also be changed.
e.g. bovine insulin, the first protein whose complete amino acid sequence analysed by Frederick Sanger in 1953.
Primary structure of bovine insulin
disulfide bond
Secondary structure1) Secondary structure of protein refers to the conformation adopted by local regions of polypeptid chain, just refers to the spatial conformation of the main chain of a peptide, regardless of the distribution of side chains.
including -helix 、 -pleated sheet 、 β-turn 、 motif.. The irregular
secondary structures are termed random coil or loop conformation.
-helixes: include right- and left-handed - helixes.
2) The force to maintain the secondary
structure of a protein is hydrogen bond.
-helixes: include right- and left-handed -
helixes.
Peptide unit
• peptide unit: peptide unit is a rigid , planar structure.The 4 members of the amide bond and other 2 α-carbons, which connect to the carboxyl carbon and the amino nitrogen respectively , are on the plane. This plane is termed peptide unit
Amide Planes
Properties of peptide unit The peptide’s C-N bond is 0.132nm.
N-Cα single bond is 0.147nm.
With ~40% double bond character, the amide C-N bonds are unable to rotate freely.
1)The six atoms of the peptide unit lie in a single plane. H and O are trans to each other.
2)The Cα-C bond and the N-Cαbonds are common
single bonds and permitted to rotate. Ψ(psi)for the Cα-C
bond and φ (phi) for the N-Cα bond,
Left- and right-handed -helixes
Right-handed -helix
left-handed -helix
-helix
0.54nm(3.6 residues)
Characters of-helix
α-helical structure is the simplest arrangement with its rigid peptide bonds
1) the peptide backbone is tightly wound around the long
axis of the molecule, and the helix is right handed with
fixed torsion angles ( = -57 = -47
2) At this right handed helix, 3.6 amino acid residues per
turn, and a pith of 5.4 nm are found. This means that the
span between two neighboring amino acids is 0.15 nm.
.
3) The hydrogen bonds are the major bonds to stabilize the
helix . The peptide C=O bond of a peptide unit points along
the helix towards N-H group of the 4th peptide unit to form a
hydrogen bonds . Each hydrogen bond closes the peptide
chain to form a ring with 13 atoms , of which the (helix can
be noted as 3.613 helix)
4) R groups of the amino acid residues protrude outward
from the helical backbone.
-pleated sheet1)The angles between peptide units are 110°, and the backbone of t
he polypeptide chain is extended into a zigzag shape.
2)The zigzag polypeptide chains are arranged side by side to form a series of pleats.
3)In -pleated sheet, the hydrogen bond occurs between neighboring segments of polypeptide chains.
4)In -pleated sheet, the neighboring hydrogen bonded polypeptide chains run in the opposite directions (antiparallel) or in the same direction(parallel )
5) R groups of adjacent amino acids protrude in opposite directions from the zigzag structure.
-pleated sheets:
hydrogen bonding
Peptide 1
Peptide 2
110 °
anti-parallel N C parallel N C C N N C
-pleated sheets: include parallel (running in the same direction, ) and antiparallel (running in the opposite direction) -pleated sheets.
the -pleated sheet
R groups of adjacent amino acids protrude in opposite directions from the zigzag structure.
-turn: -turn is a reverse turn and often occurs at protein surfaces.most-turn involve four successive amino acid residues and stabilized by a hydrogen bond located between residue 1 and 4.
-turn is formed when the polypeptide chain folds to make a 180-degree turn.
• Motif: certain arrangements of several (mostly 2-3) secondary structure elements with some specific functions can occur to form different protein structures, named motifs.
• They include the helix-turn-helix motif, zinc finger motif ,etc.
(zinc finger)(zinc finger)
N-terminal
C- terminal
Tertiary structure: describes the overall folding of a Poly- peptide chain including the spatial distribution of all atoms in the molecule. This refers to the spatial relationships between secondary structure elements. e.g. myoglobin:
The forces to maintain the tertiary structure of
a protein include hydrogen bond, hydrophobic
interaction, salt bonds, Van Der Waals force.
Generally, the hydrophobic side-chains are
buried in the interior and the polar or charged
side-chains are on the surface of the protein.
Forces to maintaining spatial conformation a hydrogen bonds , b salt bonds c hydrophobic interaction
Quaternary structure: refers to the spatial arrangement of subunits in a multisubunit protein.
A protein with quaternary structure contains at least two polypeptides (or subunits).
The forces to maintain the quaternary structure of a protein mainly are hydrogen bond, salt bond, Van Der Waals force and hydrophobic interaction
(Hemoglobin)
primary linear sequence (peptide bond) 1D
secondary local region, spatial arrangements 2-D
(hydrogen bonds)
tertiary three dimensional structure of overall fold 3-D
(hydrogen bond, salt bond, Van Der Waals force and hydrophobic interaction)
quaternary non-covalent mutimerization of subunits 4-D
(single polypeptides) into protein complexes (hydrogen bond, salt bond, Van Der Waals force and hydrophobic interaction)
Summary of Structure of Proteins
Primary structure dictates folding of higher structures
Primary structure-function relationships of Protein
• The primary structure of a protein is the material base of its three-dimensional structure.
• The spatial structure of a protein determines its biological functions.
• Proteins with homogeneous amino acid in the sequences have similar spatial structures and function.
Primary structure-function relationships of InsulinIsulins from different animal species contain two polypeptide chains and have high homogeneity and similar three dimensional structure. Isulins from different animal species show a residue identity in 22 amino acid positions.
there are great varieties in A5, A6, A10,B30, but the biological function is identical.
The 4 amino acid residues are essential for maintaining the the correct spatial structure and function (Gly-Ile-Val-Glu.)
Variation in positions in insulines from some animal species
Species Variation in positions in insulines from some animal species
A5 A6 A10 B30
Human Thr Ser Ile Thr
Pig Thr Ser Ile Ala
Dog Thr Ser Ile Ala
Rabbit Thr Ser Ile Ser
Cow Ala Ser Val Ala
Sheep Ala Gly Val Ala
Horse Thr Gly Ile Ala
Sperm whale Thr Ser Ile Ala
N-val · his · leu · thr · pro · glu · glu · · · · ·C(146)
HbS β chain
HbA β chain
N-val · his · leu · thr · pro · val · glu · · · · ·C(146)
Sickle cell hemoglobin: The hydrophilic residue glutamic acid in 6th position of β chain is substituted by a hydrophobic residue valine.the results into a high rate of hemolysis and sickle cell anemia.
This kind of disease caused by site mutation of gene coding this protein is termed “molecule disease”
Evolutionary tree of Cytochrome cCytochrome c is an evolutionary conservative. A phylogenetic tree indicate the ancestral relationships among the orgnism that produced the protein.
Structure-function relationships of Myoglobin and Hemoglobin
Structure-function relationships of Myoglobin and Hemoglobin
Myoglobin (Mb): the first protein to have its 3-d
structure solved by X-ray crystallography. It is located
in muscle.
The major functionof Mb is bind, store, and release O2
with changes in the O2 concentration in skeletalmuscle.
Mb + O2 MbO2
Structural characteristics of myoglobin: a) it consists of a single polypeptide chain o
f 153 amino acid residues and a heme prosthetic group;
b) it has a compact structure with 75% of the residues in -helix, referred to as “A” through “H”;
c) Hydrophobic groups locate in the core of the molecules,hydrophilic groups expose at the surface of the spherical molecule.
Myoglobin
•
N-terminal
C- terminal
Tertiary structure of myoglobin
F8 His
Porphyrinplane
Structure of heme
Oxygen dissociation curves of Hb and Mb
Rectangular hyperbola(myoglobin)S-shaped(hemoglobin)
Hemoglobin (Hb): hemoglobin is a tetrameric
protein which consists of two -chains (141
residues each) and two -chains (146 residues
each). The conformation of each subunit is
similar to that of myoglobin.
Function: as the oxygen transporter in RBC.
Hb + 4O2 Hb(O2)4
Salt bonds between and within subunits in deoxyHb
Conformations of hemoglobin
Oxygen dissociation curves of Hb and Mb
This shape indicates that the affinity of Hb for binding
o2 is relatively low.
Cooperativity
Cooperativity refers to the allosteric effect of
the action of one subunit on other’s biological
activity. If the action promotes the activity of
other subunits, it is said to be positive; if
otherwise inhibits other subunit’s activity,
then negative co-operativity.
O2
F8 His
F8 His
F peptid
F peptid
Porphyrin plane
Allosteric property of hemoglobin
Hb is an allosteric protein: binding of O2 to one
subunit induces conformational change of other
subunits, making them more able to bind O2.
O2 causing the changes is nanmed allosteric
Effecter.
O 2 O 2 O 2 O 2 O 2 O 2 O 2
O 2 O 2 O 2
Protein conformational disease Mad cow disease is an illness that attacks the
brain and spinal cord in adult cattle,caused by pr
ion protein(PrP) PrPsc
PrPc
α-helix
PrPsc
β-pleated sheet
normal Mad cow disease
Prion protein(PrPsc)
Prion protein(PrPsc)
Prion disease involve a novel mechanism of pathology which is the alteration of the secondary –tertiary structure of a naturally occurring protein, PrPc.
PrPc that lacks nucleic acid does not replicate itself ,but it can serve as a template upon which the α-helical structure of PrPc becomes the β-pleated sheet structure of PrPsc.
Physicochemical properties of protein
1) Isoelectric point(pI): proteins contain ionizable groups such as amino and carboxyl groups. When the net charge is zero the pH is referred to as pI of the protein.
NH3+ NH3
+ NH2
P P P
COOH COO - COO -
Cation(pH<pI) Zwitterion (pH=pI) Anion (pH>pI)
+OH- +OH-
+H+ +H+
2) Denaturation, precipitation and coagulation:Denaturation refers to the loss of native confor
mation and biological activity of the protein. Denatured proteins decrease in solubility, increase in stickiness, and are vulnerable to proteolytic enzymes.
Precipitation is the process by which protein separates out from the aqueous solution. Protein can be precipitated by removal of the two stabilizing factors: electrostatic repulsion and hydration shell.
Coagulation is the solidification change of proteins caused by physical or chemical factors.
3) Ultraviolet light absorption of proteins results from the residues that have peak absorbance at 280nm such as Trp, Tyr. This property is commonly used for quantitative determination of proteins.
4) Color reactions: such as ninhydrin and biuret reactions. Color reactions are also used for analysis of proteins.