1、 Denaturation - disruption of native conformation of a protein, with loss of biological activity Energy required is small, perhaps only equivalent to 3-4 hydrogen bonds Proteins is commonly denatured by heating Denaturation takes place over a relatively small range of temperature. Under physiologica
2、l conditions, most proteins are stable at temperatures up to 50 to 60 . However some are not. 4.6 Protein denaturation and renaturation Heat denaturation of ribonuclease A Unfolding monitored by changes in ultraviolet (blue), viscosity (red), optical rotation (green) Tm What is Tm ? Proteins can als
3、o be denatured by two types of chemicals-chaotropic agents and detergents. For example, urea, guanidinium salts, SDS. Why can the chaotropic agents and detergents result in denaturation of proteins ? The native conformation of some proteins is stabilized by disulfide bonds. Disulfide bridges in bovi
4、ne ribonuclease A (a) Location of disulfide bridges (b) Stereo view of Cys- 26 and Cys-84 a) There are four disulfide bridges in bovine ribonuclease A. (b) The bridge between Cys-26 and Cys 84 is shown in stereo. Can proteins be renatured ? Anfinsens experiments (next page). Conclusion: The conforma
5、tion of proteins are determined by their primary structure. Protein disulfide isomerase (PDI) and E.coli disulfide isomerase (DsbA) can help misfolded proteins to form correct disulfide bonds. Anfinsens experiments-Denaturation and renaturation of ribonuclease A Treatement of native ribonuclease A (
6、top) with urea in the presence of 2- mercaptoethanol unfolds the protein and disrupts disulfide bonds to produce reduced, reversibly denatured ribonuclease A (bottom). When the denatured protein is returned to physiological conditions in the absence of 2- mercaptoethanol, it refolds into its native
7、conformation and the correct disulfide bonds form. Protein folding and stability Folded proteins occupy a low-energy well that makes the native structure much more stable than alternative conformations. Many proteins can fold spontaneously to this low- energy conformation Folding of proteins is coop
8、erative. Folding of proteins is extremely rapid-in most cases the native conformation is reached in less than a second. Energy well of protein folding Funnels represent the free energy potential of folding proteins The funnels represent the free- energy poteintial of folding proteins. Rather than fo
9、llowing a series of folding steps, folding to the final structure is illustrated as a parallel process with many possible routes to the lowest energy structure. (a) In this simplified funnel showing two possible pathways to the lowest energy native structure, path A reaches the lowest energy structu
10、re directly. In path B the polypeptide enters a local low-energy minimum in the process of folding. (b) A more realistic view of the possible free-energy forms of a folding protein includes many local peaks and dips. Proteins folding and stabilization depend on several noncovalent forces and disulfi
11、de bond. Hydrophobic effect is the most important for stability of tertiary and secondary structure. Nonpolar side chains associate with each other causing a polypeptide chain to collapse to a molten globule Forces That Stabilize Protein Structure Hydrogen bond is very important for stability of sec
12、ondary structure. They are more stable in hydrophobic envirement than in hydrophilic envirement . Contributes to cooperativity of folding Helps stabilize secondary structures and native conformation Examples of hydrogen bonds The hydrogen bond donors The hydrogen bond donors and acceptors are shown.
13、 and acceptors are shown. The most common donorThe most common donor- - acceptor pair in proteins acceptor pair in proteins is the is the amideamide- -carbonylcarbonyl, , which is found between which is found between peptide groups. peptide groups. All bonds are All bonds are approximately approxima
14、tely 0.3nm 0.3nm in in length, with the shortest length, with the shortest distance for the shared distance for the shared bonds betwen the most bonds betwen the most similar bond donor similar bond donor acceptor pair.acceptor pair. Van der Waals and Charge-Charge Interactions VDW contacts occur be
15、tween nonpolar side chains and contribute to the stability of proteins Charge-charge interactions between oppositely charged side chains in the interior of a protein also may stabilize protein structure Protein Folding Is Assisted by Chaperones Molecular chaperones increase rate of correct folding a
16、nd prevent the formation of incorrectly folded intermediates particularly in very large proteins, smaller proteins need very little assistance. Chaperones can bind to unassembled protein subunits to prevent incorrect aggregation before they are assembled into a multisubunit protein Most chaperones a
17、re heat shock proteins (synthesized as temperature increases) E. coli chaperonin The core structure of chaperonin consists of two identical rings composed of seven GroEL subunits. Unfolded proteins bind to the central cavity. Bound ATP molecules can be identified by their red oxygen atoms (spacefill
18、). The quaternary structure is shown from (a) the side, and (b) the top. PDB 1DER (c) During folding, the size of the central cavity of one of the rings increases and the end is capped by a protein containing seven GroES subunits. PDB 1AON. (a) (b) Core consists of 2 identical rings (7 GroE subunits
19、 in each ring) (c) Protein folding takes place inside the central cavity Chaperonin-assisted protein folding The unfolded polypeptide enters the central cavity of chaperonin, where it folds. The hydrolysis of several ATP molecules is required for chaperonin function. The three dimensional proteins s
20、tructure is shown in Figure. The lines on the chaperonin cylinder are to represent the 7 identical GroEL subunits that make up each ring. Not shown is the end cap composed of GroES subunits. Hypothetical folding pathways are: 1. The polypeptide collapses upon itself due to the hydrophobic effect, an
21、d elements of secondary structure begin to form; 2. Subsequent steps involve rearrangement of the backbone chain to form characteristic motifs; 3. The stable native conformation. Each domain in a multidomain protein folds independently. Hypothetical protein-folding pathways During folding the polype
22、ptide collapses in upon itself due to the hydrophobic effect An intermediate “molten globule” forms with elements of secondary structure The backbone is rearranged to achieve a stable native conformation 1. -Keratin 2. -Keratin 3. Collagen 4.7 Fibrous Protein 1. -Keratin： 右手右手- 螺旋螺旋 原纤维（左旋的三原纤维（左旋的三
23、 股股- 螺旋，直径螺旋，直径 2nm） 微原纤维（直径微原纤维（直径 8nm） 大原纤维（直径大原纤维（直径 200nm） 硬角蛋白硬角蛋白含硫量高（二硫键多），如：蹄、爪、角、甲含硫量高（二硫键多），如：蹄、爪、角、甲 软角蛋白软角蛋白含硫量低（二硫键少），如：皮肤含硫量低（二硫键少），如：皮肤 2. -Keratin silk fibroin 3. Collagen 1Collagen is a major protein in connective tissue of vertebrates (25-35% of total protein in mammals) 2. Distrib
24、ution and types： type Itype XII， 3Amino acid composition: Gly, Pro, 4-OH-Pro, 3-OH-Pro, 5-OH-Lys (glycoprotein). 4-Hydroxyproline and 5-hydroxylysine are Formed by enzyme hydroxylation reactions (require vitamin C) after incorporation into collagen Vitamin C deficiency (scurvy) leads to lack of prop
25、er hydroxylation and defective triple helix (skin lesions, fragile blood vessels, bleeding gums Unlike most mammals, humans cannot synthesize vitamin C 肽链肽链( (helical helical chainschains) ) （非（非螺螺 旋，更伸展，左手）旋，更伸展，左手） 三股螺旋三股螺旋(triple (triple helices, helices, procollagenprocollagen) ) three three lef
26、tleft- -handed helical handed helical chains coiled chains coiled around each other around each other in a rightin a right- -handed handed supercoil supercoil （右手超（右手超 螺旋缆）螺旋缆） 原胶原分子原胶原分子 (Tropocollagen) (Tropocollagen) 胶原（原）纤维胶原（原）纤维 (collagen fibers)(collagen fibers) Stereo view of human Type III
27、collagen triple helix The extended view of the human collagen type III triple helix contains three identical subunits (purple, light blue and green). Three left- handed collagen helices are coiled around one another to form a right- handed supercoil. PDB 1BKV Interchain hydrogen bonding holds the th
28、ree collagen strands together. The amide hydrogen of a glycine residue in one chain is hydrogen bonded to the caronyl oxygen of a residue, often proline, in an adjacent strand. Multiple repeats of -Gly-X-Y- where X is often proline and Y is often 4-hydroxyproline Glycine residues are located along c
29、entral axis of a triple helix (other residues cannot fit) For each -Gly-X-Y- triplet, one interchain H bond forms between amide H of Gly in one chain and -C=O of residue X in an adjacent chain No intrachain H bonds exist in the collagen helix Gly , because its small size, is required at the tight ju
30、nction where the three chains are in contact (motif G-X-X). Interchain H bonding in collagen Amide H of Gly in one chain is H- bonded to C=O in another chain Covalent cross-links in collagen Collagen triple helices aggregate in a staggered fashion to form strong, insoluble fibers. The strength of co
31、llage fibrils result from covalent cross-links between collagen molecules Two allysine residues condense to form an intramolecular cross-link Glycosylation Tropocollagen assemble into collagen fibers. Crosslinks are formed between lysines through aldol condensation and dehydration. The newly transla
32、ted collagen is hydroxylated and glycosylated and triple helices are formed. The procollagen triplexes are exported and the globular domains are cut off to form the tropocollagen The newly translated collagen is hydroxylated and glycosylated and triple helices are formed. The procollagen triplexes a
33、re exported and the globular domains are cut off to form the tropocollagen Tropocollagen assemble into collagen fibers. The newly translated collagen is hydroxylated and glycosylated and triple helices are formed. The procollagen triplexes are exported and the globular domains are cut off to form th
34、e tropocollagen Tropocollagen assemble into collagen fibers. The newly translated collagen is hydroxylated and glycosylated and triple helices are formed. The procollagen triplexes are exported and the globular domains are cut off to form the tropocollagen Crosslinks are formed between lysines throu
35、gh aldol condensation and dehydration. Tropocollagen assemble into collagen fibers. The newly translated collagen is hydroxylated and glycosylated and triple helices are formed. The procollagen triplexes are exported and the globular domains are cut off to form the tropocollagen 4.8 Structure and fu
36、nction of myoglobin Myoglobin is composed of 8 helices Heme prosthetic group binds oxygen His-93 (proximal histidine) is complexed to the iron atom, and His-64 (distal histidine) forms a hydrogen bond with oxygen Interior of Mb almost all hydrophobic amino acids Heme occupies a hydrophobic cleft for
37、med by 3 helices and 2 loops Sperm whale oxymyoglobin Oxygen (red) His-93 and His-64 (green) Sperm whale (Physeter catadon) oxymyoglobin consists of eight alpha helices. The heme prosthetic group binds oxygen (red spacefill to right of heme). His-64 (green, right) forms a hydrogen bond with oxygen,
38、and His- 93 (green, left) is complexed to the iron atom of the heme. PDB 1A6M The heme is shown in spacefill in gray and red. Unlike hemoglobin, the structure of myoglobin does not change significantly upon binding or release of oxygen. The structure of myoglobin The structure of heme group Heme (pr
39、osthetic group) consists of a tetrapyrrole ring system called protoporphyrin IX complexed with iron. Porphyrin ring provides four of the six ligands surrounding iron atom Oxygen Binds Reversibly to Heme Oxymyoglobin - oxygen bearing myoglobin Deoxymyoglobin - oxygen-free myoglobin In oxymyoglobin, s
40、ix ligands are coordinated to the ferrous ion in octahedral symmetry Oxygen is coordinated between the iron and the imidazole sidechain of His-64 Binding of O2 and myoglobin Oxygen-binding site of whale oxymyoglobin The heme prosthetic group is The heme prosthetic group is represented by a parallelo
41、gram represented by a parallelogram with a nigrogen atom at each with a nigrogen atom at each corner. The blue dashed lines corner. The blue dashed lines illustrate the octahedral illustrate the octahedral geometry of the coordination geometry of the coordination plex. The hydrogen bond between The
42、hydrogen bond between HisHis- -6464 and the oxygen molecule and the oxygen molecule is shown in yellow. The is shown in yellow. The presence of Hispresence of His- -64 prevents 64 prevents oxygen and other diatomic oxygen and other diatomic molecules from binding molecules from binding perpendicular
43、ly to the heme perpendicularly to the heme plane.plane. (distal histidine) (proximal histidine) Proximal histidine residue Oxygen-binding in myoglobin Fe(II) (orange) lies in the plane of the heme group. Oxygen (green) is bound to the iron atom and the amino acid side chain of His-64. Val-68 and Phe
44、-43 contirbute to the hydrophobic environment of the oxygen binding site. PDB 1AGM His-93 complexes on the distal side of the heme from oxygen, making the 5th coordinating nitrogen around the Fe(II). The binding of oxygen in subunits of hemoglobin is almost identical. Conformation change of myoglobi
45、n to bind O2 Oxygen-binding curve of myoglobin Y O2 fractional saturation (Y) is plotted versus the partial pressure of oxygen, pO2 (oxygen concentration) Mb-O2 binding curve is hyperbolic, indicating a single equilibrium constant for binding O2 4.9 Structure and function of hemoglobin 蛋白质的结构四级结构 Th
46、e structure of hemoglobin The quaternary of hemoglobin Conformation change of hemoglobin to bind O2 Some salt bridges in hemoglobin Conformational changes in a hemoglobin chain induced by oxygenation Oxygen binding to Fe pulls the His toward ring plane Helix with His shifts position, disrupting some
47、 ion pairs between subunits (blue to red position) When the heme iron of a hemoglobin subunit is oxygenated (red), the proximal histidine residue is pulled toward the porphoryn ring. The helix containing the histidine also shifts position, disrupting ion pairs that cross-link the subunits of deoxyhe
48、moglobin. The “proximal histidine“ is His-93 that is complexed to the heme iron. His-64 is sometimes referred to as the “distal histidine.“ The movement of the Fe is only about 0.34nm but leads to a change in oxidation state (as observed in the color change) and a change in the overall structure of
49、hemoglobin through this initial movement of the proximal histidine. 0 20 40 60 80 100 120 100 80 60 40 20 0 Percent O2saturation Partial pressure of oxygen (pO2, mmHg) Muscle in exercising Relaxing muscleMyoglobin Hemoglobin Artery O2 Vein O2 Environmental Oxygen Effects Binding Affinity Adapted from Garrett & Grisham (
