1、Chapter 4 Proteins: Three- Dimensional Structures and Function 4.1 Methods for determining protein structrue 4.2 The conformation of the peptide group 4.3 Secondary structures of proteins 4.4 Tertiary structure of proteins 4.5 Quaternary structure of proteins 4.6 Protein denaturation and renaturatio
2、n 4.7 Fibrous protein 4.8 Structure and function of myoglobin 4.9 Structure and function of hemoglobin 4.10 Antibodies bind specific antigens 4.11 Measurement of protein 1. X-Ray Diffraction： 2. Nuclear Magnetic Resonance (NMR)： 3. Circular Dichroism (CD) ： 4.1 Methods for determining protein struct
3、rue (a) X-ray diffraction data is collected by sending a beam of collimated X-rays through a single protein crystal. The diffracted rays are detected on a piece of film. (b) Shown is the diffraction pattern of a crystal of adult human deoxyhemoglobin. Darker spots result from larger numbers of X- ra
4、ys being diffracted to that location. The location and intensity of the spots on the film are used to determine the three dimensional structure of the protein. Diffraction is evaluated at many different angles of incidence of the X-ray beam. Film may be cylindrical (as shown in A) or possibly spheri
5、cal. 1.X-Ray Diffraction Ribonuclease A (RNase A) is a secreted enzyme that hydrolyzes RNA during digestion. This diagram utilizes the structure of the bovine RNAse A. (a) Shown is a space-filling model of RNAse A with a bound substrate (black stick model.) (b) The same enzyme is shown with a ribbon
6、 model of the protein backbone. (c) This close-up view of the substrate binding site depicts the substrate analog (5- diphosphoadenosine- 3-phosphate) in space- filling model. The side-chains of the amino acid residues in the active site are shown in ball-and-stick model while the remainder of the p
7、rotein is represented in ribbon form. PDB 1AFK NMR (nuclear magnetic resonance) is used to analyze protein structure in solution Ribonuclease A determined by NMR (polypeptide chain backbone). The figure combines a set of very similar structures that satisfy the data on atomic interctions. Only the b
8、ackbone of the polypeptide is shown. Note the presence here of disulfide bridges (yellow) which are not shown in the X-ray derived structure. PDB code 2AAS 2. Two-dimensional NMR 3. Circular dichroism (CD) a a-helix a a-helix: 190 nm (+) 208nm, 222nm (-) q x E-3 0 200 210 220 230 240 nm b b-sheet: 1
9、95nm (+) antiparallel: red shift parallel: blue shift 215-217nm (-) b b-sheet Unordered structure: (-) below 200nm (+) 218nm weak band unordered b b-turn: 180-190nm (-) 200-205nm (+) 225nm (+) band, weak red shift Far UV（190-240nm) Protein conformation - three dimensional shape Native conformation -
10、 each protein folds into a single stable shape (physiological conditions) Biological function of a protein depends completely on its native conformation 4.2 The conformation of the peptide group 1.1. Peptide group (Peptide group (CONH ) peptide plane(amide plane) peptide plane(amide plane) Because p
11、eptide bonds are partial double bondBecause peptide bonds are partial double bond 2.2. Dihedral angle of CDihedral angle of C : (N: (N- -C C ) and (C) and (C -C)C) 3. 3. Ramachandran plotRamachandran plot =0o =0o Most of combination of and are sterically forbidden. G. N. Ramachandran and his co-work
12、ers in Madras, India, first showed that it was convenient to plot to show the distribution of allowed values in a protein or in a family of proteins. Permissible angles Incompletable permissible angles (only 20.3%) Nonpermissible angles 1. The a a Helix 2. strands and sheets 3. Loops and turns 4. Ra
13、ndom coil 4.3 Secondary structures of proteins 1. The a a-Helix Each C=O (residue n) forms a hydrogen bond with N-H of residue n+4 further towards the C-terminus Helix is stabilized by many hydrogen bonds (which are nearly parallel to long axis of the helix) All C=O groups point toward the C-terminu
14、s (entire helix is a dipole with (+) N, (-) C- termini) Pitch is 0.54nm (recurrence of equivalent positions) Rise - Each residue advances by 0.15nm along the long axis of the helix There are 3.6 amino acid residues per turn Structural feature of a a- helix The entire helix is a dipole with a positiv
15、e N- terminus and a negative C- terminus. N terminus chirality and rotation opticity of a a-helix Right-handed is more stable than left-handed. So most a a helices in proteins are right handed (backbone turns clockwise when viewed along the axis from the N terminus) Cooperativity in formation C term
16、inus Types of a a-helix: 3.613helix； 310helix； helix（4.416helix） Hydrogen bonds between AAs are especially stable in the hydrophobic interior of a protein; The average content of a helix is 26% in a total protein; The length of a helix in a protein can range from about 4 or 5 AAs to more than 40 AAs
17、, but the average is about 12 AAs; Many a a-helix amphipathic, with the hydrophilic side chains facing outward and the hydrophobic side chains facing inward. All side chains project outward from helix axis The purple ribbon indicates the shape of the polypeptide backbone. All the side chains, shown
18、as ball-and-stick models, project outward from the helix axis. This example is from residues Ile-355 (bottom) to Gly-365 (top) of horse liver alcohol dehydrogenase. Some hydrogen atoms are not shown. PDB 1ADF (a) Amino acid sequence, (b) Helical wheel diagram Highly hydrophobic residues are blue, le
19、ss hydrophobic residues are green, and highly hydrophilic residues are red in both the (a) sequence of amino acids and (b) helical wheel diagram. Although it is not obvious in the primary structure, the helical wheel diagram reveals that the hydrophobic and hydrophilic residues are on the same sides
20、 of the helix with others of the same type. In general, hydrophobic residues are more commonly found on the same side of an alpha helix, as are hydrophilic groups oriented in the same direction. If the alpha helix is located at the surface of a soluble protein, the hydrophobic side will likely be or
21、iented towards the inside, while the hydrophilic side will be oriented out. The amphipathic alpha helix is highlighted in the full structure of liver alcohol dehydrogenase from horse. The side chains of highly hydrophobic residues are shown in blue, less hydrophobic residues in green, and charged re
22、sidues are shown in red. Note that the side chains of the hydrophobic residues are directed toward the interior of the protein and that the side chains of charged residues are eposed to the surface. PDB 1ADF Leucine zipper of yeast Interactions of two alpha helixes are common The The “leucine zipper
23、“leucine zipper“ is a “ is a dimerization motif commonly dimerization motif commonly found in DNA binding proteins. found in DNA binding proteins. DNA binding region consists of two DNA binding region consists of two amphipathic a helices, one from amphipathic a helices, one from each of two protein
24、 subunits. each of two protein subunits. GCN4 is a transcription regulatory protein that binds to specific DNA sequences. The DNA binding region consists of two amphipathic alpha helices, one from each of the two subunits of the protein. The side chains of Leu residues are shown in dark blue off of
25、the lavender ribbon. Only the leucine zipper region of the yeast (Saccaromyces cerevisiae) GCN4 protein is shown in the figure. PDB 1YSA side chains of Leu residues The stability of an The stability of an a a- -helix is helix is affected by:affected by: H N C OH O 2.Size of R groups2.Size of R group
26、s 1.Same charge of AAs1.Same charge of AAs H2NCH C H OH O Pro GlyGly 2. strands and sheet -Strands - polypeptide chains that are almost fully extended -Sheets - multiple strands arranged side-by-side = most common form of arrangement in proteins -strands are not more stable. However -sheets are stab
27、ilized by hydrogen bonds between carbonyl oxygens and amide hydrogens on adjacent -strands. The -strands in -sheet can be either parallel or antiparallel. Parallel sheets are less stable than antiparallel sheets. Structural feature of -sheet b b-Sheets : (a) parallel, (b) antiparallel N端端 C端端 N端端 C端
28、端 N端端 C端端 A typical -sheet contains from 2 to as many as 15 individual -strands. Each strand has an average of 6 AAs. Some proteins are almost entirely -sheets but most proteins have a much lower -strands. The side of a -sheet facing the protein interior tends to be hydrophobic, and the side facing
29、the solvent tends to be hydrophilic. Parallel sheets are usually hydrophobic on both sides and are buried in the interior of a protein. Interactions of b b sheets b-Sheet side chains project alternately above and below the plane of the b strands One surface of a b-sheet may consist of hydrophobic si
30、de chains that can interact with other hydrophobic residues in protein interior Amphipathic a-helices have hydrophobic side chains projecting outward that can interact with hydrophobic faces of b-sheets or other helices Structure of PHL P2 protein (a) The two short two-stranded antiparallel beta she
31、ets are highlighted in blue and purple to show their orientation within the protein. (b) This close-up of just the two pairs of beta strands highlights the location of hydrophobic (blue) and polar residues (red) . An number of hydrophobic interactions connect the two sheets. Many strands that make u
32、p - sheet are twisted and the sheet is distorted and buckled. The and angles of the bonds in a strand are restricted to a broad range of values occupying a large, stable region in the upper left-hand corner of the Ramachandran plot. 3. Loops and Turns Loops and turns connect a helices and strands an
33、d allow a peptide chain to fold back on itself to make a compact structure. Loops often contain hydrophilic residues and are usually found on the surfaces of proteins, where they are exposed to solvent and form hydrogen bonds with water. Turns - loops containing 5 residues or less - Turns (reverse t
34、urns) - connect different antiparallel -strands turns (a) Type I, and (b) Type II (a) In a type 1 turn, the structure is stabilized by a hydrogen bond between the carbonyl oxygen of the first N-terminal residue (Phe) and the amide hydrogen of the fourth residue (Gly). Note the Pro at position n+1. (
35、b) Type 2 turns are also stabilized by a hydrogen bond between the carbonyl oxygen of the first N-terminal residue (Val) and the amide hydrogen of the fourth residue (Asn). Not the Gly residue at position n+2. (PDB code 1AHL from giant sea anemone neurotoxin.) turns are a common motif in antiparalle
36、l sheets as a means of making the short turns connecting adjacent strands. In type II turns, the third residue is Gly about 60% of the time; In both types of turns, Pro is often the second residue. Some of the bonds in turn residues have and angles that lie outside the “permitted” regions of a typic
37、al Ramachandran plot. -turn Pro b b Bulge 氨基酸在二级结构中出现的几率氨基酸在二级结构中出现的几率 Proteins contain stretches of nonrepetitive structure. Random coil usually are active sites of an enzyme. 4. Random coil 1. Supersecondary structure 2. Domains 3. Domain structure and function 4.4 Tertiary structures of proteins
38、Tertiary structure results from the folding of a polypeptide chain into a closely-packed three-dimensional structure An important feature of tertiary structure is that AAs that are far apart in the primary structure are brought together, permitting interactions among their side chains. Tertiary stru
39、cture is stabilized primarily by noncovalent interactions (mostly the hydrophobic effect) and disulfide bonds. 1. Supersecondary Structures (Motifs) Motifs - recurring protein structures (a) Helix-loop-helix - two helices connected by a turn (b) Coiled-coil (aaaa) - two amphipathic a helices that in
40、teract in parallel through their hydrophobic edges (c) Helix bundle (aaaa) - several a helices that associate in an antiparallel manner to form a bundle (d) babbab Unit (Rossman) - two parallel b strands linked to an intervening a helix by two loops (e) Hairpin - two adjacent antiparallel b strands
41、connected by a b turn (f) b b Meander (b b-曲折曲折) - an antiparallel sheet composed of sequential b strands connected by loops or turns (g) Greek key (回形拓扑结构回形拓扑结构) - 4 antiparallel strands (strands 1,2 in the middle, 3 and 4 on the outer edges) (h) b b Sandwich - stacked b strands or sheets Common mo
42、tifs Zinc-finger motif Three secondary structures (an a a-helix and 2 b b-strands with an anti-parallel arrangement) form a finger-like bundle held together by a zinc ion. It is often found in DNA- and RNA-binding proteins. 2. Domains 1概念： Domain - independently folded, distinctly different compact
43、units in proteins Domain size - a varies from about 25 to 30AAs to about 300AAs, with an average of about 100AAs. Domains are connected to each other by loops, bound by weak interactions between side chains Domains illustrate the evolutionary conservation of protein structure. Protieins can be group
44、ed into families (a few thousand families) according to similarities in domain strucutre and AA sequence. Pyruvate Kinase Main polypeptide chain of this common enzyme folds into three distinct domains Conservation of Cytochrome c structure. (a) Tuna (+heme), (b) Tuna , (c) Rice, (d) Yeast , (e) Bact
45、eria Cytochrome C are virtually indistinguishable in terms of tertiary structure. Structural similarity of LDH and MDH (a) B. stereothermophilus, (b) E. coli The structural similarity is apparent between (a) the lactate dehydrogenase of Bacillus stearothermophilus PDB 1LDN and (b) malate dehydrogena
46、se from Escherichia coli. PDB 1EMD The two proteins only have 23% sequence identity but obvious tertiary structure similarity. Both are approx 41% helix and 20% sheet. Protein domains can be classified: （1）All - domains consist almost entirely of a helices and loops （2）All - all domains contain only
47、 sheets and non-repetitive structures that link the strands （3）Mixed / - contain supersecondary structures such as the motif, where regions of a helix and strand alternate （4） + - domains consist of local clusters of a helices and sheet in separate, contiguous regions of the polypeptide chain Typica
48、l domain structures of globular proteins 反平行螺旋束反平行螺旋束 珠蛋白型珠蛋白型螺旋蛋白螺旋蛋白 单绕平行单绕平行桶桶 双绕平行双绕平行片片 （马鞍形扭曲片）（马鞍形扭曲片） 上下型上下型桶桶 3. Domain structure and function Often a single domain has a particular function, such as binding small molecules or catalyzing a single reaction. In many cases, binding of small mo
49、lecules and the formation of the active site of an enzyme take place at the interface between two separate domains. So in multifunctional enzymes, each catalytic activity can be associated with one of several domains found in a single polypeptide chain. 4.5 Quaternary Structure Refers to the organization of subunits i
