大学精品课件：生物化学（英文版）Chapter4 Proteins Three-Dimensional Structures and Function(part 1)

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 renaturation

2、 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 structrue

3、,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-rays be

4、ing 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 spherical,X-

5、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 model of

6、 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 protein is r

7、epresented 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 backbone of

8、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-helix,a-helix: 190 nm (+) 208nm, 222nm (,b-sheet: 195nm (+) antiparallel: red shift parallel: blue shift

9、 215-217nm (,b-sheet,Unordered structure: (-) below 200nm (+) 218nm weak band,unordered,b-turn: 180-190nm (-) 200-205nm (+) 225nm (+) band, weak red shift,Far UV（190-240nm,Protein conformation - three dimensional shape Native conformation - each protein folds into a single stable shape (physiologica

10、l conditions) Biological function of a protein depends completely on its native conformation,4.2 The conformation of the peptide group,Peptide group (CONH ) peptide plane(amide plane) Because peptide bonds are partial double bond Dihedral angle of C: (N-C) and (C-C) 3. Ramachandran plot,0o =0o,Most

11、of combination of and are sterically forbidden,G. N. Ramachandran and his co-workers 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,Nonperm

12、issible angles,The Helix strands and sheets Loops and turns Random coil,4.3 Secondary structures of proteins,1. The -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 a

13、xis of the helix) All C=O groups point toward the C-terminus (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

14、 of - helix,The entire helix is a dipole with a positive N-terminus and a negative C-terminus,N terminus,chirality and rotation opticity of -helix Right-handed is more stable than left-handed. So most helices in proteins are right handed (backbone turns clockwise when viewed along the axis from the

15、N terminus) Cooperativity in formation,C terminus,Types of -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 fro

16、m about 4 or 5 AAs to more than 40 AAs, but the average is about 12 AAs; Many -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 bac

17、kbone. All the side chains, shown 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

18、hydrophobic residues are blue, less 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 hydrophil

19、ic residues are on the same sides 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 hy

20、drophobic side will likely be oriented 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 re

21、sidues in green, and charged residues 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

22、 common,The leucine zipper is a dimerization motif commonly found in DNA binding proteins. DNA binding region consists of two amphipathic a helices, one from each of two protein subunits,GCN4 is a transcription regulatory protein that binds to specific DNA sequences. The DNA binding region consists

23、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 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 Le

24、u residues,The stability of an -helix is affected by,2.Size of R groups,1.Same charge of AAs,Pro,Gly,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 s

25、table. However -sheets are stabilized 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-Sheets : (a) parallel, (b)

26、 antiparallel,N端,C端,N端,C端,N端,C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

27、, and the side facing 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 sheets,b-Sheet side chains project alternately above and below the plane of the b strands One surface of a b-sheet may consis

28、t of hydrophobic side 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 ant

29、iparallel beta sheets 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 st

30、rands that make up -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

31、and strands and 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 - Tu

32、rns (reverse turns) - 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 pos

33、ition n+1. (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

34、antiparallel 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

35、of a typical Ramachandran plot,-turn,Pro,Bulge,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,Supersecondary structure Domains Domain structure and function,4.4 Tertiary structures of proteins,Tertiary structure

36、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 structure is stabilized

37、 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 () - two amphipathic a helices that interact in parallel throug

38、h their hydrophobic edges (c) Helix bundle () - several a helices that associate in an antiparallel manner to form a bundle (d) bab Unit (Rossman) - two parallel b strands linked to an intervening a helix by two loops,e) Hairpin - two adjacent antiparallel b strands connected by a b turn (f) b Meand

39、er (-曲折) - an antiparallel sheet composed of sequential b strands connected by loops or turns (g) Greek key (回形拓扑结构) - 4 antiparallel strands (strands 1,2 in the middle, 3 and 4 on the outer edges) (h) b Sandwich - stacked b strands or sheets,Common motifs,Zinc-finger motif,Three secondary structure

40、s (an -helix and 2 -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 units in proteins Domain size - a varies from about 25 t

41、o 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 grouped into families (a few thousand families) according to

42、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) Bacteria,Cytochrome C are virtually indistinguishable in terms

43、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 dehydrogenase from Escherichia coli. PDB 1EMD,The two proteins only have

44、 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 sheets and non-repetitive structures that link the strands （3

45、）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,Typical domain structures of globular proteins,反平行螺旋束,珠蛋白型螺旋蛋白,单绕平行桶

46、,双绕平行片 （马鞍形扭曲片,上下型桶,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 molecules and the formation of the active site of an enzyme take place at the interface between two s

47、eparate 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 in a protein with multiple subunits (an “oligomer”) The subunits of an oligomeric

48、protein may be identical or different. When they are identical, dimers and tetramers predominate. When they differ, each type often has a differnet function,Greek letters and numbers are be used to subscript oligomeric proteins. The subunits are usually held together by weak noncovalent interactions. Hydrophobic interactions are the principal forced involved, although electrostatic forces may contribute to the proper alignment of the subuni