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Protein Structure Student Edition 5/23/13 Version

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1 Protein Structure Student Edition 5/23/13 Version
Dr. Brad Chazotte 213 Maddox Hall Web Site: Original material only © B. Chazotte Pharm. 304 Biochemistry Fall 2014

2 Goals Understand the bases of & differences between primary, secondary, tertiary, & quaternary protein structure. Be familiar with basic protein purification/sequencing methods & how they depend on the physical & chemical properties of proteins. Understand the physical and chemical forces that determine secondary structure, including the peptide bond. Learn the basic types of secondary structure: -helix, -sheet, random coil & non-repetitive structures. Understand the physical & chemical forces that determine tertiary structure - protein domains and motifs. Be acquainted with techniques like NMR & X-ray crystallography that help determine protein structure. Understand the physical and chemical forces that determine quaternary structure – protein folding, denaturation, renaturation, hydropathy plots. Remember how protein structure and structural changes reflect the influence of thermodynamic concepts on structural stability.

3 Levels of Protein Structure
Primary Secondary Tertiary Quaternary Refer to the three-dimensional shape of folded polypeptide chains

4 Protein Diversity For a protein of n residues there are 20n possible sequences For 40 residue protein x 1052 For 100 residue protein x 10130 Definitions Peptides – typically less than 40 residues Dipeptide: 2 amino acids; Tripeptide: 3 amino acids Oligopeptide: many amino acids Proteins – typically polypeptides with 40 or more residues Multisubunit proteins - proteins with several identical or nonidentical subunits.

5 PRIMARY STRUCTURE & ANALYSIS
Primary Structure: the amino acid sequence of a protein’s polypeptide chain or chains. Sometimes referred to as the covalent structure.

6 Conjecture on the Limitations of Protein Size
Minimum: 40 residues – near the limit for a polypeptide to be able to fold into a discrete stable shape that permits it to carry out its basic function. Maximum: ~1000 residues – near the limit for the protein synthetic machinery to produce a peptide with minimal errors in the polypeptide, mRNA template, or gene DNA.

7 Logic of Amino Acid Sequences in Proteins
The characteristics of a protein depend more on the sequence of amino acids rather than its composition. The presence of an amino acid with its characteristic physical & chemical properties at a particular place in a protein influences the protein’s properties. (review Amino Acids lecture) The 3-D shape of a protein is a consequence of the intermolecular forces among its various residues. (review Chemical Bonding lecture) Voet, Voet & Pratt 2013 Chap. 5.1

8 Primary Structure of Bovine Insulin
Voet, Voet & Pratt 2013 Figure 5.1

9 Studying Proteins by Isolating Them

10 Protein Purification Crude Extract – 1st step whether protein is from tissue or microbe, break open the cell and release the proteins into solution. Fractionation – step where proteins are separated into different fractions bases on some chemical or physical property such as size or charge. May utilize protein solubility i.e. (pH), salt concentration, temperature, etc. .

11 Proteins Must be Stabilized after Isolation
Care must be taken to preserve protein structure and function after its is removed from its natural environment were it was stable. pH To prevent denaturation (loss of structure) or function proteins are placed in buffered solutions at or near their native pH. Temperature Protein purification is normally carried out at low temperature ~0º C. While some proteins are thermally stable at high temperatures, others may be affected by temperature a few degrees higher than the native environment. Degradative Enzymes During isolation various nucleases and proteases are released from their places in the cell and can degrade nucleic acids or proteins unless temperature, pH or inhibitory agents are added. Adsorption to Surfaces Solutions are handled to minimize foaming and are kept concentrated as interfaces (air-water, glass, plastic) can cause denaturation. Storage To maintain protein stability. Cold (-70º C or -196º C liq N2), sometime under N2(g) to remove oxygen and prevent slow oxidation. Some of the goals are to minimize microbial growth and/or oxidation. Voet, Voet & Pratt 2013 p.96

12 Assay of Purified Proteins
To purify a protein it is necessary to measure how much you have  need a specific assay. Easier for enzymes as they produce a product proportional to the amount of enzyme present. Colored or fluorescent products are especially helpful Can also use a coupled enzyme reaction, i.e. 2nd enzyme Can use immunochemical assays.

13 ELISA Enzyme-Linked Immunoabsorbent Assay
Voet, Voet & Pratt 2013 Figure 5.3

14 Some Separation Techniques
Charge Ion Exchange Chromatography Electrophoresis Polarity Hydrophobic Interaction Chromatography Size Gel Filtration Chromatography SDS-Polyacrylamide Electrophoresis Ultracentrifugation Binding Specificity Affinity Chromatography These separation techniques utilize differences in the physical and/or chemical properties that arise from the differences amino acid composition.

15 Protein Fractionation by Salting Out
Protein solubility depends on: Concentration of dissolved salts Solvent polarity pH Temperature By careful manipulation of these properties it is possible to selectively precipitate out certain proteins and leave the other soluble. Voet, Voet & Pratt 2013 Figure 5.5

16 Protein Separation by Ion Exchange Chromatography
Ion exchange chromatography makes use of the fact that opposite charges attract Polyelectrolytes such as proteins that have both negative and positive charges will bind to cation or anion exchangers depending on the protein’s net charge The binding affinity (Strength of binding) depends on the presence of other ions that compete with the protein for binding sites on the immobile phase and the pH which in terms effects the protein’s net charge. Anion exchanger: e.g., DEAE Matrix–CH2-CH2-NH(CH2CH3)2+ Cation exchanger: e.g., CM Matrix-CH2COO- Voet, Voet & Pratt 2013 Figure 5.6

17 Protein Separation by Gel Filtration Chromatography
A bead can have different pore sizes (holes) depending on the extent of cross-linking in its component polymer. The larger proteins that are excluded from the beads have a shorter path and leave the column sooner. Voet, Voet & Pratt 2013 Figure 5.7

18 Protein Separation by Affinity Chromatography
Utilize the ability of certain proteins (via biochemical properties) that are able to bind specific molecules non-covalently. Bind a specific molecule called a ligand to an inert matrix – immobile phase Column conditions are then changed, e.g. pH, ionic strength or high ligand concentration, to permit the protein to elute in a highly purified form. Voet, Voet & Pratt 2013 Figure 5.8

19 SDS-PAGE of Supernatants & Membrane Fraction from a Bacterium
Gel electrophoresis - a molecular sieving approach SDS, sodium dodecyl sulfate, when added to a protein solution binds 1 molecule of SDS per two amino acids or 1.4g per g protein. Voet, Voet & Pratt Figure 5.9 & 2013 Fig 5.9 & 5.10

20 Separation by Zonal Centrifugation
Gradient preparation Berg, Tymoczko, & Stryer 2012 Fig 3.16 Voet, Voet & Pratt 2013 Figure 5.12

21 Protein Sequencing A protein must be broken down into fragments small enough to be individually sequenced. The fragments are used to reconstruct the protein by analyzing the fragment overlaps. Sequence is a prerequisite for determining protein’s 3-D structure and understanding its molecular mechanism. Sequence comparisons among analogous proteins from different species yield insights into protein function as well as reveal evolutionary relationships among proteins Many inherited diseases are caused by point mutations in the amino acid sequence. Sequence analysis can assist with diagnostic testing and therapy development. Voet, Voet & Pratt 2006 Chapter 5

22 Sanger Method for Protein Sequencing
Bind to terminal amino groups to form a yellow dinitrophenyl derivative Hydrolyze protein Identify terminal amino acid chromatographically Also today nucleic acids sequencing is frequently used to determine protein sequences Voet, Voet & Pratt p.108 Box 5.1

23 Basic Logic of Protein Sequencing
1. polypeptide chains that are linked by disulfide bonds are separated by reduction of the sulfhydral groups of cysteine. 2. chemical or enzymatic means are used to cleave the resultant polypeptide chains into smaller fragments. 3. Each small fragment is sequenced. 4. Compare overlapping sequences produced by different enzymes or chemical degradations to logically reconstruct original protein sequence 5. Repeat process without cleaving the disulfide bonds to determine where those bonds are located Voet, Voet & Pratt 2013 Figure 5.13

24 End Group Analysis Used to determine the number of distinct polypeptide chains in a protein (if end groups not chemically blocked). There are several procedures for the N-terminus. No reliable chemical procedure for C-terminus, an enzymatic approach uses carboxypeptidases.

25 Protein Sequencing End Group Analysis with Danzyl Chloride
Conjugate the fluorophore to primary amine(s). Perform an acid hydrolysis Identify via chromatography the labeled amino acid. Voet, Voet & Pratt 2013 Figure 5.14

26 Disulfide Bond Cleavage
Need to cleave to separate polypeptide chains. Two methods: oxidative cleavage with performic acid disadvantage: destroys met and Trp indol side chain reductive cleavage with mercaptan e.g. 2-mercaptoethanol. Usually alkylate product with iodoacetic acid to prevent disulfide bond reformation.

27 Peptide Hydrolysis Chemical approach Acid hydrolysis
disadvantages – destroys Ser, Thr, Tyr & Trp converts Asn & Gln to Asp & Glu, respectively Base Hydrolysis disadvantages – destroys Cys, Ser, Thr & Arg Biochemical Approach Enzymatic Hydrolysis disadvantages – often incomplete some autodigestion

28 Amino Acid Analysis Separation by HPLC
Complete hydrolysis will yield the composition but not the sequence Voet, Voet & Pratt 2006 Figure 5.15

29 Molecular Mass Determination by Mass Spectrometry
Electrospray ionization mass spectrometry Peptide in solution sprayed from capillary tube at high voltage to produce highly charged droplets Solvent soon evaporates to give peptide ions in gas phase – Yield to +2.0 charge per kilodalton from, e.g., Arg & Lys protonation Measures mass/charge (m/z) ratio Electrospray ionization (ESI) does NOT destroy proteins as earlier mass spec techniques did. Matrix-assisted Laser desorption/ionization –time of flight MALDI-TOF Permits the ionization of proteins that formerly could not be efficiently ionized due to their high MW and low volatility. Laser vaporizes solvent – some protein enter gas phase too. Protein ionizes and is separated on a mass/charge ratio. ESI mass spectrum of horse heart apomyolobin Mass spectrum: series of peaks of ions differing by a single charge and mass of 1 proton. Each peak corresponds to an m/z ratio of an (M + nH)n+ ion Can take two adjacent peaks and solve two linear equations to get MW. Berg, Tymoczko, & Stryer 2012 Fig 3.34 Voet, Voet, & Pratt 2013 Fig 17a,b

30 Tandem Mass Spectroscopy for Peptide Sequencing
Tandem refers to two mass spectrometers in series Ions of proteins, i.e. precursor ions, from the 1st mass spec are broken into smaller peptide chains, i.e. product ions, by bombardment with atoms of an inert gas. These are in turn passed to a 2nd mass analyzer. Product ions can be formed such that individual amino acids are cleaved from the precursor ion such that a family of ions can be produced Each ion represents the original peptide minus one or more amino acids from the end. The mass difference between the peaks in the plot represent the sequence of the amino acids. “By comparing molecular masses of successively larger members of a family of fragments, the molecular masses and therefore the identities of the corresponding amino acids can be determined” Berg, Tymoczko, & Stryer 2012 Fig 3.36 Voet, Voet & Pratt 2013, Fig. 5-18; & p. 113

31 Polypeptide Cleavage Endopeptidases
Trypsin Rn-1 = pos chg res: Arg, Lys; Rn≠Pro (C-side) Chymotrypsin Rn-1 = bulky hydroph res Phe, Trp, Tyr; Rn≠Pro (C-side) Elastase Rn-1 = small neut. Res: Ala, Gly, Ser, Val; Rn≠Pro Thermolysin Rn = Ile, Met, Phe, Try, Val Rn≠Pro (N-Side) Pepsin Rn = Leu, Phe, Trp, Typ; Rn≠Pro (N-side) Endopeptidase V8 Rn-1 = Glu Cyanogen Bromide (CNbr) Rn = Met (C side) Voet, Voet, & Pratt 2013 Table 5.4 Endopeptidases hydrolyze internal peptide bonds and are used to fragment polypeptides but require certain adjacent side chains.

32 Edman Degradation of Proteins
Use repeated (sequential) cycles of the Edman degradation. Trifluoroacetic acid cleavage of the N-terminal amino acid does NOT hydrolyze the other peptides bonds. Identify PTH-amino acid by chromatographic techniques. Voet, Voet & Pratt Figure 5.16

33 Protein Sequence Determination using Overlapping Fragments
Voet, Voet & Pratt 2013 Figure 5.119

34 Determining Disulfide Bond Location(s)
Voet, Voet & Pratt 2013 Figure 5.20

35 Sites to Find Sequence Data
Voet, Voet & Pratt 2013 Table 5.5 Voet, Voet & Pratt 2013 Fig 5.21

36 Cytochrome c Sequence Analyses
Voet, Voet & Pratt 2013 Table 5.6

37 Cytochrome c Phylogenetic Tree
Voet, Voet & Pratt Figure 5.22

38 Protein Evolution, Gene Duplication & Protein Modules
Protein evolution rates The rate at which mutations are incorporated into a protein are dependent on the degree to which a change in an amino acid effects a protein’s function Gene duplication Proteins with similar functions tend to have similar sequences. New related function can arise by gene duplication. An aberrant genetic recombination in which one chromosome acquires both copies of a primordial gene. Protein modules New proteins (and functions) can also be generated by incorporation of various amino acid module or motifs.

39 Sample Protein Evolution Rates
Proteins mutate at different rates over time. But mutations in the DNA typically occur at the same rate Differences due to the rate at which functionally or structurally acceptable changes occurs. That is those changes that are NONLETHAL Voet, Voet & Pratt 2013 Figure 5.24

40 SECONDARY STRUCTURE The local spatial arrangement of a polypeptide’s backbone atoms without regard to the conformation of its side chains.

41 Levels of Protein Structure
Voet, Voet & Pratt 2013 Figure 6.1

42 The (trans) Peptide Bond - Structure
In most cases in the protein backbone the peptide bond is in the trans configuration Means -carbons of adjacent amino acids are on opposite sides So less steric hindrance of adjacent amino acids side chains Find ~ 8 kJ greater stability of the trans vs the cis configuration Bond angles and lengths effect to a large extent the freedom of movement and the configuration of the protein. (Important!) Resonance give rise to 40% double bond character Voet, Voet & Pratt Figure 6.2

43 Polypeptide: Extended Conformation & Torsion Angles
“peptide group” Definition:  and  = 180º when the polypeptide chain is fully extended. They increase clockwise when looking from Cα Voet, Voet & Pratt 2013 Figure 6.3; 6.4

44 Peptide Bonds: Steric Interference
Amide hydrogen Carbonyl oxygen Steric interference Voet, Voet & Pratt Figure 6.5

45 Ramachandran Diagram (Allowed Bond Angles )
↑↑ -pleated sheet α-helix (left handed) Sterically allowed angles for all aa except Gly & Pro van der Waals radii, the attractive and repulsive forces we covered in earlier lectures have a significance for protein structure. . α-helix Note: Gly is less sterically hindered Voet, Voet & Pratt 2013 Figure 6.6

46 Protein α-Helix Structure
Amide H The alpha helix structures is one of the most stable and is therefore one of the most abundant biological structures. H-bond every 4th residue Carbonyl O The carbonyl oxygen on residue N is hydrogen bonded to the amide hydrogen on residue N+4 an optimum bond length of 2.8Å. This is a source of great thermodynamic stability. 5.4Å Right-handed helix Helix core is tightly packed such that the atoms are at or near their van der Waals radii. left right Voet, Voet & Pratt 2013 Figure 6.7 Lehninger Box 6.1

47 -Helix Stability and Amino Acid Sequence
Interactions between amino acids can stabilize or destabilize the helix. e.g. a long block of Glu residues will not form an -helix at pH 7.0 due to the negatively charged carboxyl groups overpowering H-bonds Many adjacent Lys and/or Arg residues with pos. charges will repel each other at pH 7.0 The bulk & shape of Asn, Ser, Thr and Leu can also destabilize a helix if close together in the backbone sequence The twist of the helix ensures that critical interactions occur between a side chain (R-group) and another 3 or 4 residues away. Positively charged amino acids are often found three residues away from a negatively charge amino acid – supports ion-pair formation Aromatic residues are often 3 residues apart to support hydrophobic interactions. Proline (N in rigid ring structure) causes a kink in -helix. Rarely found in helix

48 Constraints on -Helix Stability (Summary)
Electrostatic repulsion or attraction between successive amino acids with charged R groups. Bulkiness of adjacent R groups. Interactions between amino acid side chains spaced 3 (or 4) residues apart. The occurrence of Pro or Gly residues. Interaction between amino acid residues at the ends of the helical segment and the inherent electric dipole of the helix.

49 Electric Dipole of the Peptide Bond & Interactions Between -Helix Residues Three Apart
Arg103 side chain Asp100 side chain amino carbonyl The electric dipole of the peptide bond is transmitted along an -helical segment via the intrachain hydrogen bonds and this results in an overall helix dipole. Troponin c protein segment Lehninger Figure 6.6 Lehninger Figure 6.5

50 Protein -Sheet Structures
-sheet makes full use of the hydrogen bonding capacity of the polypeptide backbone H-bonding occurs between neighboring polypeptide chains, i.e. interchain, rather than intrachain. -Sheets: parallel vs Antiparallel Space-filling Antiparallel -Sheet Pleated -Sheet Voet, Voet & Pratt 2013 Figures 6.9, 6.10, 6.11

51 Historical Classification of Proteins
Globular polypeptide chains folded in to spherical or globular shape. These often contain several types of secondary structure. Typically most enzymes and regulatory proteins. Fibrous polypeptide chains arranged in long chains or sheets. Usually consist of a single type of secondary structure. Typically provide support, shape and external protection to vertebrates. Alberts et al 2004 Fig 4.9

52 Fibrous Proteins Fibrous proteins share properties that convey strength and/or flexibility to structures in which they are part. In each case the fundamental structural unit is a simple repeating element of secondary structure. All fibrous proteins are insoluble in water as a result of the high concentration of hydrophobic residues on the protein surface and interior. The hydrophobic residues are largely buried via packing many similar polypeptides chains together to form elaborate supramolecular complexes.

53 -Keratin Structure: coiled coil
Rich in Ala, Val, Leu, Ile, Met and Phe – hydrophobic residues A “Permanent Wave” A coiled coil - composed of two parallel -helices that are twisted around each other to form a left-handed supertwisted coiled coil. Voet, Voet & Pratt 2013 Figure 6.15 Lehninger Box 6.2

54 Silk fibroin -Sheets in Side View
Has great strength Not very extensible (would break polypeptide chain covalent bonds) Very flexible (Neighboring sheets associate with weak van der Waals forces). Ala or Ser Gly Typical repeat: (Gly-Ser-Gly-Ala-Gly-Ala)n Voet, Voet & Pratt 2002 Figure 6.16

55 Collagen Triple Helix Structure
Composition: ~33% Gly ~15-30% Pro, Hyp and Hyl most abundant vertebrate protein occurring fibers form the major stress bearing components of connective tissues Three parallel, left-handed helical polypeptide chains with three residues per turn twisted together to form a right-handed superhelical structure. Repeating Sequence: Gly-X-Y where: X is often Pro, Y is often Hyp. Hyl is sometimes at Y Hyp = 4-hydroxyprolyl Hyl = 5-hydroxylysyl Voet, Voet & Pratt 2013 Figure 6.17

56 Collagen’s Molecular Interactions
Every third polypeptide residue passes through the very crowded center of the superhelix, hence the repeated gly every third residue. H-Bond Space-filling model Ball & stick model H-bonding in collagen triple helix Voet, Voet & Pratt 2002 Figure 6.18 Voet, Voet & Pratt 2013 Figure 6.18a

57 Secondary Structure & Properties Table
Lehninger Table 6.1

58 Nonrepetitive Protein Structure
Native, folded proteins can have nonrepetitive structures that are also ordered like helices or -sheets but they are irregular and therefore more difficult to give a clear, simple description Globular proteins (majority of proteins in nature) can contain a number of secondary structure types. They may have these irregular structures in addition to coils and sheets. The appearance of certain residues outside an α-helix or β-sheet may be nonrandom. Helix capping: Asn and Gln often flank the ends of an α-helix since their side chains can fold back to H-bond with the 4 terminal residues of the helix. β-bulge: a distortion in a β-sheet where a polypeptide strand may have an extra, non H-bonded residue which produces a structural distortion.

59 Turn & Loop Structures in Polypeptides
Reverse turn types  Loop in space-filling model found in most proteins with 60 are more residues and are composed on 6 to 16 residues. Voet, Voet & Pratt 2013 Figure 6.14 Voet, Voet & Pratt 2006 Figure 6.20  Loop almost always located on the protein surface. May be involved in recognition processes.

60 Structure of -turns hydrogen oxygen Type II always Gly at 3
Connecting elements that link successive runs of an alpha helix or a beta sheet. A 180° turn of four amino acids Most common type of turn Lehninger Figure 4.8

61 Relative Probability of an AA Being in These Secondary Structures
Take home message: chemical and physical characteristics of an amino acid (charge, bond angles, etc.) influence its ability to participate in particular secondary structures (for illustrative, informational purposes only) Lehninger Figure 4.10

62 TERTIARY STRUCTURE The three-dimensional structure of an entire polypeptide including its side chains Tertiary structure describes the folding of the protein’s secondary structure elements and also specifies the position of each atom in the protein.

63 Myoglobin Tertiary Structure: View Types
ribbon mesh Surface contour Myoglobin is composed of eight relatively straight alpha helices interrupted by bends and some of these are beta turns. Space-filling w/ side chains Ribbon w/ side chains Lehninger Figure 4.16

64 Protein 3-D structure & X-ray crystallography
Protein crystal: flavodoxin from Desulfovbrio vulgaris X-ray diffraction pattern of sperm whale myoglobin crystal 3-D electron density of human rhino virus crystal Voet, Voet & Pratt 2013 Figure 6.21 Voet, Voet & Pratt 2002 Figure 6.23 Voet, Voet & Pratt 2013 Figure 6.20b

65 3-D Protein Structure Determination X-Ray Crystallography
Generate a good protein crystal (not easy). Detector “sees” a pattern of spots called reflections from X-ray beam. EACH atom makes a contribution to EACH spot. Massive calculations to produce an electron density map. Nuclei have greatest density. Yields map of structure Lehninger Box 4.4

66 Myoglobin, Globular Proteins, & Tertiary Structure
Positioning of amino acid side chains reflects a structure that derives much of its stability from hydrophobic interactions A dense hydrophobic core is typical of globular proteins. In dense, closely packed environment weak interactions, e.g. van der Waals, strengthen and reinforce one another.

67 NMR in Protein Structure Determination
Only certain atoms such as 1H, 13C, 15N, 19F, and 31P give rise to an NMR signal. 1-D NMR used to identify nuclei and their immediate chemical environment. Also use NOE signals provide information about the distance between atoms #1 2-D Lehninger Box 4.4 Fig 2

68 NMR in Protein Structure Determination of Full Structure from 2D Spectrum
Backbone showing possible constraints Part of reason for the multiple structures shown is that proteins are dynamic molecules with molecular vibrations occurring in solution. Src protein SH3 domain – 64 residue polypeptide 20 possible structures shown w/ backbone in white Voet, Voet & Pratt Fig 6.25

69 Protein Structural Motifs and Domains
In globular proteins the amino acid side chains are distributed according to their polarities to achieve the most energetically favorable conditions. Val, Leu, Ile, Met & Phe occur mostly in the protein interior away from aqueous solvent molecules. Arg, His, Lys, Asp, & Glu are typically located at the proteins surface where their charges can be solvated Ser, Thr, Asn, Gln, & Tyr (uncharged polar) are found on the protein surface but also in the protein’s interior where they are almost always hydrogen bonded.

70 Side Chain Locations Seen in Space-filling Models
sperm whale myoglobin concanavalin A -sheet interior this side Nonpolar side chains Polar side chains backbone -Sheet α-Helix Voet, Voet & Pratt 2008 Figure 6.26

71 Horse Heart Cytochrome c Structure
Fe atom & heme Hydrophillic side chains Hydrophobic side chains Hydrophilic sides chains are shown in green and can be seen to be at the protein’s surface. The hydrophobic side chains are in orange and are closer to the protein’s interior and near the porphyrin ring. Voet, Voet & Pratt 2013 Figure 6.27

72 Protein Motifs (Supersecondary Structures)
There are grouping of certain secondary structural elements that occur in many unrelated globular proteins. Most common motif is an -helix connecting two parallel strands of a -sheet.  motif Antiparallel strands connected by relatively tight reverse turns  hairpin Two successive antiparallel helices pack against each other with their axis inclined  motif. Extended  sheet can role up to form three different types of barrels.  barrel

73 Protein Structural Motifs
Different proteins combine these structures in various ways to achieve their function. Certain successful and energetically favorable designs are preserved in many diverse proteins. α   hairpin α α  barrels Voet, Voet & Pratt 2008 Figure 6.28

74 The Rossman Fold Nucleotide binding site
A prime example of a structure function-relationship Binds dinucleotides such as NAD+. Utilizes  strands which form a parallel sheet with -helical connections Two such  units are shown. Voet, Voet & Pratt 2013 Figure 6.31 Voet, Voet & Pratt 2002 Figure 6.29

75 Protein Domains Polypeptide chains > ~200 residues usually fold into two or more globular clusters. Typical domain 100 – 200 residues with an 25 Å avg. diameter. Neighboring domains are usually connected by one or two polypeptide segments. Many domains are structurally independent units with characteristics of a small globular protein Domains generally consist of two or more layers of secondary structures – seals off a domain’s hydrophobic core from aqueous environment. Domains often have a specific function, e.g. nucleotide binding.

76 Domains in Evolution Common protein structures likely arose and persisted because of their ability to: Form stable folding patterns. Tolerate amino acid deletions, substitutions, & insertions which makes them better able to survive evolutionary changes. Support essential biological functions. Studies of proteins support the concept that essential structural and functional elements of proteins rather than their amino acid residues are conserved during evolution, e.g. changes in like residues that do not appreciably change structure are not dysfunctional.

77 QUATERNARY STRUCTURE The spatial arrangement of the subunits of a multisubunit protein Subunits typically associate via noncovalent interactions. Contact regions between subunits resemble the interior of single subunit proteins. Design Benefits: Easier to synthesize multiple smaller subunits than one large polypeptide chain free from error. A subunit with an error can more easily replaced than a single large polypeptide – more efficient. Definitions: Oligomer – proteins with more than one subunit Protomer – repeating structural subunits of a protein

78 Quaternary Structure of Hemoglobin
An oligomeric protein Each of the four subunits 1212 is shown in a different color. (Heme is red) Voet, Voet & Pratt 2008 Figure 6.33

79 Oligomeric Protein Symmetry Examples
Related by single axis of rotation Proteins can only have rotational symmetry When n-fold rotation axis intersects a 2-fold rotation axis at 90° Other types of symmetry based on geometrical objects Voet, Voet & Pratt 2013 Figure 6.34

80 Protein Folding & Stability
Normally for biological structures, the molecules exist in conformations that are at energy minimums, i.e. the most thermodynamically stable. Hydrophobic effects, electrostatic interactions, and hydrogen bonding, the noncovalent interactions, each provide energies of thousands of kJ/mol over an entire protein. Thermodynamic studies of native proteins revealed that native protein are only marginally stable under physiological conditions as the free energy required to denature them is approximately 0.4 kcal/mol per amino acid residue. For 100 residue protein ONLY 40kJ/mol more stable than unfolded Conclude: a protein’s structure is in fact a delicate balance of counteracting forces.

81 Stabilizing Forces Hydrophobic effect - Nonpolar molecules seek thermodynamically (entropically) to minimize their contact with water. This is the major determinant of native protein structure – greatest contribution to stability

82 Hydropathy Plot Protein interior
Plot combines hydrophobic and hydrophilic tendencies of individual amino acids. The greater a side chain’s hydropathy the more like it is to be in a protein’s interior. Protein exterior Voet, Voet & Pratt 2008 Figure 6.35

83 Electrostatic Interactions
The association of two ionic protein groups of opposite charge is called an ion pair or salt bridge. Approximately 75% of charged residues are involved in ion pairs and are mostly on the protein surface.

84 Chemical Cross-Linking
Disulfide Bonds Thought that disulfide bonds are not so much a stabilizing force, but they may function to lock in a particular conformation of a polypeptide backbone. Since the cell cytoplasm is a reducing environment most intracellular proteins do not have disulfide bridges. Metal Ions can also internally link proteins

85 Electrostatic Contributions to Protein Stability
Van der Waals interactions are an important stabilizing force in the closely packed protein interior where they act over short distances and are lost when the protein is unfolded. Hydrogen bonds make a minor contribution because those groups can H-bond with water as a protein unfolds. H-bonds do “select” the unique native structure of a protein from a small number of hydrophobically stabilized conformations. Ions pairs also make minor contributions because the free energy of the pair does not compensate for the loss of side chain entropy and the loss of the free energy of solvation.

86 Protein Denaturation/Renaturation
Proteins can be denatured by a variety of conditions & substances. Heating (adding energy) Most proteins exhibit a sharp transition over a narrow temperature range indicative of the protein unfolding in a cooperative manner. pH variation Alters the ionization of amino acid side chains which results in changes in charge distributions and H-bonding needs. Detergents (amphipathic molecules) Associated with nonpolar residues and disrupt the hydrophobic interactions Chaotropic agents (urea, guanidinium chloride) Ions or small organic molecules that at high 5- 10M concentration disrupt hydrophobic interactions

87 Protein Denaturation Curves
Lehninger Figure 4.26

88 Protein Renaturation Anfinsen classic experiment with RNAse A (4 disulfide bonds must reform): Proteins can spontaneously fold into their native conformation under physiological conditions – protein’s primary structure dictates its three dimensional structure.

89 Protein Denaturation/Renaturation
Voet, Voet & Pratt 2008 Figure 6.39

90 Thermodynamics of Protein Folding
Number of possible conformations is large and therefore the conformation energy is large. At this point only a small percentage of the intramolecular interactions found in the native state are present native min Energy max Decreasing number of possible states, i.e. the entropy Amount of protein in native state increasing Free energy is decreasing as we head to a free energy minimum at the bottom, i.e., native state. Energy min Lehninger Figure 6.27

91 Protein Misfolding: Prions & Disease
Brain becomes riddled with holes Caused by a single 28 kd protein called a prion protein Normal Prp is mostly alpha helical. Illness occurs when an altered form of Prp called PrpScr is present which has mixed alpha helix and beta sheets Protein Misfolding: Prions & Disease Prion: proteinaceous infectious particle Lehninger Box 6.4

92 Alzheimers and -Amyloid Protein
-Amyloid protein is normally present in the human brain but its function is unknown In Alzheimers a 40 residue (shorter) segment of this protein forms fibrous deposits or plaques. This cleaved protein tends to aggregate only with itself. This protein does not fold properly! Amyloid plaques w/ Aβ protein in brain tissue of human Alzheimers patient Native (N) <=> Unfolded (U) Keq = [U]/[N] = e-∆G˚’/RT As the ∆G˚’ for unfolding decreases the portion unfolded proteins increases.

93 Proteins that Help Native Protein Folding
Molecular chaperones – proteins that bind to unfolded and partially folded polypeptide chains to prevent improper folding by prevent improper association of hydrophobic regions that could lead to polypeptide aggregation, precipitation, or non-native folding. Important for multisubunit and multidomain proteins. First described as heat shock proteins (Hsp) Most are ATPases Voet, Voet & Pratt 2012 Figure 6.45

94 Proteins are Dynamic Structures Myoglobin
Remember that proteins are in fact dynamic molecules with normal structural fluctuations These fluctuations are important for function particularly in enzymes. Snapshots are for the structure of myoglobin seen over 4 x seconds Voet, Voet & Pratt 2013 Figure 6.39

95 End of Lectures


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