Paul D. Adams University of Arkansas Mary K. Campbell Shawn O. Farrell Chapter Four The Three-Dimensional Structure of Proteins

Protein Structure Many _________________ are possible for proteins: Due to flexibility of amino acids linked by peptide bonds At least one major _________________ has biological activity, and hence is considered the protein’s _______________ _________________

Levels of Protein Structure ___ structure: the ________________ of amino acids in a polypeptide chain, read from the N-terminal end to the C-terminal end ___ structure: the ______________ ______________ arrangements (conformations) in localized regions of a polypeptide chain; refers only to interactions of the peptide backbone e. g.,  -helix and  -pleated sheet ___ structure: 3-D arrangement of all atoms ___ structure: arrangement of monomer subunits with respect to each other

1˚ Structure The 1˚ sequence of proteins determines its 3-D conformation Changes in just one amino acid in sequence can alter biological function, e.g. hemoglobin associated with sickle-cell anemia Determination of 1˚ sequence is routine biochemistry lab work (See Ch. 5).

2˚ Structure 2˚ of proteins is hydrogen-bonded arrangement of _________________ of the protein Two bonds have _____________ ____________: 1)Bond between _________________ and _________________ in residue 2)Bond between the _________________ and _________________ of residue See Figure 4.1

 -Helix Coil of the helix is ___________ or _____________ There are ______ amino acids per turn Repeat distance is _____ Å Each peptide bond is _________ and ________ C=O of each peptide bond is _________________ _________ to the N-H of the fourth amino acid away C=O----H-N hydrogen bonds are ________________ helical axis All R groups point _________________ from helix

 -Helix (Cont’d)

Several factors can _______________ an  -helix _______ creates a bend because of (1) the restricted rotation due to its cyclic structure and (2) its  -amino group has no N-H for hydrogen bonding strong _________________ _________________ caused by the proximity of several side chains of like charge, e.g., Lys and Arg or Glu and Asp _________________ _________________ caused by the proximity of bulky side chains, e.g., Val, Ile, Thr

 -Pleated Sheet Polypeptide chains lie adjacent to one another; may be _________________ or _________________ R groups ________, first above, then below _______ Each peptide bond is ________ and ________ C=O and N-H groups of each peptide bond are ____________ to axis of the sheet C=O---H-N hydrogen bonds are between adjacent sheets and ____________ to the direction of the sheet

 -Pleated Sheet (Cont’d)

 -bulge- a common nonrepetive irregular 2˚ motif in ____________ structure  -Pleated Sheet (Cont’d)

____________ found in reverse turns Spatial (steric) reasons Polypeptide changes direction ____________ also encountered in reverse turns. Why? Structures of Reverse Turns

 -Helices and  -Sheets structures: __________________ structures: the combination of  - and  -sections, as for example  unit:  unit: two parallel strands of  -sheet connected by a stretch of  -helix  unit:  unit: two antiparallel  -helices  -meander:  -meander: an antiparallel sheet formed by a series of tight reverse turns connecting stretches of a polypeptide chain Greek key: Greek key: a repetitive supersecondary structure formed when an antiparallel sheet doubles back on itself  -barrel:  -barrel: created when  -sheets are extensive enough to fold back on themselves

Schematic Diagrams of Supersecondary Structures

Collagen Triple Helix Consists of three polypeptide chains wrapped around each other in a ropelike twist to form a triple helix called __________________; MW approx. 300,000 30% of amino acids in each chain are Pro and Hyp (hydroxyproline); hydroxylysine also occurs Every third position is Gly and repeating sequences are X-Pro-Gly and X-Hyp-Gly Each polypeptide chain is a helix but not an  -helix The three strands are held together by hydrogen bonding involving hydroxyproline and hydroxylysine With age, collagen helices become cross linked by covalent bonds formed between Lys and His residues

Fibrous Proteins : contain polypeptide chains organized approximately parallel along a single axis. They Fibrous proteins: contain polypeptide chains organized approximately parallel along a single axis. They consist of long fibers or large sheets consist of long fibers or large sheets tend to be mechanically strong tend to be mechanically strong are insoluble in water and dilute salt solutions are insoluble in water and dilute salt solutions play important structural roles in nature play important structural roles in nature Examples are Examples are of hair and wool ____________ of hair and wool of connective tissue of animals including cartilage, bones, teeth, skin, and blood vessels ____________ of connective tissue of animals including cartilage, bones, teeth, skin, and blood vessels

Globular Proteins Globular proteins: proteins which are folded to a more or less spherical shape they tend to be soluble in ____________ and ____________ solutions most of their polar side chains are on the outside and interact with the aqueous environment by hydrogen bonding and ion-dipole interactions most of their nonpolar side chains are ______ ______ nearly all have substantial sections of _____________ and ____________

Comparison of Shapes of Fibrous and Globular Proteins

3˚ Structure The ____________ arrangement of atoms in the molecule. In ____________ protein, backbone of protein does not fall back on itself, it is important aspect of 3˚ not specified by 2˚ structure. In ____________ protein, more information needed. 3k structure allows for the determination of the way helical and pleated-sheet sections fold back on each other. Interactions between ______ ______ also plays a role.

Forces in 3˚ Structure _________ interactions, including _________ _________ between polar side chains, e.g., Ser and Thr _________ interaction between nonpolar side chains, e.g., Val and Ile _________ _________ between side chains of opposite charge, e.g., Lys and Glu _________ _________ between side chains of like charge, e.g., Lys and Arg, Glu and Asp _________ interactions: Disulfide (-S-S-) bonds between side chains of _________

Forces That Stabilize Protein Structure

3° and 4° Structure Tertiary (3°) structure: Tertiary (3°) structure: the arrangement in space of all atoms in a polypeptide chain it is not always possible to draw a clear distinction between _________ and _________ structure Quaternary (4°) structure: Quaternary (4°) structure: the association of polypeptide chains into _________ Proteins are divided into two large classes based on their three-dimensional structure _________ proteins

Determination of 3° Structure X-ray crystallography uses a perfect crystal; that is, one in which all individual protein molecules have the same 3D structure and orientation exposure to a beam of x-rays gives a series of diffraction patterns information on molecular coordinates is extracted by a mathematical analysis called a Fourier series 2-D Nuclear magnetic resonance can be done on protein samples in aqueous solution

High resolution method to determine 3˚ structure of proteins (from crystal) Diffraction pattern produced by electrons scattering X-rays Series of patterns taken at different angles gives structural information Determines solution structure Structural info. Gained from determining distances between nuclei that aid in structure determination X-Ray and NMR Data

Myoglobin A single polypeptide chain of ____ amino acids A single ______ group in a _____________ pocket 8 regions of  -helix; no regions of  -sheet Most _______ side chains are on the __________ ________ side chains are folded to the __________ Two His side chains are in the interior, involved with interaction with the heme group Fe(II) of heme has 6 coordinates sites; 4 interact with N atoms of heme, 1 with N of a His side chain, and 1 with either an O 2 molecule or an N of the second His side chain

The Structure of Myoglobin

Oxygen Binding Site of Myoglobin

Denaturation Denaturation: Denaturation: the loss of the structural order (2°, 3°, 4°, or a combination of these) that gives a protein its biological activity; that is, the loss of biological activity Denaturation can be brought about by heat large changes in pH, which alter charges on side chains, e.g., -COO - to -COOH or -NH  + to -NH  detergents such as sodium dodecyl sulfate (SDS) which disrupt hydrophobic interactions urea or guanidine, which disrupt hydrogen bonding mercaptoethanol, which reduces disulfide bonds

Denaturation of a Protein

Several ways to denature proteins Heat pH Detergents Urea Guanadine hydrochloride Denaturation and Refolding in Ribonuclease

Quaternary Structure Quaternary (4°) structure: Quaternary (4°) structure: the association of polypepetide ________ into _____________ proteins dimers trimers tetramers Noncovalent interactions electrostatics, hydrogen bonds, hydrophobic

Oxygen Binding of Hemoglobin (Hb) A _________ of two  -chains (141 amino acids each) and two  -chains (153 amino acids each);  2  2 Each chain has 1 heme group; hemoglobin can bind up to 4 molecules of O 2 Binding of O 2 exhibited by _________ ___________; when one O 2 is bound, it becomes easier for the next O 2 to bind The function of hemoglobin is to transport oxygen The structure of oxygenated Hb is different from that of unoxygenated Hb H +, CO 2, Cl -, and 2,3-_______________ (BPG) affect the ability of Hb to _________ & ________ oxygen

Structure of Hemoglobin

Conformation Changes That Accompany Hb Function Structural changes occur during binding of small molecules Characteristic of __________________ behavior Hb exhibits different 4˚ structure in the bound and unbound oxygenated forms Other _________ are involved in cooperative effect of Hb can affect protein’s affinity for O 2 by altering structure

Oxy- and Deoxyhemoglobin

Protein Folding Dynamics Can 3˚ structure of protein be predicted? Yes, within limitations The integration of biochemistry and computing has led to bioinformatics Protein structure prediction is one of the principal application of bioinformatics First step to predict protein structure is to search for sequence homology

Predicting Protein Structure

Hydrophobic Interactions Hydrophobic interactions are major factors in protein folding Folds so that nonpolar hydrophobic side chains tend to be on inside away from water, and polar side chains on outside accessible to aqueous environment Hydrophobic interactions are __________________

Hydrophobic and Hydrophilic Interactions in Proteins

Protein Folding Chaperones In the protein-dense environment of a cell, proteins may begin to fold _________ or may associate with other proteins before folding is completed Special proteins called _________ aid in the correct and timely _________ of many proteins hsp70 were the first chaperone proteins discovered Chaperones exist in organisms from prokaryotes to humans