Paul D. Adams • University of Arkansas

Mary K. CampbellShawn O. Farrellhttp://academic.cengage.com/chemistry/campbell

Chapter FourThe 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)

-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

• __________________ structuresstructures:: the combination of - and -sections, as for example• unitunit:: two parallel strands of -sheet connected by

a stretch of -helix• unitunit:: two antiparallel -helices• -meander-meander:: an antiparallel sheet formed by a series of

tight reverse turns connecting stretches of a polypeptide chain

• Greek keyGreek 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

• Fibrous proteins:: contain polypeptide chains contain polypeptide chains organized approximately parallel along a single axis. organized approximately parallel along a single axis. TheyThey• consist of long fibers or large sheetsconsist of long fibers or large sheets• tend to be mechanically strongtend to be mechanically strong• are insoluble in water and dilute salt solutionsare insoluble in water and dilute salt solutions• play important structural roles in natureplay important structural roles in nature

• Examples areExamples are• ____________ of hair and woolof hair and wool• ____________ of connective tissue of animals of connective tissue of animals

including cartilage, bones, teeth, skin, and blood including cartilage, bones, teeth, skin, and blood vesselsvessels

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°) structureTertiary (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°) structureQuaternary (4°) structure:: the association of polypeptide chains into _________

• Proteins are divided into two large classes based on their three-dimensional structure• _________ proteins• _________ 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 O2 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°) structureQuaternary (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); 22

• Each chain has 1 heme group; hemoglobin can bind up to 4 molecules of O2

• Binding of O2 exhibited by _________ ___________; when one O2 is bound, it becomes easier for the next O2 to bind

• The function of hemoglobin is to transport oxygen• The structure of oxygenated Hb is different from that

of unoxygenated Hb

• H+, CO2, 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 O2 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