CHEM 4311 General Biochemistry I Fall 2012

Chapters 4, 5 and 6

Chapter 4 Amino Acids

Instructor: Office: Phone: email: Office hours

Dr. Khairul I Ansari 316CPB 817-272-0616 kansari@uta.edu 12 am 1:30 pm Tuesday &.Thursday

Amino acids
Amino acids are the building blocks of proteins
Proteins are the most versatile macromolecule in living organism Crucial functions: Catalyst, Transport, Store other molecules like oxygen, Mechanical support Immune protection Generate body movement Transmit nerve impulse Cell growth and differentiation

Few Examples Proteins

There are 20 commonly occurring amino acids in nature that form all these diverse classes of proteins

Amino acids
What are the structures and properties of amino acids? What are the acid-base properties of amino acids? What reactions do amino acids undergo? What are the optical and stereochemical properties of amino acids? What are the spectroscopic properties of amino acids? How are amino acid mixtures separated and analyzed? What is the fundamental structural pattern in proteins?

Figure 5.6 Bovine pancreatic ribonuclease A contains 124 amino acid residues,

Structures and Properties of Amino Acids, the Building Blocks of Proteins? Amino acids contain a central tetrahedral carbon atom There are 20 common amino acids Amino acids can join via peptide bonds Several amino acids occur only rarely in proteins Some amino acids are not found in proteins

Amino Acids Building Blocks of Proteins

Figure 4.1 Anatomy of an amino acid. Except for proline and its derivatives, all of the amino acids commonly found in proteins possess this type of structure.

What Are the Structures and Properties of Amino Acids?

Figure 4.6 The ionic forms of the amino acids, shown without consideration of any ionizations on the side chain. The cationic form is the low pH form, and the titration of the cationic species with base yields the zwitterion and finally the anionic form.

Figure 4.2 Two amino acids can react with loss of a water molecule to form a covalent bond. The bond joining the two amino acids is called a peptide bond.

20 Common Amino Acids

The 20 Common Amino Acids

You should know names, structures, pKa values, 3-letter and 1-letter codes Aliphatic amino acids Aromatic amino acids Polar, uncharged amino acids Acidic amino acids Basic amino acids
Figure 4.3 Some of the nonpolar (hydrophobic) amino acids.

The 20 Common Amino Acids

The 20 Common Amino Acids

Figure 4.3 Some of the polar, uncharged amino acids. Figure 4.3 Some of the nonpolar (hydrophobic) amino acids.

The 20 Common Amino Acids

The 20 Common Amino Acids

Figure 4.3 The acidic amino acids. Figure 4.3 Some of the polar, uncharged amino acids.

The 20 Common Amino Acids

Several Amino Acids Occur Rarely in Proteins

We'll see some of these in later chapters Selenocysteine in many organisms Pyrrolysine in several archaeal species Hydroxylysine, hydroxyproline - collagen Carboxyglutamate - blood-clotting proteins Pyroglutamate in bacteriorhodopsin GABA, epinephrine, histamine, serotonin act as neurotransmitters and hormones Phosphorylated amino acids a signaling device

Figure 4.3 The basic amino acids.

Several Amino Acids Occur Rarely in Proteins

Several Amino Acids Occur Rarely in Proteins

Figure 4.4 (b) Some amino acids are less common, but nevertheless found in certain proteins. Hydroxylysine and hydroxyproline are found in connective-tissue proteins; carboxyglutamate is found in blood-clotting proteins; pyroglutamate is found in bacteriorhodopsin (see Chapter 9).

Several Amino Acids Occur Rarely in Proteins

Amino acids
Properties of Amino Acids Acid-Base Properties What Reactions Do Amino Acids Undergo? Peptide bond formation Optical and Stereochemical Properties Spectroscopic Properties

Figure 4.4 (c) Several amino acids that act as neurotransmitters and hormones.

How Are Amino Acid Mixtures Separated and Analyzed?

4.2 What Are Acid-Base Properties of Amino Acids?

4.2 What Are Acid-Base Properties of Amino Acids?

Amino Acids are Weak Polyprotic Acids The degree of dissociation depends on the pH of the medium H2A+ + H2O = HA0 + H3O+

The second dissociation (the amino group in the case of glycine): HA0 + H2O = A + H3O+

K a1 =

[HA ][H 3O ] [H 2 A + ]

Ka2

[A ][H 3O + ] = [HA 0 ]

pKa Values of the Amino Acids

You should know these numbers and know what they mean! Alpha carboxyl group - pKa = 2 Alpha amino group - pKa = 9 These numbers are approximate, but entirely suitable for our purposes.
Figure 4.6 The ionic forms of the amino acids, shown without consideration of any ionizations on the side chain. The cationic form is the low pH form, and the titration of the cationic species with base yields the zwitterion and finally the anionic form.

pKa Values of the Amino Acids

You should know these numbers and know what they mean Arginine, Arg, R: pKa(guanidino group) = 12.5 Aspartic Acid, Asp, D: pKa = 3.9 Cysteine, Cys, C: pKa = 8.3 Glutamic Acid, Glu, E: pKa = 4.3 Histidine, His, H: pKa = 6.0

pKa Values of the Amino Acids

You should know these numbers and know what they mean! Lysine, Lys, K: pKa = 10.5 Serine, Ser, S: pKa = 13 Threonine, Thr, T: pKa = 13 Tyrosine, Tyr, Y: pKa = 10.1

Figure 4.7 Titration of glycine, a simple amino acid. The isoelectric point, pI, the pH where the molecule has a net charge of 0, is defined as (pK1+ pK2)/2.

Figure 4.8 Titration of glutamic acid.

A Sample Calculation

What is the pH of a glutamic acid solution if the alpha carboxyl is 1/4 dissociated? pH = 2 + log10 [1] [3] pH = 2 + (-0.477) pH = 1.523
Figure 4.8 Titration of lysine.

Another Sample Calculation

What is the pH of a lysine solution if the side chain amino group is 3/4 dissociated?

pH = 10.5 + log10

[3] [1]

pH = 10.5 + (0.477) pH = 10.977 = 11.0

Reactions of Amino Acids

Carboxyl groups form amides & esters Amino groups form Schiff bases and amides Side chains show unique reactivities Cys residues can form disulfides and can be easily alkylated Few reactions are specific to a single kind of side chain

Figure 4.9 Typical reactions of the common amino acids (see text for details).

Figure 4.11 Reactions of amino acid side-chain functional groups.

Figure 4.10 The pathway of the ninhydrin reaction, which produces a colored product called Ruhemanns Purple that absorbs light at 570 nm. Note that the reaction involves and consumes two molecules of ninhydrin.

Cysteine

N-Ethylmaleimide

Iodoacetate

Acrylonitrile

Ellmans reagent

p-Hydroxy

Lysine

Stereochemistry of Amino Acids

All but glycine are chiral L-amino acids predominate in nature D,L-nomenclature is based on D- and Lglyceraldehyde R,S-nomenclature system is superior, since amino acids like isoleucine and threonine (with two chiral centers) can be named unambiguously

Figure 4.12 Enantiomeric molecules based on a chiral carbon atom. Enantiomers are nonsuperimposable mirror images of each other.

Figure 4.13 The configuration of the common L-amino acids can be related to the configuration of L(-)glyceraldehyde as shown. These drawings are known as Fischer projections. The horizontal lines of the Fischer projections are meant to indicate bonds coming out of the page from the central carbon, and vertical lines represent bonds extending behind the page from the central carbon atom.

Figure 4.14 The stereoisomers of isoleucine and threonine. The structures at the far left are the naturally occurring isomers.

Spectroscopic Properties
All amino acids absorb at infrared wavelengths Only Phe, Tyr, and Trp absorb UV Absorbance at 280 nm is a good diagnostic device for amino acids NMR spectra are characteristic of each residue in a protein, and high resolution NMR measurements can be used to elucidate three-dimensional structures of proteins

The assignment of (R) and (S) notation for glyceraldehyde and L-alanine .

Figure 4.15 The ultraviolet absorption spectra of the aromatic amino acids at pH 6. (From Wetlaufer, D.B., 1962. Ultraviolet spectra of proteins and amino acids. Advances in Protein Chemistry 17:303 390.)

Figure 4.16 Proton NMR spectra of several amino acids. Zero on the chemical shift scale is defined by the resonance of tetramethylsilane (TMS). (Adapted from Aldrich Library of NMR Spectra.)

Separation of Amino Acids

Mikhail Tswett, a Russian botanist, first separated colorful plant pigments by chromatography Many chromatographic methods exist for separation of amino acid mixtures Ion exchange chromatography High-performance liquid chromatography

Figure 4.18 Cation (a) and anion (b) exchange resins commonly used for biochemical separations.

Figure 4.19 Operation of a cation exchange column, separating a mixture of Asp, Ser, and Lys. (a) The cation exchange resin in the beginning, Na+ form. (b) A mixture of Asp, Ser, and Lys is added to the column containing the resin. (c) A gradient of the eluting salt (e.g., NaCl) is added to the column. Asp, the least positively charged amino acid, is eluted first. (d) As the salt concentration increases, Ser is eluted. (e) As the salt concentration is increased further, Lys, the most positively charged of the three amino acids, is eluted last.

Figure 4.20 The separation of amino acids on a cation exchange column.

Peptide Bond formation

Figure 4.21 Chromatographic fractionation of a synthetic mixture of amino acids on ion exchange columns using Amberlite IR-120, a sulfonated polystyrene resin similar to Dowex-50. A second column with different buffer conditions is used to resolve the basic amino acids. (Adapted from Moore, S., Spackman, D., and Stein, W., 1958. Chromatography of amino acids on sulfonated polystyrene resins. Analytical Chemistry 30:11851190.)

Figure 5.1 Anatomy of an amino acid. Except for proline and its derivatives,

all of the amino acids commonly found in proteins possess this type of structure.

The Peptide Bond

is usually found in the trans conformation has partial (40%) double bond character is about 0.133 nm long - shorter than a typical single bond but longer than a double bond Due to the double bond character, the six atoms of the peptide bond group are always planar! N partially positive; O partially negative

Amino Acids Can Join Via Peptide Bonds

Figure 4.2

The -COOH and NH3+ groups of two amino acids can react with the resulting loss of a water molecule to form a covalent amide bond.

Figure 5.3 The -COOH and -

NH3+ groups of two amino acids can react with the resulting loss of a water molecule to form a covalent amide bond.

Figure 5.2 Anatomy of an amino acid. Except for proline and its derivatives,

all of the amino acids commonly found in proteins possess this type of structure.

The Coplanar Nature of the Peptide Bond Six atoms of the peptide group lie in a plane!

Peptides
Short polymers of amino acids Each unit is called a residue 2 residues - dipeptide 3 residues - tripeptide 12-20 residues - oligopeptide many - polypeptide

Figure 5.4

Anatomy of an amino acid. Except for proline and its derivatives, all of the amino acids commonly found in proteins possess this type of structure.

Peptide bond
By Convention: The amino end is taken to be the beginning of polypeptide chain Ala-Gly-Trp (AGW) is tripeptide Trp-Gly-Ala (WGA) is also a tripeptide AGW and WGA are different tripeptide

Protein
One or more polypeptide chains One polypeptide chain - a monomeric protein More than one - multimeric protein Homomultimer - one kind of chain Heteromultimer - two or more different chains Hemoglobin, for example, is a heterotetramer It has two alpha chains and two beta chains

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Protein
One or more polypeptide chains One polypeptide chain - a monomeric protein More than one - multimeric protein Homomultimer - one kind of chain Heteromultimer - two or more different chains

Proteins - Large and Small

Insulin - A chain of 21 residues, B chain of 30 residues total mol. wt. of 5,733 Glutamine synthetase - 12 subunits of 468 residues each total mol. wt. of 600,000 dalton RNA polymerase II -12 subunit protein complex Mol wt. about 550,000 Connectin proteins - alpha - MW 2.8 million!

The Sequence of Amino Acids in a Protein

unique characteristic of every protein encoded by the nucleotide sequence of DNA The central Dogma in life DNA to RNA to Protein a form of genetic information is read from the amino terminus to the carboxyl terminus

Chapter 5
Proteins: Their primary structure and Biological functions

Outline
What is the fundamental structural pattern in proteins? What architectural arrangements characterize protein structure? How are proteins isolated and purified from cells? How is the amino acid analysis of proteins performed? How is the primary structure of a protein determined? Can polypeptides be synthesized in the laboratory? What is the nature of amino acid sequences? Do proteins have chemical groups other than amino acids? What are the many biological functions of proteins?

Primary structure: The amino acids sequence of the protein is,

by definition, the primary structure

Bovine pancreatic ribonuclease A contains 124 amino acid residues,. Four intrachain disulfide bridges (S-S) form crosslinks in this polypeptide between

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Secondary Structures
Through H- bonding interactions between adjacent amino acids residues, polypeptide chain arrange themselves in a characteristic helical or pleated sheet architecture Extend along one dimension, like coils of a spring
Alpha- hlix Beta-Sheet

Local structures Both of these structures owe their stability to the formation of hydrogen bonds between NH and O=C functions along the polypeptide backbone.

Tertiary Structures
Polypeptide chains bend and fold to obtain a more compact three dimensional shape- Tertiary (3o) structure is generated Globular structures are generated when 3o is formed Globular structures: lowest surface to volume ratio, minimizing the interaction of the protein with surrounding environment

(a) The primary structure and (b) a representation of the tertiary

structure of chymotrypsin, a proteolytic enzyme, The ribbon diagram depicts the

three-dimensional track of the polypeptide in space.

The Quaternary Level of Protein Structure

Important to note
Primary structure is determined by the covalently linked amino acid residues in the polypeptide backbone 2o, 3o and 4o structures are determined by the non covalent forces such as H-bonds, ionic interactions, van der Waals and hydrophobic interactions

Figure 5.5 Hemoglobin is a tetramer consisting of two and two polypeptide chains.

All the information necessary for the protein molecule to achieve its intricate architecture is contained within its primary structure

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A Proteins Conformation Can Be Described as Its Overall Three-Dimensional Structure

Distinguish conformation and configuration A configuration change require the breaking of a bond. A protein, or any molecule, can change its conformation by changing shape without breaking a bond. Under physiological condition, only few energetically favorable conformations are possible

How Are Proteins Isolated and Purified from Cells?

The thousands of proteins in cells can be separated and purified on the basis of size and electrical charge Proteins tend to be least soluble at their isoelectric point The pH value where the sum of their positive and negative charges is zero Increasing ionic strength at first increases the solubility of proteins (salting-in), then decreases it (salting-out)

The solubility is markedly influenced by pH and ionic strength. solubility of a typical protein as a function of pH and salt concentrations.

How Is the Amino Acid Analysis of Proteins Performed?

Peptides bonds of proteins are hydrolyzed either by strong acids or strong base Acids hydrolysis is of choice, Typically 6N HCl at 110oC for 24 hrs Acid hydrolysis liberates the amino acids of a protein Note that some amino acids are partially or completely destroyed by acid hydrolysis Chromatographic methods are used to separate the amino acids The amino acid compositions of different proteins are different Amino acid analysis does not give the sequence information

Increasing ionic strength at first increases the solubility of proteins (salting-in), then decreases it (salting-out)

How is the Primary Structure of a Protein Determined?

The sequence of amino acids in a protein is distinctive Both chemical and enzymatic methodologies are used in protein sequencing

Frederick Sanger was the first - in 1953, he sequenced the two chains of insulin. Sanger's results established that all of the molecules of a given protein have the same sequence. Proteins can be sequenced in two ways: - real amino acid sequencing - sequencing the corresponding DNA in the gene

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The hormone insulin consists of two polypeptide chains, A and B, held together by two disulfide cross-bridges (SS). The A chain has 21 amino acid residues and an intrachain disulfide; the B polypeptide contains 30 amino acids. The sequence shown is for bovine insulin.

Determining the Sequence: an Eight Step Strategy

1. If more than one polypeptide chain, separate. 2. Cleave (reduce) disulfide bridges 3. Determine composition of each chain 4. Determine N- and C-terminal residues 5. Cleave each chain into smaller fragments and determine the sequence of each chain 6. Repeat step 5, using a different cleavage procedure to generate a different set of fragments.
7. Reconstruct the sequence of the protein from the sequences of overlapping fragments 8. Determine the positions of the disulfide crosslinks

Step 1:
Separation of chains Subunit interactions depend on weak forces Separation is achieved with: - extreme pH - 8M urea - 6M guanidine HCl - high salt concentration (usually ammonium sulfate)

Step 2:
Cleavage of Disulfide bridges Performic acid oxidation Sulfhydryl reducing agents - mercaptoethanol - dithiothreitol or dithioerythritol - to prevent recombination, follow with an alkylating agent like iodoacetate

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Step 3A:
Identify N- and C-terminal residues
N-terminal analysis: Edman's reagent Phenylisothiocyanate derivatives are phenylthiohydantoins ( PTH derivatives)

Chromatographic method can be used to identify the PTH derivative Importantly, in this procedure, rest of the polypeptide remains intact And can be re-subjected to further round of Edmans degradation to identify successive amino acids Automated Edman sequenator Up to 50 cycles For larger protein less 10-20 cycles

Step 3B: :
C-terminal analysis Enzymatic analysis (carboxypeptidase)
Carboxypeptidase A cleaves any residue except Pro, Arg, and Lys Carboxypeptidase B only works on Arg and Lys Carboxypeptidase C and Y works on any C-terminal residue

Steps 4 and 5:
Fragmentation of the chains
Enzymatic fragmentation trypsin, chymotrypsin, clostripain, staphylococcal protease Chemical fragmentation cyanogen bromide

Chemical Fragmentation
Cyanogen bromide
CNBr acts only on methionine residues CNBr is useful because proteins usually have only a few Met residues a peptide with a C-terminal homoserine lactone

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Enzymatic Fragmentation
Trypsin Chymotrypsin Clostripain Staphylococcal protease

Trypsin - cleavage on the C-side of Lys, Arg

Enzymatic Fragmentation
Trypsin - cleavage on the C-side of Lys, Arg Chymotrypsin - C-side of Phe, Tyr, Trp
Trypsin - cleavage on the C-side of Lys, Arg

Clostripain - like trypsin, but attacks Arg more than Lys Staphylococcal protease C-side of Glu, Asp in phosphate buffer specific for Glu in acetate or bicarbonate buffer

Reconstructing the Sequence

Steps 6:
Reconstructing the Sequence Use two or more fragmentation agents in separate fragmentation experiments Sequence all the peptides produced (usually by Edman degradation) Compare and align overlapping peptide sequences to learn the sequence of the original polypeptide chain

Tryptic peptide Ala-Ala-Trp-Gly-Lys Thr-Asn-Val-Lys

Chimotryptic peptide Val-Lys-Ala-Ala-Trp

Tryptic peptide

Tryptic peptide

Thr-Asn-Val-Lys-Ala-Ala-Trp-Gly-Lys Chimotryptic peptide

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Figure 5.18 Summary of the sequence analysis of catrocollastatinC, a 23.6-kD protein found in the venom of the western diamondback rattlesnake Crotalus atrox. Sequences shown are given in the one-letter amino acid code.
(Adapted from Shimokawa, K., et al., 1997. Archives of Biochemistry and Biophysics 343:3543.)

Reconstructing the Sequence

Compare cleavage by trypsin and staphylococcal protease on a typical peptide: Trypsin cleavage: A-E-F-S-G-I-T-P-K L-V-G-K Staphylococcal protease: F-S-G-I-T-P-K L-V-G-K-A-E

Reconstructing the Sequence

L-V-G-K A-E-F-S-G-I-T-P-K A-E F-S-G-I-T-P-K Correct sequence: L-V-G-K-A-E-F-S-G-I-T-P-K L-V-G-K-

Step 7:
Location of Disulfide Cross-Bridges Peptide fragments may be linked by disulfide bridges Diagonal electrophoresis may be used to identify links Off-diagonal peptide fragments can be isolated and sequenced

Amino Acid Sequence Can Be Determined by Mass Spectrometry

Mass spectrometry separates particles on the basis of mass-to-charge ratio Fragments of proteins can be generated in various ways MS can also separate these fragments Two most common method of mass spectrometry for protein analysis (a): Electrospray Ionisation (ESI-MS) (b) Matrix assisted laser desorption-time of flight (MALDITOF-MS)

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Laboratory Synthesis of Peptides

Strategies are complex because of the need to control side chain reactions Blocking groups must be added and later removed du Vigneauds synthesis of oxytocin in 1953 was a milestone Bruce Merrifields solid phase method was even more significant

Amino Acid Sequences provides many kinds of insights?

Sequences and composition reflect the function of the protein Membrane proteins have more hydrophobic residues, whereas fibrous proteins may have atypical sequences Homologous proteins from different organisms have homologous sequences Molecular evolution e.g., cytochrome c is highly conserved Structural Mottifs

Apparently Different Proteins May Share a Common Ancestry

Evolutionary relatedness can be inferred from sequence homology

Consider lysozyme and human milk -lactalbumin These proteins are identical at 48 positions (out of 129 in lysozyme and 123 in human milk -lactalbumin Functions of these two are not related

Figure 5.30 The tertiary structures of hen egg white lysozyme and human -lactalbumin are very similar. (Adapted from Acharya, K. R., et al., 1990. Journal of Protein Chemistry 9:549563; and
Acharya, K. R., et al., 1991. Journal of Molecular Biology 221:571581.)

5.8 Do Proteins Have Chemical Groups Other Than Amino Acids?

Proteins may be "conjugated" with other chemical groups If the non-amino acid part of the protein is important to its function, it is called a prosthetic group. Be familiar with the terms: glycoprotein, lipoprotein, nucleoprotein, phosphoprotein, metalloprotein, hemoprotein, flavoprotein.

Chapter 6
Proteins: Secondary, Tertiary, and Quaternary Structure

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Outline
What Are the Noncovalent Interactions That Dictate and Stabilize Protein Structure? What Role Does the Amino Acid Sequence Play in Protein Structure? What Are the Elements of Secondary Structure in Proteins, and How Are They Formed? How Do Polypeptides Fold into Three-Dimensional Protein Structures? How Do Protein Subunits Interact at the Quaternary Level of Protein Structure?

All of the information necessary for folding the peptide chain into its "native structure is contained in the primary amino acid structure of the peptide.

Noncovalent Interactions Dictate and Stabilize Protein Structures:

6.1 - What Are the Noncovalent Interactions That Dictate and Stabilize Protein Structures?
What are they? What are the relevant numbers? van der Waals: 0.4 - 4 kJ/mol hydrogen bonds: 12-30 kJ/mol ionic bonds: 20 kJ/mol hydrophobic interactions: <40 kJ/mol
Figure 6.1 An electrostatic interaction between the -amino group of a lysine and the carboxyl group of a glutamate residue.

6.2 - What Role Does the Amino Acid Sequence Play in Protein Structures?

How do proteins recognize and interpret the folding information? Certain loci along the chain may act as nucleation points Protein chain must avoid local energy minima Chaperones may help

All of the information necessary for folding the peptide chain into its "native structure is contained in the primary amino acid structure of the peptide.

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6.3 - What Are the Elements of Secondary Structure in Proteins, and How Are They Formed?
The atoms of the peptide bond lie in a plane The resonance stabilization energy of the planar structure is 88 kJ/mol A twist about the C-N bond involves a twist energy of 88 kJ/mol times the square of the twist angle. Twists can occur about either of the bonds linking the alpha carbon to the other atoms of the peptide backbone

Consequences of the Amide Plane

Two degrees of freedom per residue for the peptide chain Angle about the C(alpha)-N bond is denoted phi Angle about the C(alpha)-C bond is denoted psi The entire path of the peptide backbone is known if all phi and psi angles are specified Some values of phi and psi are more likely than others.

The angles phi and psi are shown here

Figure 6.2 The amide or peptide bond planes are joined by the tetrahedral bonds of the -carbon. The rotation parameters are and . The conformation shown corresponds to = 180and = 180 . Note that positive values of and correspond to clockwise rotation as viewed from C . Starting from 0 a , rotation of 180in the clockwise direction (+180 is equivalent to a ) rotation of 180in the counterclockwise direction (-180 (Illustration: Irving Geis. ).
Rights owned by Howard Hughes Medical Institute. Not to be reproduced without permission.)

Steric Constraints on phi & psi

Unfavorable orbital overlap precludes some combinations of phi and psi phi = 0 psi = 180is unfavorable , phi = 180 psi = 0is unfavorable , phi = 0 psi = 0is unfavorable ,

Steric Constraints on phi & psi

G. N. Ramachandran was the first to demonstrate the convenience of plotting phi,psi combinations from known protein structures The sterically favorable combinations are the basis for preferred secondary structures
Figure 6.3 Many of the possible conformations about an -carbon between two peptide planes are forbidden because of steric crowding. Several noteworthy examples are shown here. Note: The formal IUPAC-IUB Commission on Biochemical Nomenclature convention for the definition of the torsion angles and in a polypeptide chain (Biochemistry 9:34713479, 1970) is different from that used here, where the C atom serves as the point of reference for both rotations, but the result is the same. (Illustration:
Irving Geis. Rights owned by Howard Hughes Medical Institute. Not to be reproduced without permission.)

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Classes of Secondary Structure

Figure 6.4 A Ramachandran diagram showing the sterically reasonable values of the angles and . The shaded regions indicate particularly favorable values of these angles. Dots in purple indicate actual angles measured for 1000 residues (excluding glycine, for which a wider range of angles is permitted) in eight proteins. The lines running across the diagram (numbered +5 through 2 and -5 through -3) signify the number of amino acid residues per turn of the helix; + means righthanded helices; - means left-handed helices. ( , . ., 1981,

34:167 339.) 34:

All these are local structures that are stabilized by hydrogen bonds Alpha helix Other helices Beta sheet (composed of "beta strands") Tight turns (aka beta turns or beta bends) Beta bulge

The Alpha Helix

Read the box on page 162 First proposed by Linus Pauling and Robert Corey in 1951 Identified in keratin by Max Perutz A ubiquitous component of proteins Stabilized by H bonds
Figure 6.6 Four different graphic representations of the -helix. (a) As it originally appeared in Paulings 1960 The Nature of the Chemical Bond. (b) Showing the arrangement of peptide planes in the helix. EACH peptide carbonyl is hydrogen bonded to the peptide N-H group four residue farther up the chain

The Alpha Helix

Know these numbers Residues per turn: 3.6 Rise per residue: 1.5 Angstroms Rise per turn (pitch): 3.6 x 1.5 = 5.4 Angstroms The backbone loop that is closed by any Hbond in an alpha helix contains 13 atoms phi = -60 degrees, psi = -45 degrees The non-integral number of residues per turn was a surprise to crystallographers

Figure 6.7 The three-dimensional structures of two proteins that contain substantial amounts of -helix in their structures. The helices are represented by the regularly coiled sections of the ribbon drawings. Myohemerythrin is the oxygen-carrying protein in certain invertebrates, including Sipunculids, a phylum of marine worm. (Jane Richardson)

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Figure 6.8 The arrangement of NH and C=O groups (each with an individual dipole moment) along the helix axis creates a large net dipole for the helix. Numbers indicate fractional charges on respective atoms.

Figure 6.9 Four NH groups at the N-terminal end of an -helix and four C=O groups at the C-terminal end cannot participate in hydrogen bonding. The formation of Hbonds with other nearby donor and acceptor groups is referred to as helix capping. Capping may also involve appropriate hydrophobic interactions that accomodate nonpolar side chains at the ends of helical segments.

The Beta-Pleated Sheet

Composed of beta strands Also first postulated by Pauling and Corey, 1951 Strands may be parallel or antiparallel Rise per residue: 3.47 Angstroms for antiparallel strands 3.25 Angstroms for parallel strands Each strand of a beta sheet may be pictured as a helix with two residues per turn

Figure 6.10 A pleated sheet of paper with an antiparallel -sheet drawn on it. (Irving Geis)

The Beta Turn

(aka beta bend, tight turn)
Figure 6.11 The arrangement of hydrogen bonds in (a) parallel and (b) antiparallel pleated sheets.

allows the peptide chain to reverse direction carbonyl C of one residue is H-bonded to the amide proton of a residue three residues away proline and glycine are prevalent in beta turns

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Figure 6.12 The structures of two kinds of -turns (also called tight turns or -bends) (Irving Geis) Figure 6.13 Three different kinds of -bulge structures involving a pair of adjacent polypeptide chains. (Adapted from Richardson, J. S., 1981. Advances in Protein Chemistry 34:167339.)

6.4 How Do Polypeptides Fold into ThreeDimensional Protein Structures? Several important principles: Secondary structures form wherever possible (due to formation of large numbers of H bonds) Helices and sheets often pack close together

Proteins folding determinants and pathways

Certain loci along the chain may act as nucleation points Protein chain must avoid local energy minima Chaperones may help The atoms of the peptide bond lie in a plane

The resonance stabilization energy of the planar structure is 88 kJ/mol A twist about the C-N bond involves a twist energy of 88 kJ/mol times the square of the twist angle. Twists can occur about either of the bonds linking the alpha carbon to the other atoms of the peptide backbone

Consequences of the Amide Plane

Two degrees of freedom per residue for the peptide chain Understand phi and psi well Study Ramachandran Plot

Steric Constraints on phi & psi

Unfavorable orbital overlap precludes some combinations of phi and psi phi = 0 psi = 180is unfavorable , phi = 180 psi = 0is unfavorable , phi = 0 psi = 0is unfavorable ,

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Classes of Secondary Structure

All these are local structures that are stabilized by hydrogen bonds Alpha helix Other helices Beta sheet (composed of "beta strands") Tight turns (aka beta turns or beta bends) Beta bulge

Protein Folding into Three-Dimensional Protein Structures, tertiary structures

Several important principles: Secondary structures form wherever possible (due to formation of large numbers of H bonds) Helices and sheets often pack close together The backbone links between elements of secondary structure are usually short and direct Proteins fold to make the most stable structures (make H bonds and minimize solvent contact

Fibrous and globular Proteins

Fibrous proteins: Primarily structural role
Much or most of the polypeptide chain is organized approximately parallel to a single axis Fibrous proteins are often mechanically strong Fibrous proteins are usually insoluble Alpha Keratin: Found in hair, fingernails, claws, horns and beaks Beta-Keratin: Found in silk fibers Collagen - A Triple Helix Principal component of connective tissue (tendons, cartilage, bones, teeth)

Globular Proteins
Some design principles
Figure 6.18 Poly(Gly-Pro-Pro), a collagen-like righthanded triple helix composed of three left-handed helical chains. (Adapted from Miller, M. H., and Scheraga, H. A., 1976, Calculation of the structures of collagen models. Role of interchain interactions in determining the triple-helical coiled-coil conformation. I. Poly(glycyl-prolyl-prolyl). Journal of Polymer Science Symposium 54:171200.)

Most polar residues face the outside of the protein and interact with solvent Most hydrophobic residues face the interior of the protein and interact with each other Packing of residues is close However, ratio of vdw volume to total volume is only 0.72 to 0.77, so empty space exists The empty space is in the form of small cavities

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An amphiphilic helix in flavodoxin:

A nonpolar helix in citrate synthase:

A polar helix in calmodulin:

Figure 6.23 The three-dimensional structure of bovine ribonuclease A, showing the -helices as ribbons. (Jane Richardson)

Globular Proteins
More design principles
"Random coil" is not random Structures of globular proteins are not static Various elements and domains of protein move to different degrees Some segments of proteins are very flexible and disordered Know the kinds and rates of protein motion

Globular Proteins
The Forces That Drive Folding Peptide chain must satisfy the constraints inherent in its own structure Peptide chain must fold so as to "bury" the hydrophobic side chains, minimizing their contact with water Peptide chains, composed of L-amino acids, have a tendency to undergo a "right-handed twist"

A New Way to Look at Globular Proteins

Look for "layer structures Helices and sheets often pack in layers Hydrophobic residues are sandwiched between the layers Outside layers are covered with mostly polar residues that interact favorably with solvent

Figure 6.27 The natural right-handed twist exhibited by polypeptide chains, and the variety of structures that arise from this twist.

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Figure 6.28 Examples of protein domains with different numbers of layers of backbone structure. (a) Cytochrome c' with two layers of -helix. (b) Domain 2 of phosphoglycerate kinase, composed of a -sheet layer between two layers of helix, three layers overall. (c) An unusual five-layer structure, domain 2 of glycogen phosphorylase, a -sheet layer sandwiched between four layers of -helix. (d) The concentric layers of -sheet (inside) and -helix (outside) in triose phosphate isomerase. Hydrophobic residues are buried between these concentric layers in the same manner as in the planar layers of the other proteins. The hydrophobic layers are shaded yellow. (Jane Richardson)

Classes of Globular Proteins

Jane Richardson's classification Antiparallel alpha helix proteins Parallel or mixed beta sheet proteins Antiparallel beta sheet proteins Metal- and disulfide-rich proteins

Figure 6.29 Several examples of antiparallel -proteins. (Jane Richardson)

Figure 6.30 Parallel -array proteinsthe eight-stranded -barrels of triose phosphate isomerase (a, side view, and b, top view) and (c) pyruvate kinase. (Jane Richardson) Figure 6.31 Several typical doubly wound parallel -sheet proteins. (Jane Richardson)

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Figure 6.33 Examples of the so-called Greek key antiparallel barrel structure in proteins.

Figure 6.32 Examples of antiparallel -sheet structures in proteins. (Jane Richardson)

Thermodynamics of Folding
Read the box on page 185
Separate the enthalpy and entropy terms for the peptide chain and the solvent Further distinguish polar and nonpolar groups The largest favorable contribution to folding is the entropy term for the interaction of nonpolar residues with the solvent

Molecular Chaperones
Why are chaperones needed if the information for folding is inherent in the sequence? to protect nascent proteins from the concentrated protein matrix in the cell and perhaps to accelerate slow steps Chaperone proteins were first identified as "heat-shock proteins" (hsp60 and hsp70)

Protein Modules
An important insight into protein structure Many proteins are constructed as a composite of two or more "modules" or domains Each of these is a recognizable domain that can also be found in other proteins Sometimes modules are used repeatedly in the same protein There is a genetic basis for the use of modules in nature

Figure 6.36 Ribbon structures of several protein modules utilized in the construction of complex multimode proteins. (a) The complement control protein module. (b) The immunoglobulin module. (c) The fibronectin type I module. (d) The growth factor module. (e) The kringle module. (Adapted from
Baron, M., Norman, D., and Campbell, I., 1991, Protein modules. Trends in Biochemical Sciences 16:13-17.)

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Predictive Algorithms
Figure 6.37 A sampling of proteins that consist of mosaics of individual protein modules. The modules shown include CG, a module containing carboxyglutamate residues; G, an epidermal growth-factorlike module; K, the kringle domain, named for a Danish pastry; C, which is found in complement proteins; F1, F2, and F3, first found in fibronectin; I, the immunoglobulin superfamily domain; N, found in some growth factor receptors; E, a module homologous to the calcium-binding EF hand domain; and LB, a lectin module found in some cell surface proteins.
(Adapted from Baron, M., Norman, D., and Campbell, , 1991, Protein modules. Trends in Biochemical Sciences 16:1317.)

If the sequence holds the secrets of folding, can we figure it out? Many protein chemists have tried to predict structure based on sequence Chou-Fasman: each amino acid is assigned a "propensity" for forming helices or sheets Chou-Fasman: is only modestly successful and doesn't predict how sheets and helices arrange

Quaternary Structure
What are the forces driving quaternary association? Typical Kd for two subunits: 10-8 to 10-16M! These values correspond to energies of 50-100 kJ/mol at 37 C Entropy loss due to association - unfavorable Entropy gain due to burying of hydrophobic groups - very favorable!
Figure 6.43 The quaternary structure of liver alcohol dehydrogenase. Within each subunit is a six-stranded parallel sheet. Between the two subunits is a two-stranded antiparallel sheet. The point in the center is a C2 symmetry axis. (Jane Richardson)

Figure 6.44 Isologous and heterologous associations between protein subunits. (a) An isologous interaction between two subunits with a twofold axis of symmetry perpendicular to the plane of the page. (b) A heterologous interaction that could lead to the formation of a long polymer. (c) A heterologous interaction leading to a closed structurea tetramer. (d) A tetramer formed by two sets of isologous interactions.

Figure 6.45 The polypeptide backbone of the prealbumin dimer. The monomers associate in a manner that continues the msheets. A tetramer is formed by isologous interactions between the side chains extending outward from sheet DAGHHGAD in both dimers, which pack together nearly at right angles to one another. (Jane Richardson)

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What are the structural and functional advantages driving quaternary association?

Know these!
Figure 6.47 Schematic drawing of an immunoglobulin molecule showing the intramolecular and intermolecular disulfide bridges. (A space-filling model of the same molecule is shown in Figure 1.11.)

Stability: reduction of surface to volume ratio Genetic economy and efficiency Bringing catalytic sites together Cooperativity

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