chem 4311- chapter 4-5-6 -animo acid and proteins

7/29/2019 Chem 4311- Chapter 4-5-6 -Animo Acid and Proteins

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Instructor: Dr. Khairul I AnsariOffice: 316CPBPhone: 817-272-0616email: [email protected] hours 12 am 1:30 pm Tuesday &.Thursday

CHEM 4311General Biochemistry I

Fall 2012

Chapters 4, 5 and 6 Chapter 4Amino Acids

Amino acids

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

Amino acids are the building blocks of proteins

Few Examples Proteins

Figure 5.6 Bovine pancreatic ribonuclease A contains 124 amino acidresidues,

There are 20 commonly occurring amino acids in naturethat 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 ofamino acids?

How are amino acid mixtures separated andanalyzed?

What is the fundamental structural pattern inproteins?


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Structures and Properties of Amino Acids, the

Building Blocks of Proteins?

Amino acids contain a central tetrahedralcarbon 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

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

Amino AcidsBuilding Blocks of Proteins

Figure 4.6 The ionic forms of the am ino acids, shown without consideration of any

ionizations on the side chain. The cationic form is the low pH form, and the titration ofthe cationic species with base yields the zwitterion and f inally the anionic form.

What Are the Structures and Properties of Amino Acids?

Figure 4.2 Twoamino acids canreact with loss of awater molecule toform a covalentbond. The bond

joining the twoamino acids iscalled a peptidebond.

20 Common Amino Acids

You should know names, structures, pKavalues, 3-letter and 1-letter codes

Aliphatic amino acids Aromatic amino acids Polar, uncharged amino acids Acidic amino acids Basic amino acids

The 20 Common Amino Acids

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


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


Figure 4.3 Some of the polar, uncharged amino acids.


Figure 4.3 Some of the polar, uncharged amino acids.


Figure 4.3 The acidic amino acids.


Figure 4.3 The basic 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


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Several Amino Acids Occur Rarely in Proteins Several Amino Acids Occur Rarely in Proteins

Figure 4.4 (b) Some amino acids are less common, butnevertheless found in certain proteins. Hydroxylysine andhydroxyproline are found in connective-tissue proteins; carboxy-glutamate is found in blood-clotting proteins; pyroglutamate isfound in bacteriorhodopsin (see Chapter 9).

Several Amino Acids Occur Rarely in Proteins

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

Amino acids

Properties of Amino Acids

Acid-Base Properties

What Reactions Do Amino Acids Undergo?

Peptide bond formation

Optical and Stereochemical Properties

Spectroscopic Properties

How Are Amino Acid Mixtures Separated and

Analyzed?

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+

0

3

1

2

[HA ][H O ]

[ H A ]

+

+=

aK

4.2 What Are Acid-Base Properties of Amino Acids?

The second dissociation (the amino group in thecase of glycine):

HA0

+ H2O = A

+ H3O+

3

2 0

[A ][H O ]

[HA ]

+

=a

K


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Figure 4.6 The ionic forms of the am ino acids, shown without consideration of anyionizations on the side chain. The cationic form is the low pH form, and the titration ofthe cationic species with base yields the zwitterion and f inally the anionic form.

pKa Values of the Amino Acids

You should know these numbers and knowwhat they mean!

Alpha carboxyl group - pKa = 2 Alpha amino group - pKa = 9 These numbers are approximate, but entirely

suitable for our purposes.


You should know these numbers and knowwhat 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


You should know these numbers and knowwhat 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. Theisoelectric point, pI, the pHwhere the molecule has a netcharge of 0, is defined as

(pK1+ pK2)/2.

Figure 4.8 Titration ofglutamic acid.


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A Sample Calculation

What is the pH of a glutamic acid solutionif 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 solutionif 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 andcan be easily alkylated

Few reactions are specific to a singlekind of side chain

Figure 4.9Typicalreactionsof thecommonaminoacids (seetext fordetails).


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Figure 4.10The pathway of theninhydrin reaction,which produces acolored productcalled RuhemannsPurple that absorbslight at 570 nm. Notethat the reactioninvolves andconsumes twomolecules ofninhydrin.

Figure 4.11 Reactions of aminoacid side-chain functional groups.

Cyste ine N-E thylma le im ide

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 L-

glyceraldehyde R,S-nomenclature system is superior,

since amino acids like isoleucine andthreonine (with two chiral centers) can benamed unambiguously

Figure 4.12 Enantiomericmolecules based on a chiralcarbon atom. Enantiomers arenonsuperimposable mirrorimages of each other.

Figure 4.13 Theconfiguration of thecommon L-amino acidscan be related to theconfiguration of L(-)-glyceraldehyde as shown.These drawings areknown as Fischerprojections. The horizontallines of the Fischerprojections are meant toindicate bonds coming outof the page from thecentral carbon, andvertical lines representbonds extending behindthe page from the centralcarbon atom.

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


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The assignment of (R) and (S) notation for glyceraldehyde and

L-alanine .

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 aprotein, and high resolution NMR measurements canbe used to elucidate three-dimensional structures ofproteins

Figure 4.15The ultravioletabsorption spectra ofthe aromatic aminoacids at pH 6. (FromWetlaufer, D.B.,1962. Ultravioletspectra of proteinsand amino acids.

Advances in ProteinChemistry 17:303390.)

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

Separation of Amino Acids

Mikhail Tswett, a Russian botanist, firstseparated colorful plant pigments bychromatography

Many chromatographic methods exist forseparation of amino acid mixtures Ion exchange chromatography High-performance liquid

chromatography

Figure 4.18 Cation (a) andanion (b) exchange resinscommonly used forbiochemical separations.


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Figure 4.19 Operation of a cation exchange column,separating a mixture of Asp, Ser, and Lys. (a) Thecation exchange resin in the beginning, Na+ form. (b) Amixture of Asp, Ser, and Lys is added to the columncontaining the resin. (c) A gradient of t he eluting salt(e.g., NaCl) is added to the column. Asp, the leastpositively charged amino acid, is eluted first. (d) As thesalt concentration increases, Ser is eluted. (e) As thesalt concentration is increased further, Lys, the mostpositively charged of the three amino acids, is elutedlast.

Figure 4.20 The separation of amino acidson a cation exchange column.

Figure 4.21Chromatographicfractionation of a syntheticmixture of amino acids onion exchange columns usingAmberlite IR-120, asulfonated polystyrene resinsimilar to Dowex-50. Asecond column with differentbuffer conditions is used toresolve the basic aminoacids. (Adapted from Moore,S., Spackman, D., and Stein,W., 1958. Chromatographyof amino acids on sulfonatedpolystyrene 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.

Peptide Bond formation

The Peptide Bond

is usually found in the transconformationhas partial (40%) double bond characteris about 0.133 nm long - shorter than a typical

single bond but longer than a double bondDue to the double bond character, the six atomsof the peptide bond group are always planar!N partially positive; O partially negative

Figure 4.2The -COOH and -NH

3

+ groups of twoamino acids canreact with theresulting loss of awater molecule toform a covalentamide bond.

Amino Acids Can Join Via Peptide Bonds


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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 ofstructure.

Figure 5.3 The -COOH and -NH3+ groups of two amino acidscan react with the resulting lossof a water molecule to form acovalent amide bond.

Figure 5.4Anatomy of anamino acid.Except for prolineand itsderivatives, all ofthe amino acidscommonly foundin proteinspossess this typeof 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

Peptide bond

By Convention: The amino end is taken to be thebeginning of polypeptide chain

Ala-Gly-Trp (AGW) is tripeptideTrp-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 complexMol wt. about 550,000

Connectin proteins - alpha - MW 2.8 million!

The Sequence of Amino Acidsin a Protein

unique characteristic of every protein

encoded by the nucleotide sequence of DNA

The central Dogma in lifeDNA 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 r esidues,.Four intrachain disulfide bridges (S-S) form crosslinks in this polypeptidebetween


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Secondary Structures

Through H- bonding interactions between adjacent amino

acids residues, polypeptide chain arrange themselves in acharacteristic helical or pleated sheet architecture

Extend along one dimension, like coils of a spring

Local structures

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

Alpha- hlixBeta-Sheet

Tertiary Structures

Polypeptide chains bend and fold to obtain a morecompact 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 withsurrounding 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

Figure 5.5Hemoglobin is atetramer consistingof two and two polypeptide chains.

Primary structure is determined by the covalently linked aminoacid residues in the polypeptide backbone

2o, 3o and 4o structures are determined by the non covalentforces such as H-bonds, ionic interactions, van der Waals andhydrophobic interactions

All the information necessary for the protein molecule toachieve its intricate architecture is contained within its primarystructure

Important to note


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A Proteins Conformation Can Be Described as ItsOverall Three-Dimensional Structure

Distinguish conformation and configuration

A configuration change require the breaking of a bond.

A protein, or any molecule, can change its conformationby changing shape without breaking a bond.

Under physiological condition, only few energeticallyfavorable conformations are possible

How Are Proteins Isolated and Purified fromCells?

The thousands of proteins in cells can be separated andpurified 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 andnegative charges is zero

Increasing ionic strength at first increases the solubilityof 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.

Increasing ionic strength at firstincreases the solubility ofproteins (salting-in), thendecreases it (salting-out)

Peptides bonds of proteins are hydrolyzed either by strong acids orstrong 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 byacid 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

How Is the Amino Acid Analysis of ProteinsPerformed?

How is the Primary Structure of aProtein Determined?

The sequence of amino acids in a protein is distinctive

Both chemical and enzymatic methodologies are used inprotein sequencing

Sanger's results established that all of themolecules of a given protein have thesame sequence.

Proteins can be sequenced in two ways:- real amino acid sequencing- sequencing the corresponding DNA in

the gene

Frederick Sanger was the first - in 1953, hesequenced the two chains of insulin.


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The hormone insulin consists of twopolypeptide chains, A and B, held together

by two disulfide cross-bridges (SS). The A

chain has 21 amino acid residues and anintrachain disulfide; the B polypeptide

contains 30 amino acids. The sequence

shown is for bovine insulin.

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 togenerate a different set of fragments.

7. Reconstruct the sequence of the protein from the sequencesof overlapping fragments

8. Determine the positions of the disulfide crosslinks

Determining the Sequence: an Eight Step Strategy

Step 1:Separation of chains

Subunit interactions depend on weakforces

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 analkylating 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 ( PTHderivatives)

Chromatographic method can be used to identify thePTH derivative

Importantly, in this procedure, rest of the polypeptideremains intact

And can be re-subjected to further round of Edmansdegradation 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 exceptPro, 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 afew 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


Chymotrypsin - C-side of Phe, Tyr, Trp

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

Tryptic peptide

Ala-Ala-Trp-Gly-Lys

Thr-Asn-Val-Lys

Chimotryptic peptideVal-Lys-Ala-Ala-Trp

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

Tryptic peptide Tryptic peptide

Chimotryptic peptide

Reconstructing the SequenceSteps 6:

Reconstructing the Sequence

Use two or more fragmentation agents in separatefragmentation experiments

Sequence all the peptides produced (usually by Edmandegradation)

Compare and align overlapping peptide sequences tolearn the sequence of the original polypeptide chain


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Figure 5.18Summary of thesequenceanalysis ofcatrocollastatin-C, a 23.6-kDprotein found inthe venom ofthe westerndiamondbackrattlesnakeCrotalus atrox.Sequencesshown aregiven in theone-letter aminoacid code.(Adapted fromShimokawa, K., etal., 1997. Archivesof Biochemistryand Biophysics343:3543.)


Compare cleavage by trypsin and staphylococcalprotease 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


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

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 BeDetermined by Mass Spectrometry

Mass spectrometry separates particles on the basis ofmass-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 forprotein analysis

(a): Electrospray Ionisation (ESI-MS)(b) Matrix assisted laser desorption-time of flight (MALDI-

TOF-MS)


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Laboratory Synthesis of Peptides

Strategies are complex because of the needto 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 providesmany kinds of insights?

Sequences and composition reflect the function ofthe protein Membrane proteins have more hydrophobic residues,

whereas fibrous proteins may have atypicalsequences

Homologous proteins from different organisms havehomologous sequencesMolecular evolutione.g., cytochrome c is highly conserved

Structural Mottifs

Apparently Different Proteins MayShare a Common Ancestry

Evolutionary relatedness can be inferred from sequencehomology

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 verysimilar. (Adapted from Acharya, K. R., et al., 1990. Journal of Protein Chemistry9:549563; andAcharya, K. R., et al., 1991. Journal of Molecular Biology221:571581.)

5.8 Do Proteins Have Chemical GroupsOther Than Amino Acids?

Proteins may be "conjugated" with otherchemical groups

If the non-amino acid part of the protein is

important to its function, it is called aprosthetic 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 ProteinStructure?

What Are the Elements of Secondary Structure in Proteins,and How Are They Formed?

How Do Polypeptides Fold into Three-Dimensional ProteinStructures?

How Do Protein Subunits Interact at the Quaternary Level ofProtein 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 InteractionsThat 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:


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6.3 - What Are the Elements of SecondaryStructure in Proteins, and How Are They

Formed?The atoms of the peptide bond lie in a plane

The resonance stabilization energy of the planarstructure is 88 kJ/mol

A twist about the C-N bond involves a twistenergy of 88 kJ/mol times the square of thetwist angle.

Twists can occur about either of the bondslinking the alpha carbon to the other atoms ofthe peptide backbone

Consequences of the Amide Plane

Two degrees of freedom per residue for thepeptide chain

Angle about the C(alpha)-N bond is denoted phiAngle about the C(alpha)-C bond is denoted psiThe entire path of the peptide backbone isknown if all phi and psi angles are specifiedSome values of phi and psi are more likely thanothers.

Figure 6.2The amide or peptide bond planes arejoined by the tetrahedral bonds of the-carbon. The rotation parameters are and . The conformation showncorresponds to = 180and =180.Note that positive values of and correspond to clockwise rotation asviewed from C . Starting from 0, arotation of 180in the clockwisedirection (+180) is equivalent to arotation of 180in the counterclockwisedirection (-180). (Illustration: Irving Geis.Rights owned by Howard Hughes Medical

Institute. Not to be reproduced withoutpermission.)

The angles phiand psi areshown here

Steric Constraints on phi & psi

Unfavorable orbital overlap precludes somecombinations of phi and psi

phi = 0, psi = 180is unfavorablephi = 180, psi = 0is unfavorablephi = 0, psi = 0is unfavorable

Figure 6.3 Many of the possible conformations about an -carbon between two peptideplanes are forbidden because of steric crowding. Several noteworthy examples areshown here.

Note: The formal IUPAC-IUB Commission on Biochemical Nomenclatureconvention for the definition of the t orsion angles and in a polypeptide chain(Biochemistry9:34713479, 1970) is different from that used here, where the Catomserves as the point of reference for both rotations, but t he result is the same. (Illustration:Irving Geis. Rights owned by Howard Hughes Medical Institute. Not to be reproduced without permission.)


G. N. Ramachandran was the first todemonstrate the convenience of plottingphi,psi combinations from known proteinstructures

The sterically favorable combinations are thebasis for preferred secondary structures


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Figure 6.4

A Ramachandran diagram showing t he stericallyreasonable v alues of the angles and . Theshaded regions indicate particularly favorablevalues of these angles. Dots in purple indicateactual angles measured for 1000 residues(excluding glycine, for which a wider range ofangles is permitted) in eight proteins. The linesrunning across the diagram (numbered +5 through2 and -5 through -3) signify the number of aminoacid residues per turn of the helix; + means right-handed helices; - means left-handedhelices. ( , . ., 1981, 34:34:34:34:167 339.)

Classes of Secondary Structure

All these are local structures that arestabilized 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 RobertCorey 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 originallyappeared in Paulings 1960 The Nature of the Chemical Bond. (b) Showing thearrangement of peptide planes in the helix.

EACH peptide carbonyl is hydrogen bonded to the peptide N-H group four residue fartherup 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 H-bond 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 substantialamounts of -helix in their structures. The helices are represented by the regularly coiledsections of the ribbon drawings. Myohemerythrin is the oxygen-carrying protein in certaininvertebrates, including Sipunculids, a phylum of marine worm. (Jane Richardson)


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Figure 6.8The arrangement of NH and C=O groups (each withan individual dipole moment) along the helix axiscreates a large net dipole for the helix. Numbersindicate fractional charges on respective atoms.

Figure 6.9Four NH groups at the N-terminal end of an -helixand four C=O groups at the C-terminal end cannotparticipate in hydrogen bonding. The formation of H-bonds with other nearby donor and acceptor groups isreferred to as helix capping. Capping may alsoinvolve appropriate hydrophobic interactions thataccomodate nonpolar side chains at the ends of helicalsegments.

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 turnFigure 6.10 A pleated sheet of paper with an antiparallel-sheet drawn on it. (IrvingGeis)

Figure 6.11 The arrangementof hydrogen bonds in (a)parallel and (b) antiparallel-pleated sheets.

The Beta Turn

(aka beta bend, tight turn)

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 6.13Three different kinds of-bulge structures involving a pair of adjacent polypeptidechains. (Adapted from Richardson, J. S., 1981. Advances in Protein Chemistry 34:167339.)

6.4 How Do Polypeptides Fold into Three-Dimensional Protein Structures?

Several important principles:

Secondary structures form whereverpossible (due to formation of largenumbers of H bonds)

Helices and sheets often pack closetogether

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 is88 kJ/mol

A twist about the C-N bond involves a twist energy of 88kJ/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


Unfavorable orbital overlap precludes somecombinations of phi and psi

phi = 0, psi = 180is unfavorablephi = 180, psi = 0is unfavorablephi = 0, psi = 0is unfavorable


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Classes of Secondary Structure

All these are local s tructures that are stabilized byhydrogen 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 ProteinStructures, tertiary structures

Several important principles:

Secondary structures form wherever possible (due toformation of large numbers of H bonds)

Helices and sheets often pack close together

The backbone links between elements of secondarystructure are usually short and direct

Proteins fold to make the most stable structures (make Hbonds and minimize solvent contact

Fibrous and globular Proteins

Fibrous proteins: Primarily structural role

Much or most of the polypeptide chain is organizedapproximately parallel to a single axis

Fibrous proteins are often mechanically strong Fibrous proteins are usually insoluble

Alpha Keratin: Found in hair, fingernails, claws, hornsand beaks

Beta-Keratin: Found in silk fibers

Collagen - A Triple Helix Principal component ofconnective tissue (tendons, cartilage, bones, teeth)

Figure 6.18 Poly(Gly-Pro-Pro), a collagen-like right-handed triple helix composed of three left-handedhelical chains. (Adapted from Miller, M. H., andScheraga, H. A., 1976, Calculation of the st ructures of

collagen models. Role of interchain interactions indetermining the triple-helical coiled-coil conformation. I.Poly(glycyl-prolyl-prolyl).Journal of Polymer Science

Symposium 54:171200.)

Some design principles

Most polar residues face the outside of the protein andinteract with solvent

Most hydrophobic residues face the interior of the proteinand interact with each other

Packing of residues is close

However, ratio of vdw volume to total volume is only 0.72to 0.77, so empty space exists

The empty space is in the form of small cavities

Globular Proteins


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

An amphiphilic helixin flavodoxin:

A nonpolar helix incitrate synthase:

A polar helixin calmodulin:

Globular Proteins

More design principles

"Random coil" is not random

Structures of globular proteins are not static

Various elements and domains of protein move todifferent degrees

Some segments of proteins are very flexible anddisordered

Know the kinds and rates of protein motion

Globular ProteinsThe Forces That Drive Folding

Peptide chain must satisfy the constraintsinherent in its own structure

Peptide chain must fold so as to "bury" thehydrophobic side chains, minimizing their contactwith water

Peptide chains, composed of L-amino acids,have a tendency to undergo a "right-handedtwist"

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

A New Way to Look at GlobularProteins

Look for "layer structures

Helices and sheets often pack in layers

Hydrophobic residues are sandwichedbetween the layers

Outside layers are covered with mostly polarresidues that interact favorably with solvent


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Figure 6.28 Examples of proteindomains with different numbersof layers of backbone structure.(a) Cytochrome c' with twolayers of -helix. (b) Domain 2of phosphoglycerate kinase,

composed of a-sheet layerbetween two layers of helix,three layers overall. (c) Anunusual five-layer structure,domain 2 of glycogenphosphorylase, a-sheet layersandwiched between four layersof -helix. (d) The concentriclayers of-sheet (inside) and-helix (outside) in triosephosphate isomerase.Hydrophobic residues areburied between theseconcentric layers in the samemanner as in the planar layersof the other proteins. Thehydrophobic layers are shadedyellow. (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 phosphateisomerase (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 6.32 Examples of antiparallel-sheet structures in proteins. (Jane Richardson)

Figure 6.33Examples of theso-called Greekkey antiparallel-barrel structure in

proteins.

Thermodynamics of Folding

Read the box on page 185

Separate the enthalpy and entropy terms for thepeptide chain and the solvent

Further distinguish polar and nonpolar groups

The largest favorable contribution to folding is theentropy term for the interaction of nonpolar residueswith the solvent

Molecular Chaperones

Why are chaperones needed if theinformation for folding is inherent in thesequence?

to protect nascent proteins from theconcentrated protein matrix in the celland 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 ormore "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 sameprotein

There is a genetic basis for the use of modules in nature

Figure 6.36 Ribbonstructures of severalprotein modulesutilized in theconstruction ofcomplex multimodeproteins. (a) Thecomplement controlprotein module. (b)The immunoglobulinmodule. (c) Thefibronectin type I

module. (d) Thegrowth factor module.(e) The kringlemodule. (Adapted fromBaron, M., Norman, D.,and Campbell, I., 1991,Protein modules. Trendsin Biochemical Sciences16:13-17.)


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Figure 6.37 A sampling of proteinsthat consist of mosaics of indivi dualprotein modules. The modules shown

include CG, a module containing -carboxyglutamate residues; G, anepidermal growth-factorlike module;K, the kringle domain, named for aDanish pastry; C, which is found incomplement proteins; F1, F2, and F3,first found in fibronectin; I, theimmunoglobulin superfamily domain;N, found in some growth factorreceptors; E, a module homologousto the calcium-binding EF handdomain; and LB, a lectin m odulefound in some cell surface proteins.(Adapted from Baron, M., Norman, D., andCampbell, , 1991, Protein modules. Trendsin Biochemical Sciences16:1317.)

Predictive AlgorithmsIf the sequence holds the secrets of folding, can we

figure it out?

Many protein chemists have tried to predictstructure 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 helicesarrange

Quaternary Structure

What are the forces driving quaternaryassociation?

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 li ver alcohol dehydrogenase. Wi thin eachsubunit is a six-stranded parallel sheet. Between the two subunits is a two-strandedantiparallel sheet. The point in the center is a C2 symmetry axis. (Jane Richardson)

Figure 6.44 Isologousand heterologousassociations betweenprotein subunits. (a)An isologousinteraction betweentwo subunits with atwofold axis ofsymmetryperpendicular to theplane of the page. (b)

A heterologousinteraction that couldlead to the formationof a long polymer. (c)A heterologousinteraction leading toa closed structureatetramer.(d) Atetramer formed bytwo sets of isologousinteractions.

Figure 6.45 Thepolypeptidebackbone of theprealbumin dimer.The monomersassociate in amanner thatcontinues the m-sheets. A tetrameris formed byisologousinteractions

between the sidechains extendingoutward from sheetDAGHHGADin both dimers,which pack togethernearly at rightangles to oneanother. (JaneRichardson)


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Figure 6.47 Schematic drawingof an immunoglobulin moleculeshowing the intramolecular andintermolecular disulfide bridges.(A space-filling model of thesame molecule is shown inFigure 1.11.)

What are the structural and functional advantagesdriving quaternary association?

Know these!

Stability: reduction of surface to volume ratio

Genetic economy and efficiency

Bringing catalytic sites together

Cooperativity

chem 4311- chapter 4-5-6 -animo acid and proteins

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