Proteins: Function & Structure

Proteins1. Cellular Overview

1. Functions2. Key Properties

2. Core Topics1. Amino Acids: properties, classifications,

pI

2. Primary Structure, Secondary Structure, and Motifs

3. Tertiary Structure1. Fibrous vs. Globular

4. Quaternary Structure

Amazing Proteins: Function1. Catalysts (Enzymes)

2. Transport & Storage

• The largest class of proteins, accelerate rates of reactions

DNA Polymerase

Hemoglobin

Catalase CK2 Kinase

Ovalbumin CaseinSerum albumin Ion channels

Amazing Proteins: Function4. Structural

5. Generate MovementCollagen Keratin Silk Fibroin

Actin Myosin

Amazing Proteins: Function5. Regulation of Metabolism and Gene

Expression

6. Protection

Insulin Lac repressor

Immunoglobulins Thrombin and Fibrinogen

RicinVenom Proteins

Amazing Proteins: Function7. Signaling and response (inter and

intracellular)

Apoptosis

Membrane proteinsSignal transduction

Amazing Proteins: Properties•Biopolymers of amino

acids•Contains a wide range

of functional groups•Can interact with

other proteins or other biological macromolecules to form complex assemblies

•Some are rigid while others display limited flexibility

a-Amino Acids: Protein Building Blocks

C

O

C

R

H O-

NH3+

Carboxyl group

R-group or side-chain

a-amino group

a-carbon

Amino acids are zwitterionic•“Zwitter” = “hybrid” in German

•Fully protonated forms will have specific pKa’s for the different ionizable protons

•Amino acids are amphoteric (both acid and base)

C

O

C

R1

H OH

NH2 C

O

C

R1

H O-

NH3+

C

O

C

R1

H O-

NH3+

Stereochemistry of amino acids

•AA’s synthesized in the lab are racemic mixtures. AA’s from nature are “L” isomers

•These are all optically-active except for glycine (why?)

Stereochemistry of amino acids (AA)

Synthesis of Proteins

C

O

C

R1

H O-

NH3+

C

O

C

R1

H

NH3+

C

O

C

R2

H O-

NH

C

O

C

R2

H O-

NH3++

peptide bond

+ H2O

Synthesis of Proteins

+ C

O

C

R3

H O-

NH3+

C

O

C

R1

H

NH3+

C

O

C

R2

H O-

NH

C

O

C

R1

H

NH3+

C

O

C

R2

H

NHC

O

C

R3

H O-

NH

Synthesis of Proteins

C

O

C

R1

H

NH3+

C

O

C

R2

H

NHC

O

C

R3

H O-

NH + C

O

C

R4

H O-

NH3+

C

O

C

R1

H

NH3+

C

O

C

R2

H

NHC

O

C

R3

H

NH

C

O

C

R4

H O-

NHN-Terminal End

C-Terminal End

Synthesis of Proteins

C

O

C

R1

H

NH3+

C

O

C

R2

H

NH C

O

C

R3

H

NH C

O

C

R4

H O-

NH

C

O

C

R4

H

NH3+

C

O

C

R2

H

NH C

O

C

R3

H

NH C

O

C

R1

H O-

NH

=≠

Synthesis of Proteins

C

O

C

R1

H

NH3+

C

O

C

R2

H

NH C

O

C

R3

H

NH C

O

C

R4

H O-

NH

=≠

C

O

C

R1

H

NH3+

C

O

C

R3

H

NH C

O

C

R2

H

NH C

O

C

R4

H O-

NH

Synthesis of Proteins

C

O

C

R1

H

NH3+

C

O

C

R2

H

NH C

O

C

R3

H

NH C

O

C

R4

H O-

NH

≠

C

O

C

R1

H

NH3+

C

O

C

R3

H

NH C

O

C

R2

H

NH C

O

C

R4

H O-

NH

C

O

C

R4

H

NH3+

C

O

C

R2

H

NH C

O

C

R3

H

NH C

O

C

R1

H O-

NH

≠

COMMON AMINO ACIDS20 common amino acids make up the multitude of proteins we know of

Amino Acids With Aliphatic Side Chains

Amino Acids With Aliphatic Side Chains

Amino Acids With Aliphatic Side Chains

Amino Acids With Aromatic Side Chains

Amino Acids with Aromatic Side Chains Can Be Analyzed by UV Spectroscopy

Amino Acids With Hydroxyl Side Chains

Amino Acid with a Sulfhydryl Side Chain

Disulfide Bond Formation

Amino Acids With Basic Side Chains

Amino Acids With Acidic Side Chains and Their Amide Derivatives

There are some important uncommon amino acids

pH and Amino Acids

Net charge: +1 Net charge: 0 Net charge: -1

Characteristics of Acidic and Basic Amino Acids

•Basic amino acids▫High pKa ▫Function as bases

at physiological pH

▫Side chains with N

•Acidic amino acids▫Low pKa ▫Negatively

charged at physiological pH

▫Side chains with –COOH

▫Predominantly in unprotonated form

Isoelectic point (pI)

•the pH at which the compound is electrically neutral▫Equal number of (+) and (-) charge

•At pH < pI amino acid is (+)•At pH > pI amino acid is (-)

•CRITICAL FOR: protein analysis, purification, isolation, crystallization

Protein StructureWe use different “levels” to fully describe the structure

of a protein.

Primary Structure•Amino acid sequence•Standard: Left to Right means N to C-

terminal

•Eg. Insulin (AAA40590)

•The info needed for further folding is contained in the 1o structure.

MAPWMHLLTVLALLALWGPNSVQAYSSQHLCGSNLVEALYMTCGRSGFYRPHDRRELEDLQVEQAELGLEAGGLQPSALEMILQKRGIVDQCCNNICTFNQLQNYCNVP

Secondary Structure•The regular local structure based on the

hydrogen bonding pattern of the polypeptide backbone▫α helices▫β strands (β sheets)▫Turns and Loops

•WHY will there be localized folding and twisting? Are all conformations possible?

Consequences of the Amide PlaneTwo 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

See blackboard for explanation why the peptide bond is planar

Unfavorable orbital overlap precludes some combinations of phi and psi

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

Sasisekharan

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

α Helix

• First proposed by Linus Pauling and Robert Corey in 1951.• 3.6 residues per turn, 1.5 Angstroms rise per residue• Residues face outward

α Helix

•α-helix is stabilized by H-bonding between CO and NH groups

•Except for amino acid residues at the end of the α-helix, all main chain CO and NH are H-bonded

α Helix representation

β strand

•Fully extended•β sheets are formed by linking 2 or more

strands by H-bonding

• Beta-sheet also proposed by Corey and Pauling in 1951.

PARALLEL

ANTIPARALLEL

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

Mixed β Sheets

Twisted β Sheets

Loops

What Determines the Secondary Structure?

•The amino acid sequence determines the secondary structure

•The α helix can be regarded as the default conformation – Amino acids that favor α helices:

Glu, Gln, Met, Ala, Leu – Amino acids that disrupt α helices:

Val, Thr, Ile, Ser, Asx, Pro

What Determines the Secondary Structure?

•Branching at the β-carbon, such as in valine, destabilizes the α helix because of steric interactions

•Ser, Asp, and Asn tend to disrupt α helices because their side chains compete for H-bonding with the main chain amide NH and carbonyl

•Proline tends to disrupt both α helices and β sheets

•Glycine readily fits in all structures thus it does not favor α helices in particular

Can the Secondary Structure Be Predicted?•Predictions of secondary structure of

proteins adopted by a sequence of six or fewer residues have proved to be 60 to 70% accurate

•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 ▫George Rose may be much closer to solving the

problem. See Proteins 22, 81-99 (1995)

Modeling protein folding with Linus (George Rose)

Tertiary Structure•The overall 3-D fold of the polypeptide

chain •The amino acid sequence determines the

tertiary structure (Christian Anfinsen)•The polypeptide chain folds so that its

hydrophobic side chains are buried and its polar charged chains are on the surface▫Exception : membrane proteins▫Reverse : hydrophobic out, hydrophilic in

•A single polypeptide chain may have several folding domains

•Stabilized by H-bonding, LDF, noncovalent interactions, dipole interactions, ionic interactions, disulfide bonds

Fibrous and Globular Proteins

Fibrous Proteins•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

•Usually play a structural role in nature

Examples of Fibrous Proteins•Alpha Keratin: hair, nails, claws, horns,

beaks

•Beta Keratin: silk fibers (alternating Gly-Ala-Ser)

Examples of Fibrous Proteins•Collagen: connective

tissue- tendons, cartilage, bones, teeth▫Nearly one residue out

of three is Gly ▫Proline content is

unusually high ▫Unusual amino acids

found: (4-hydroxyproline, 3-hydroxyproline , 5-hydroxylysine)

▫Special uncommon triple helix!

Globular Proteins•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 but empty spaces exist in the form of small cavities

•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

An amphiphilic helix in flavodoxin:

A nonpolar helix in citrate synthase:

A polar helix in calmodulin:

Quaternary Structures•Spatial arrangement of subunits and the

nature of their interactions. Can be hetero and/or homosubunits

•Simplest example: dimer (e.g. insulin)

ADVANTAGES of 4o Structures▫ Stability: reduction of surface to volume ratio ▫ Genetic economy and efficiency ▫ Bringing catalytic sites together ▫ Cooperativity

Protein Folding• The largest

favorable contribution to folding is the entropy term for the interaction of nonpolar residues with the solvent

•CHAPERONES assist protein folding▫ to protect nascent

proteins from the concentrated protein matrix in the cell and perhaps to accelerate slow steps