Biochemistry 301

Principles of Protein Structure

Walter Chazin5140 BIOSCI/MRBIIIE-mail: Walter.Chazin

http://structbio.vanderbilt.edu/chazin

Jan. 8-10, 2003

Text Books

Branden and ToozeIntroduction to Protein Structure

Voet, Voet and PrattFundamentals of Biochemistry

StryerBiochemistry

Proteins: Polymers of Amino Acids

• 20 different amino acids: many combinations

• Proteins are made in the RIBOSOME

Amino Acid Chemistry

NH2 C

R1

CO

H

NH C

R2

COOH

H

NH2 C

R

COOH

H

amino acid

20 different types

Amino acid Polypeptide Protein

NH2 C

R1

COOH

H

NH2 C

R2

COOH

H

Amino Acid Chemistry

NH2 C

R

COOH

H

amino acid

The free amino and carboxylic acid groups have pKa’s

COOH COO-

pKa ~ 2.2

NH2NH3+

pKa ~ 9.4

At physiological pH, amino acids are zwitterions

+NH3 C

R

COO-

H

Amino Acid Chemistry

Note the axesAlso titratable

groups in side chain

Glycine Gly - G                                      

2.4 9.8

Alanine Ala - A                                          

2.4 9.9

Valine Val - V

                                                        

2.2 9.7

Leucine Leu - L

                                                                       

2.3 9.7

Isoleucine

Ile - I

                                                                 

2.3 9.8

Amino Acids with Aliphatic R-Groups

pKa’s

Amino Acids with Polar R-GroupsNon-Aromatic Amino Acids with Hydroxyl R-Groups

Serine Ser - S                                                            

2.2 9.2 ~13

Threonine Thr - T

                                                        

2.1 9.1 ~13

Amino Acids with Sulfur-Containing R-Groups

Cysteine Cys - C                                                           

1.9 10.8 8.3

Methionine

Met-M

                                                             

2.1 9.3

Aspartic Acid Asp - D                                                                  

2.0 9.9 3.9

Asparagine Asn - N                                                                    

2.1 8.8

Glutamic Acid

Glu - E                                                                             

2.1 9.5 4.1

Glutamine Gln - Q                                                                                 

2.2 9.1

Acidic Amino Acids and Amide Conjugates

Basic Amino Acids

Arginine Arg - R

                                                                                    

1.8 9.0 12.5

Lysine Lys - K                                                                              

2.2 9.2 10.8

Histidine

His - H                                                                  

1.8 9.2 6.0

Aromatic Amino Acids and Proline

Phenylalanine

Phe - F

                                                             2.2 9.2

Tyrosine Tyr - Y

                                                                        2.2 9.1 10.1

Tryptophan Trp-W

                                                                      2.4 9.4

Proline Pro - P                              

2.0 10.6

Hierarchy of Protein Structure

• 20 different amino acids: many combinations

The order of amino acids: Protein sequencePrimary Structure

Local conformation, depends on sequenceSecondary Structure

Overall structure of the chain(s) in full 3DTertiary/Quaternary Structure

Beyond Primary Structure:The Peptide Bond

-C - N-

O

=

-H-C = N-

O-

-

-H

Resonance structures

Peptide plane is flat angle ~180º

Partial double-bond:

Peptide bond

Implications of Peptide Planes

angle varies little, and angles vary alot

Many / combinations cause atoms to collide

Each residue is sandwiched between two planes

C

HR

Peptide planes

C

H R

C

Polypeptide Backbone

Backbone restricted limited conformations

Collisions with side chain groups further limit /combinations

C

HR

C

H R

C

H R

Secondary StructureLocal Conformation of Consecutive Residues

• Three low energy backbone combinations

1. Right-hand helix: -helix (-40°, -60°)

2. Extended: antiparallel -sheet (140°, -140°)

3. Left-hand helix (rarerare): -helix (45°, 45°)Glycine: special it has no side chain!

• Hydrogen bonds between backbone atoms provides stability to secondary structures

• Amino acids have specific preferences

Secondary Structure- Helix

H-bond

Secondary Structure- Sheet

Oxygen Nitrogen

R Group

Hydrogen

Carbon Carbonyl C

H Bond

Secondary Structure- Turn

1

43

2

Reverses direction of the chain

Ribbon and Topology DiagramsRepresentations of Secondary Structures

Sheets (arrows), Helices (cylinders)

B/T- Figure 2.17

Ribbon and Topology DiagramsOrganization of Secondary Structures

helix

B/T- Figure 2.11

Beyond Secondary StructureSupersecondary structure (motifs): small, discrete, commonly observed aggregates of secondary structures

sheet

helix-loop-helix

Domains: independent units of structure barrel four-helix bundle

*Domains and motifs sometimes interchanged*

Protein Motifs

V/V/P- Figure 6.28

Hairpin Motif

B/T- Figure 2.14

Helix-Loop-Helix (H-L-H) Motif

B/T- Figure 2.12

EF-Hand H-L-H Motif

B/T- Figure 2.13

Greek Key Motif

B/T- Figure 2.15

Multi-Domain (Modular) Proteins

EGF

Protease

Kringle

Ca-binding

Protein

Domain

Tertiary StructureDefinition: Overall 3D form of a molecule

Organization of the secondary structures/ motifs/domains

Optimization of interactions between residues

A specific 3D structure is formed

All proteins have multiple secondary structures, almost always multiple motifs, and

in some cases multiple domains

Tertiary Structure

Specific structures result from long-range interactions

Electrostatic (charged) interactions

Hydrogen bonds (OH, N H, S H)

Hydrophobic interactions

Soluble proteins have an inside (core) and outside

Folding driven by water- hydrophilic/phobic

Side chain properties specify core/exterior

Some interactions inside, others outside

Tertiary Structure

I. Ionic Interactions (exterior)

Forms between 2 charged side chains:

1 Negative – Glu,Asp 1 Positive – Lys,Arg,His  

Also called “salt bridges”.

Ionic interactions are pH-dependent (pKa).

Occurs at the exterior

NOTE: pKs for in the interior of a protein may be

very different from free amino acid.

Tertiary Structure

II. Hydrogen bonds (interior and exterior)

Forms between side chains/backbone/water:

Charged side chains: Glu,Asp,His,Lys,Arg

Polar chains: Ser,Thr,Cys,Asn,Gln,[Tyr,Trp] 

Not a specific covalent bond – lower energy.

Occurs inside, at the exterior, and with water.

Tertiary Structure

III. Hydrophobic Interactions (interior)

Forms between side chains of non-polar residues:

Aliphatic (Ala,Val,Leu,Ile,Pro,Met)

Aromatic (Phe,Trp,[Tyr])

Clusters of side chains- but no requirement for a specific orientation like an H-bond

In the protein interior, away from water

Not pH dependent

Tertiary Structure

IV. Disulfide Bonds (interior and exterior)

Forms between Cys residues:

Cys-SH + HS-Cys Cys-S-S-Cys

Catalyzed by specific enzymes, oxidizing agents

Restricts flexibility of the protein

Usually within a protein, less for linking proteins

Disulfide Bonding

V/V/P- Figure 16.6

Quaternary Structure

Definition: Organization of multiple chain associations

Oligomerization- Homo (self), Hetero (different)

Used in organizing single proteins and protein

machines

Specific structures result from long-range interactions

Electrostatic (charged) interactions

Hydrogen bonds (OH, N H, S H)

Hydrophobic interactions

Disulfides only VERY infrequently

Quaternary Structure

The classic example- hemoglobin 2-2

B/T- Figure 3.7 END OF PART 1

Protein Structure from SequenceProtein Structure from SequenceThe pattern of amino acid side chains determines the local conformation and the global structure

*Pattern is more important than exact sequence*

A T V R L L E W E D L

Reporting/Comparing Protein Sequences

A T V R L L E Y K D L5 10

h-CaM

b-CaM

conservative non-conservative

Proteins Fold To TheirProteins Fold To TheirNative StructureNative Structure

Folded proteins are only marginally stable!!

~0.4 kJ•mol-1 required to unfold (cf. ~20/H-bond)

Balance loss of entropy vs. stabilizing forces

Protein fold is specified by sequence

Reversible reaction- denature (fold)/renature

Even single mutations can cause changes

Recent discovery that amyloid diseases (eg.

CJD, Alzheimer) are due to unstable protein folding

How Does a Protein Find It’s Fold? How Does a Protein Find It’s Fold?

A protein of n residues: 20n possible sequences!

100 residue protein has 10020 possibilities 1.3 X 10130!

The latest estimates indicate < 40,000 sequences in the human genome

THERE MUST BE RULES!

• 20 different amino acids: many combinations

N C

1 2 3 4

Amino terminus Carboxyl terminus

Residue number

Limitations on Protein SequenceLimitations on Protein Sequence

Minimum length based on ability to perform a

biochemical function: ~40 residues (e.g. inhibitors)

Maximum length based on complexity of assembly:

Conversion of DNA code and production of proteins

is carried out by molecular machines that are not

perfect. If the sequence gets too long, too many

errors will build up.

*Length is generally 100-1000 residues*

Protein FoldingProtein FoldingThe hydrophobic effect is the major driving force

Hydrophobic side chains cluster/exclude water

Release of water cages in unfolded state

Other forces providing stability to the folded state

Hydrogen bonds

Electrostatic interactions

Chemical cross links- Disulfides, metal ions

Protein FoldingProtein Folding

Random folding has too many possibilities

• Backbone restricted but side chains not

• A 100 residue protein would require 1087 s to search all conformations (age of universe < 1018 s)

• Most proteins fold in less than 10 s!!

Proteins must fold along specific pathways!!

Protein Folding PathwaysProtein Folding PathwaysUsual order of folding events

Secondary structures formed quickly (local)

Secondary structures aggregate to form motifs

Hydrophobic collapse to form domains

Coalescence of domains

Molecular chaperones assist folding in-vivo

Complexity of large chains/multi-domains

Cellular environment is rich in interacting molecules Chaperones sequester proteins and allow time to fold

Progressive Folding of ProteinsProgressive Folding of ProteinsFrom Disordered to Native StateFrom Disordered to Native State

Protein Folding Funnel

V/V/P- Figures 6.37/38

Functional Classes of ProteinsFunctional Classes of Proteins

• Receptors- sense stimuli, e.g. in neurons

• Channels- control cell contents

• Transport- e.g. hemoglobin in blood

• Storage- e.g. ferritin in liver

• Enzyme- catalyze biochemical reactions

• Cell function- multi-protein machines

• Structural- collagen in skin

• Immune response- antibodies

Structural Classes of ProteinsStructural Classes of Proteins

1. Globular proteins (enzymes, molecular machines)

Variety of secondary structures

Approximately spherical shape

Water soluble

Function in dynamic roles (e.g. catalysis,

regulation, transport, gene processing)

Globular ProteinsGlobular Proteins

V/V/P- Figure 6.27

Hemoglobin Conconavalin A Triose Phosphate isomerase

Structural Classes of ProteinsStructural Classes of Proteins

2. Fibrous Proteins (fibrils, structural proteins)

One dominating secondary structure

Typically narrow, rod-like shape

Poor water solubility

Function in structural roles (e.g. cytoskeleton,

bone, skin)

Collagen: A Fibrous ProteinCollagen: A Fibrous Protein

V/V/P- Figures 6.17/18

Triple Helix

Gly-Pro-Pro Repeat

StabilizingInter-strand

H-bonds

Structural Classes of ProteinsStructural Classes of Proteins

3. Membrane Proteins (receptors, channels)

Inserted into (through) membranes

Multi-domain- membrane spanning,

cytoplasmic, and extra-cellular domains

Poor water solubility

Function in cell communication (e.g. cell

signaling, transport)

Photosynthetic Reaction CenterPhotosynthetic Reaction Center

B/T Figure 13.6

Extracellular

Intracellular(cytoplasmic)

Membrane-spanning

In the physical sense, the progression of living organisms results from the communication

between molecules.

Interaction between molecules is determined by binding affinities.

Binding Classification of ProteinsBinding Classification of Proteins

• Structural- other structural proteins

• Receptors- regulatory proteins, transmitters

• Toxins- receptors

• Transport- O2/CO2, cholesterol, metals, sugars

• Storage- metals, amino acids,

• Enzymes- substrates, inhibitors, co-factors

• Cell function- proteins, RNA, DNA, metals, ions

• Immune response- foreign matter (antigens)

Surface Determines What BindsSurface Determines What Binds

1. Steric access

2. Shape

3. Hydrophobic accessible surface

4. Electrostatic surface

Sequence and structure optimized to generate surface properties for requisite binding event(s)

Determinants of Protein SurfaceDeterminants of Protein Surface

Function requires specific amino acid properties

Not all amino acids are equally useful

Abundant: Leu, Ala, Gly, Ser, Val, Glu

Rare: Trp, Cys, Met, His

Post-translational modifications Addition of co-factors- metals, hemes, etc. Chemical modification- phosphorylation,

glycosylation, acetylation, ubiquination, sumoylation

Binding Alters Protein StructureBinding Alters Protein StructureMechanisms of Achieving Functional PropertiesMechanisms of Achieving Functional Properties 1. Allosteric Control- binding at one site effects changes

in conformation or chemistry at a point distant in space

2. Stimulation/inhibition by control factors- proteins, ions, metals control progression of a biochemical process (e.g. controlling access to active site)

3. Reversible covalent modification- chemical bonding, e.g. phosphorylation (kinase/phosphatase)

4. Proteolytic activation/inactivation- irreversible, involves cleavage of one or more peptide bonds

Calcium Signal TransductionCalcium Signal TransductionAllostery & Stimulation by Control FactorAllostery & Stimulation by Control Factor

Target

Ca2+

Calmodulin

SequenceSequenceStructureStructureFunctionFunction

Many sequences can give same structure Side chain pattern more important than

sequence When homology is high (>50%), likely to have same

structure and function (Structural Genomics) Cores conserved Surfaces and loops more variable

*3-D shape more conserved than sequence*

*There are a limited number of structural frameworks*

I. Homologous: similar sequence (cytochrome c) Same structure Same function Modeling structure from homology

Varied Relationships Between Varied Relationships Between Sequence, Structure and Sequence, Structure and

FunctionFunction

V/V/P Figure 6.31

C-Type CytochromesC-Type CytochromesSame structure/function- Different SequenceSame structure/function- Different Sequence

Heme

Constant structural elements and basic architecture

Varied Relationships Between Varied Relationships Between Sequence, Structure and Sequence, Structure and

FunctionFunctionI. Homologous: very similar sequence (cytochrome c)

Same structure Same function Modeling structure from homology

II. Similar function- different sequence (dehydrogenases) One domain same structure One domain different

B/T Figure 10.8

NAD-Binding DomainsNAD-Binding DomainsConserved Domains/Functional ElementsConserved Domains/Functional Elements

Lactate DehydrogenaseAlcohol Dehydrogenase

Varied Relationships Between Varied Relationships Between Sequence, Structure and Sequence, Structure and

FunctionFunctionI. Homologous: very similar sequence (cytochrome c)

Same structure Same function Modeling structure from homology

II. Similar function- different sequence (dehydrogenases) One domain same structure One domain different

III. Similar structure- different function (cf. thioredoxin) Same 3-D structure Not same function

B/T Figures 10.8/2.7

NADH-Binding and RedoxNADH-Binding and RedoxSame structure- Different FunctionSame structure- Different Function

Alcohol Dehydrogenase Lactate Dehydrogenase

Thioredoxin