protein structure bioch301.1-2

8/11/2019 Protein Structure Bioch301.1-2

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Biochemistry 301

Principles of Protein Structure

Walter Chazin

5140 BIOSCI/MRBIII

E-mail: Walter.Chazin

http://structbio.vanderbilt.edu/chazin

Jan. 8-10, 2003


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Text Books

Branden and Tooze

Introduction to Protein Structure

Voet, Voet and Pratt

Fundamentals of Biochemistry

Stryer

Biochemistry


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Proteins: Polymers of Amino Acids

20 different amino acids: many combinations

Proteins are made in the RIBOSOME


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Amino Acid Chemistry

NH2 Ca

R1

CO

H

NH Ca

R2

COOH

H

NH2 Ca

R

COOH

H

amino acid

20 different types

Amino acid Polypeptide Protein

NH2 Ca

R1

COOH

H

NH2 Ca

R2

COOH

H


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NH2 Ca

R

COOH

H

amino acid

The free amino and carboxylic acid groups have pKas

COOH COO-

pKa ~ 2.2

NH2NH3+

pKa ~ 9.4

At physiological pH, amino acids are zwitterions

+NH3 Ca

R

COO-

H


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Note the axesAlso titratable

groups in side chain


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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

pKas


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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


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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


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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


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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


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Hierarchy of Protein Structure


The order of amino acids: Protein sequence

Primary Structure

Local conformation, depends on sequenceSecondary Structure

Overall structure of the chain(s) in full 3D

Tertiary/Quaternary Structure


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Beyond Primary Structure:

The Peptide Bond

-C - N-

O=

-H

-C = N-

O--

-H

Resonance structures

Peptide plane is flatwangle ~180

Partial double-bond:

Peptide bond


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Implications of Peptide Planes

wangle varies little, f andangles vary alot

Many f/combinations cause atoms to collide

Each residue is sandwiched between two planes

Ca

HRf

Peptide planes

Ca

H R

Ca


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Polypeptide Backbone

Backbone restricted limited conformations

Collisions with side chain groups further limit f/combinations

Ca

HRf

Ca

H R

Ca

H R


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Secondary StructureLocal Conformation of Consecutive Residues

Three low energy backbone f combinations

1. Right-hand helix: a-helix (-40

, -60

)

2. Extended: antiparallelb

-sheet (140

, -

140

)

3. Left-hand helix (rare): a-helix (45, 45)

Glycine: special it has no side chain!

Hydrogen bondsbetween backbone atoms

provides stability to secondary structures

Amino acids have specific preferences


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Secondary Structure-a

Helix

H-bond


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Secondary Structure-b

Turn

1

43

2

Reverses direction of the chain


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Ribbon and Topology DiagramsRepresentations of Secondary Structures

Sheets (arrows), Helices (cylinders)

B/T- Figure 2.17


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Ribbon and Topology DiagramsOrganization of Secondary Structures

helix

B/T- Figure 2.11


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Beyond Secondary Structure

Supersecondary structure (motifs): small,discrete, commonly observed aggregates ofsecondary structures

bsheet

helix-loop-helix

bab

Domains: independent units of structure

bbarrelfour-helix bundle

*Domains and motifs sometimes interchanged*


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Protein Motifs

V/V/P- Figure 6.28


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Hairpin Motif

B/T- Figure 2.14


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

B/T- Figure 2.12


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EF-Hand H-L-H Motif

B/T- Figure 2.13


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Greek Key Motif

B/T- Figure 2.15


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Multi-Domain (Modular) Proteins

EGF

Protease

Kringle

Ca-binding

Protein

Domain


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Tertiary Structure

Definition: 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


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Tertiary Structure

Specific structures result from long-range interactions

Electrostatic (charged) interactions

Hydrogen bonds (OH, NH, S H)

Hydrophobic interactions

Soluble proteins have an inside (core) and outside

Folding driven by water- hydrophilic/phobic

Side chain properties specify core/exteriorSome interactions inside, others outside


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Tertiary Structure

I. Ionic Interactions (exterior)

Forms between 2 charged side chains:

1 NegativeGlu,Asp 1 PositiveLys,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.


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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 bondlower energy.

Occurs inside, at the exterior, and with water.


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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 fora specific orientation like an H-bond

In the protein interior, away from waterNot pH dependent


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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


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Disulfide Bonding

V/V/P- Figure 16.6


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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, NH, S H)Hydrophobic interactions

Disulfides only VERY infrequently


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Quaternary Structure

The classic example- hemoglobin a2-b2

B/T- Figure 3.7 END OF PART 1


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Protein Structure from Sequence

The pattern of amino acid side chains determinesthe local conformation and the global structure

*Pattern is more impo rtant 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 L

5 10

h-CaM

b-CaM

conservative non-conservative


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How Does a Protein Find Its Fold?

A protein of n residues: 20npossible sequences!

100 residue protein has 10020possibilities 1.3 X 10130!

The latest estimates indicate < 40,000

sequences in the human genomeTHERE MUST BE RULES!


N C

1 2 3 4

Amino terminus Carboxyl terminus

Residue number


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Limitations 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 manyerrors will build up.

*Leng th is general ly 100-1000 resid ues*


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Protein Folding

The hydrophobic effect is the major driving forceHydrophobic side chains cluster/exclude water

Release of water cages in unfolded state

Other forces providing stability to the folded stateHydrogen bonds

Electrostatic interactions

Chemical cross links- Disulfides, metal ions


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Protein Folding

Random folding has too many possibilities

Backbone restricted but side chains not

A 100 residue protein would require 1087s to

search all conformations (age of universe < 1018s)

Most proteins fold in less than 10 s!!

Proteins must fold along specific pathways!!


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Protein Folding Pathways

Usual order of folding eventsSecondary structures formed quickly (local)

Secondary structures aggregate to form motifs

Hydrophobic collapse to form domains

Coalescence of domains

Molecular chaperones assist folding in-v ivo

Complexity of large chains/multi-domains

Cellular environment is rich in interactingmoleculesChaperones sequester proteins andallow time to fold


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Progressive Folding of ProteinsFrom Disordered to Native State

Protein Folding Funnel

V/V/P- Figures 6.37/38


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Functional 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


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Structural 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)


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Globular Proteins

V/V/P- Figure 6.27

Hemoglobin a Conconavalin A Triose Phosphate isomerase


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Structural 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)


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Photosynthetic Reaction Center

B/T Figure 13.6

Extracellular

Intracellular(cytoplasmic)

Membrane-spanning


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I n the physical sense, the

progression of l iving organisms

results from the communication

between molecules.

I nteraction between molecules is

determined by binding aff inities.


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Binding 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)


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Surface Determines What Binds

1. Steric access

2. Shape

3. Hydrophobic

accessible surface

4. Electrostatic surface

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


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Determinants 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

Bi di Alt P t i St t


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Binding Alters Protein Structure

Mechanisms 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 Transduction


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Calcium Signal TransductionAl lostery & Stimulation by Control Factor

Target

Ca2+

Calmodulin


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SequenceStructureFunction

Many sequences can give same structure

Side chain pattern more important thansequence

When homology is high (>50%), likely to have samestructure 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*


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I. Homologous: similar sequence (cytochrome c)

Same structure

Same function

Modeling structure from homology

Varied Relationships Between

Sequence, Structure and Function


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V/V/P Figure 6.31

C-Type CytochromesSame structure/function- Different Sequence

Heme

Constant structural elements and basic architecture


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I. Homologous: very similar sequence (cytochrome c)

Same structure

Same function


II. Similar function- different sequence (dehydrogenases)

One domain same structure

One domain different


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B/T Figure 10.8

NAD-Binding DomainsConserved Domains/Functional Elements

Lactate DehydrogenaseAlcohol Dehydrogenase

V i d R l i hi B


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I. Homologous: very similar sequence (cytochrome c)

Same structure

Same function


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

A i i


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NADH-Binding and RedoxSame structure- Different Function

Alcohol Dehydrogenase Lactate Dehydrogenase

Thioredoxin

protein structure bioch301.1-2

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