Amino Acids Polymerize to Form Peptides
Amino acids → pep des → proteins• serylglycyltyrosylalanylleucine• Ser-Gly-Tyr-Ala-Leu• SGYALFavorable Interactions in Proteins• Hydrophobic effect– The release of water molecules from the structured solvation layer
around the molecule as protein folds increases the net entropy.

• Hydrogen bonds– Interac on of N−H and C=O of the peptide bond leads to local regularstructures such as  helices and  sheets.

• Van der Waals force– Attraction between all atoms contributes significantly to the stability inthe interior of the protein.

• Electrostatic interactions– long-range strong interactions between permanently charged groups– Salt bridges, especially those buried in the hydrophobic environment,strongly stabilize the protein.

Four Levels of Protein Structure

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Primary Structure: The Peptide Bond
• The structure of the protein is partially dictated by theproperties of the peptide bond.
• The peptide bond is a resonance hybrid of two canonicalstructures.

• The resonance causes the peptide bonds:– to be less reactive compared with esters, for example– to be quite rigid and nearly planar– to exhibit a large dipole moment in the favored transconfiguration

The Rigid Peptide Plane and
the Partially Free Rotations• Rotation around the peptide bond is not permitted due toresonance structure.
• Rotation around bonds connected to the  carbon is permitted.
–  (phi): angle around the  carbon—amide nitrogen bond–  (psi): angle around the  carbon—carbonyl carbon bond
• In a fully extended polypeptide, both  and  are 180°.

The organization around the peptide bond, paired with the identityof the R groups, determines the secondary structure of the protein.

The Polypeptide Is Made Up of a Series of Planes Linked
at α Carbons

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(-139, +135)(-51, +153)(-119, +113)(-57, -47)

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Secondary Structures• Secondary structure refers to a local spatial arrangement ofthe polypeptide backbone.
• Two regular arrangements are common:• the  helix– stabilized by hydrogen bonds between nearby residues• the  sheet– stabilized by hydrogen bonds between adjacentsegments that may not be nearby• Irregular arrangement of the polypeptide chain is called therandom coil.

The  Helix• Helical backbone is held together
by hydrogen bonds between thebackbone amides of an n andn + 4 amino acids.
• It is a right-handed helix with 3.6
residues (5.4 Å) per turn.

n+4• Peptide bonds are aligned
roughly parallel with the helicalaxis.
• Side chains point out and areroughly perpendicular with thehelical axis.
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nThe  Helix: Top View
• The inner diameter of the helix (no sidechains) is about 4–5 Å.
• too small for anything to fit “inside”• The outer diameter of the helix (withside chains) is 10–12 Å.
• happens to fit well into the major

groove of dsDNA• Amino acids #1 and #8 align nicely on
top of each other.

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Sequence Affects Helix Stability• Not all polypeptide sequences adopt -helical structures.
• Small hydrophobic residues such as Ala and Leu arestrong helix formers.
• Pro acts as a helix breaker because the rotation aroundthe N-Ca (φ-angle) bond is impossible.
• Gly acts as a helix breaker because the tiny R groupsupports other conformations.
• Attractive or repulsive interactions between side chains 3
to 4 amino acids apart will affect formation.

Parallel and Antiparallel  Sheets• Multi -strand interactions are called sheets.
• Sheets are held together by the hydrogen bonding of
amide and carbonyl groups of the peptide bond from
opposite strands.
• Two major orientations of  sheets are determined by
the directionality of the strands within:– Parallel sheets have strands that are oriented in thesame direction.
– Antiparallel sheets have strands that are oriented in
opposite directions.

In parallel  sheets, the H-bonded strands run in thesame direction.
• Hydrogen bonds between strands are bent (weaker).

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In antiparallel  sheets, the H-bonded strands run in
opposite directions.
• Hydrogen bonds between strands are linear (stronger).

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 Turns•  turns occur frequently whenever strands in  sheets change
the direction.
• The 180° turn is accomplished over four amino acids.
• The turn is stabilized by a hydrogen bond from a carbonyl
oxygen to amide proton three residues down the sequence.
• Proline in position 2 or glycine in position 3 are common in turns.

Protein Tertiary Structure• Tertiary structure refers to the overall spatial arrangement ofatoms in a protein.
• Stabilized by numerous weak interactions between aminoacid side chains largely hydrophobic and polar interactions
 can be stabilized by disulfide bonds• Interacting amino acids are not necessarily next to each otherin the primary sequence.
• Two major classes:– fibrous:– globular:

Fibrous Proteins
TABLE 4-3

Secondary Structures and Properties of Some Fibrous Proteins

Structure

Characteristics

Examples of occurrence

α Helix, cross-linked bydisulfide bonds

Tough, insoluble protective structures ofvarying hardness and flexibility

α-Keratin of hair, feathers, nails

β Conformation

Soft, flexible filaments

Silk fibroin

Collagen triple helix

High tensile strength, without stretch

Collagen of tendons, bone matrix

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Structure of -Keratin in Hair

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Chemistry of Permanent Waving

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Structure of Collagen• Collagen is an important constituent of connective tissue:
tendons, cartilage, bones, cornea of the eye.
• Each collagen chain is a long Gly- and Pro-rich left-handed
helix.
• Three collagen chains intertwine into a right-handed
superhelical triple helix.
• The triple helix has higher tensile strength than a steel wireof equal cross section.
• Many triple-helices assemble into a collagen fibril.

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4-Hydroxyproline in CollagenThom Robert Alan（1959） James Lind-conqueror of scurvy Parke, Davis & Company

collagen diseases!

• Forces the proline ring to be astructure favorable to fold.
• Offers more hydrogen bonds
between the three strands ofcollagen.
• The posttranslational
processing is catalyzed by prolylhydroxylase and requires αketoglutarate, molecularoxygen, and ascorbate (vitaminC).
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Vitamin C in Prolyl 4-Hydroxylase Restores Fe2+
State: Homework*

Collagen Fibrils• Collagen superstructures areformed by cross-linking ofcollagen triple-helices to formcollagen fibrils.
• Crosslinks are covalent bonds
between Lys or HyLys, or His
amino acid residues.

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Directed by Peter Jackson

Water-Soluble Globular Proteins:

X-ray diffraction of myoglobin by John Kendrew et al, 1950

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ribbonrepresentation
surface contour image• compact!
4 H2O H2Oa Protein or DNA •(hydrophobic) •VW force!

ribbonrepresentation
with s-c of LIVF

H2O

H2O
H2O

H2O

H2O

space filling model

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X-ray Diffraction of Myoglobin Confirms:• All the peptide bonds are in the planar trans configuration– the first evidence of such structure!
• Three of the 4 Pro residues are found at bends.
• The fourth Pro residue occurs within an alpha helix, where
it creates a kink necessary for tight helix packing.
• The Fe in the heme group binds to His 93.

Repeated Motifs Contribute to Final Fold
• Motifs: Specific arrangementof several secondary structureelements– all  helix– all  sheet– both• Motifs can be found asrecurring structures innumerous proteins.
• Globular proteins arecomposed of different motifs
folded together.
• Domains: independentfunctionally and structurally.

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Intrinsically Disordered Proteins• Contain protein segments composed of amino acids whose
higher concentration forces less-defined structure (K, R, E, P)• Disordered regions can conform to many different proteins,facilitating interaction with numerous different partner
proteins.

predictor of natural
disordered regions

Quaternary Structure
A quaternary structure is formed by the assembly of individual
polypeptides into a larger functional cluster: Subunits, why?

2α (141 aa) + 2β (146 aa)

Protein Structural Determination Methods:
X-Ray Crystallography
Steps needed• purify the protein• crystallize the protein• collect diffraction data
• calculate electron density• fit known amino acid residues
into density
Pros• no size limits• well establishedCons• difficult for membrane proteins• cannot resolve (see) hydrogens

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Protein Structural Determination Methods:
Biomolecular NMR
Steps needed• purify the protein• dissolve the protein• collect NMR data
• assign NMR signals• calculate the structurePros• no need to crystallize theprotein• can see many hydrogens
Cons• difficult for insoluble proteins• works best with small proteins

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Proteostasis: The life of a protein

Protein Stability and Folding• Loss of structural integrity with accompanying loss of activity
is called denaturation.
• Proteins can be denatured by: temp, pH, organic solvents,chaotropic agents (urea, guanidine hydrochloride)?

Ribonuclease Refolding• Ribonuclease is a small proteinthat contains eight cysteines
linked via four disulfide bonds.
• Urea in the presence of 2mercaptoethanol fully denaturesribonuclease.
• When urea and 2mercaptoethanol are removed,
the protein spontaneously
refolds, and the correct disulfide
bonds are reformed.
• The sequence alone determines
the native conformation.
• 1972 Chemistry Nobel Prize(Christian B. Anfinsen)

renaturation

How Can Proteins Fold So Fast?
• Proteins fold to the lowest-energy fold in the microsecond tosecond time scales. How can they find the right fold so fast?
• It is mathematically impossible for protein folding to occur byrandomly trying every conformation until the lowest-energy
one is found (Levinthal’s paradox).
• Search for the minimum is not random because the direction
toward the native structure is thermodynamically mostfavorable.

Proteins Folding Follow a Distinct Path1.
2.

local secondary structures form
longer-ranger interaction

Chaperones Prevent Misfolding and Aggregation ofUnfolded Peptides•••triggers ATP hydrolysis

interact with partially- ormis-folded protein
facilitate folding pathways
or provide themicroenvironments
prevent aggregation

Chaperonins

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Protein Misfolding Is the Basis of Numerous
Human Diseases• Native (correctly folded) amyloid is a soluble globular
protein,• Misfolded  amyloid promotes
aggregation at newly exposed
protein-protein interface.
• Correctly folded helices are lostand peptides form  strands, helices, and  sheets.
Homework!
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Summary• the two most important secondary structures–  helices–  sheets• Properties and function of fibrous proteins are related bytheir structures.
• Three-dimensional structures of proteins: protein folding
and denaturation – one of the largest unsolved questions inbiochemistry