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Biochemistry 2/e - Garrett & Grisham
CHAPTER 5
Proteins: Their Biological
Functions and Primary Structure
to accompany
Biochemistry, 2/e
by
Reginald Garrett and Charles Grisham
All rights reserved. Requests for permission to make copies of any part of the work
should be mailed to: Permissions Department, Harcourt Brace & Company, 6277
Sea Harbor Drive, Orlando, Florida 32887-6777
Copyright © 1999 by Harcourt Brace & Company
Outline
• 5.1 Proteins - Linear Polymers of Amino Acids
• 5.2 Architecture
• 5.3 Many Biological Functions
• 5.4 May be Conjugated with Other Groups
• 5.7 Primary Structure Determination
• 5.8 Consider the Nature of Sequences

5.1 Proteins are Linear Polymers of Amino Acids

The Peptide Bond

• is usually found in the trans conformation
• has partial (40%) double bond character
• is about 0.133 nm long - shorter than a
typical single bond but longer than a
double bond
• Due to the double bond character, the six
atoms of the peptide bond group are
always planar!
• N partially positive; O partially negative


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

“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


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
• Connectin proteins - alpha - MW 2.8 million!
• beta connectin - MW of 2.1 million, with a
length of 1000 nm -it can stretch to 3000
nm!

The Sequence of Amino Acids

in a Protein
• is a unique characteristic of every
protein
• is encoded by the nucleotide sequence
of DNA
• is thus a form of genetic information
• is read from the amino terminus to the
carboxyl terminus
The sequence of ribonuclease A

5.2 Architecture of Proteins

• Shape - globular or fibrous
• The levels of protein structure
- Primary - sequence
- Secondary - local structures - H-bonds
- Tertiary - overall 3-dimensional shape
- Quaternary - subunit organization


What forces determine the

structure?
• Primary structure - determined by
covalent bonds
• Secondary, Tertiary, Quaternary structures -
all determined by weak forces
• Weak forces - H-bonds, ionic interactions,
van der Waals interactions, hydrophobic
interactions


How to view a protein?

• backbone only
• backbone plus side chains
• ribbon structure
• space-filling structure


Configuration and
conformation are
not the same

5.3 Biological Functions of

Proteins
Proteins are the agents of biological function
• Enzymes - Ribonuclease
• Regulatory proteins - Insulin
• Transport proteins - Hemoglobin
• Structural proteins - Collagen
• Contractile proteins - Actin, Myosin
• Exotic proteins - Antifreeze proteins in fish

The tetrameric structure of hemoglobin

5.4 Other Chemical Groups in

Proteins
Proteins may be "conjugated" with other
chemical groups
• If the non-amino acid part of the protein is
important to its function, it is called a
prosthetic group.
• Be familiar with the terms: glycoprotein,
lipoprotein, nucleoprotein, phosphoprotein,
metalloprotein, hemoprotein, flavoprotein.

5.7 Sequence Determination

Frederick Sanger was the first - in 1953, he
sequenced the two chains of insulin.
• Sanger's results established that all of the
molecules of a given protein have the
same sequence.
• Proteins can be sequenced in two ways:
- real amino acid sequencing
- sequencing the corresponding DNA in
the gene

Insulin consists of two

polypeptide chains, A
and B, held together by
two disulfide bonds.
The A chain has 21
residues and the B
chain has 30 residues.
The sequence shown is

that of bovine insulin.

Determining the Sequence

An Eight Step Strategy
• 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 to generate a
different set of fragments.


• 7. Reconstruct the sequence of the

protein from the sequences of
overlapping fragments
• 8. Determine the positions of the
disulfide crosslinks

Step 1:
Separation of chains
• Subunit interactions depend on weak
forces
• 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 an
alkylating agent like iodoacetate


Step 3:
Determine Amino Acid Composition
• described on pages 112,113 of G&G
• results often yield ideas for
fragmentation of the polypeptide chains
(Step 5, 6)

Step 4:
Identify N- and C-terminal residues
• N-terminal analysis:
– Edman's reagent
– phenylisothiocyanate
– derivatives are phenylthiohydantions
– or PTH derivatives


Step 4:
Identify N- and C-terminal residues
• C-terminal analysis
– Enzymatic analysis (carboxypeptidase)
– Carboxypeptidase A cleaves any residue
except Pro, Arg, and Lys
– Carboxypeptidase B (hog pancreas) only
works on Arg and Lys

Steps 5 and 6:
Fragmentation of the chains
• Enzymatic fragmentation
– trypsin, chymotrypsin, clostripain,
staphylococcal protease
• Chemical fragmentation
– cyanogen bromide

Enzymatic Fragmentation
• Trypsin - cleavage on the C-side of Lys, Arg
• 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


Chemical Fragmentation
Cyanogen bromide
• CNBr acts only on methionine residues
• CNBr is useful because proteins usually
have only a few Met residues
• see Fig. 5.21 for mechanism
• be able to recognize the results!
– a peptide with a C-terminal homoserine
lactone







Step 7:
Reconstructing the Sequence
• Use two or more fragmentation agents in
separate fragmentation experiments
• Sequence all the peptides produced
(usually by Edman degradation)
• Compare and align overlapping peptide
sequences to learn the sequence of the
original polypeptide chain


Compare cleavage by trypsin and
staphylococcal protease 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


• The correct overlap of fragments:
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

Sequence analysis of catrocollastatin-C, a 23.6 kD

protein from the venom of Crotalus atrox


Nature of Protein Sequences

• Sequences and composition reflect the
function of the protein
• Membrane proteins have more
hydrophobic residues, whereas fibrous
proteins may have atypical sequences
• Homologous proteins from different
organisms have homologous sequences
• e.g., cytochrome c is highly conserved



Phylogeny of Cytochrome c
• The number of amino acid differences
between two cytochrome c sequences is
proportional to the phylogenetic difference
between the species from which they are
derived
• This observation can be used to build
phylogenetic trees of proteins
• This is the basis for studies of molecular
evolution





Laboratory Synthesis of
Peptides
• Strategies are complex because of the
need to control side chain reactions
• Blocking groups must be added and later
removed
• du Vigneaud’s synthesis of oxytocin in
1953 was a milestone
• Bruce Merrifield’s solid phase method was
even more significant

Solid Phase Synthesis

• Carboxy terminus of a nascent peptide is
covalently anchored to an insoluble resin
• After each addition of a residue, the
resin particles are collected by filtration
• Automation and computer control now
permit synthesis of peptides of 30
residues or more


Chapter 6
Proteins: Secondary, Tertiary, and
Quaternary Structure
to accompany
Biochemistry, 2/e
by
Reginald Garrett and Charles Grisham
All rights reserved. Requests for permission to make copies of any part of the work
should be mailed to: Permissions Department, Harcourt Brace & Company, 6277
Sea Harbor Drive, Orlando, Florida 32887-6777
Outline
• 6.1 Forces Influencing Protein Structure
• 6.2 Role of the Amino Acid Sequence in
Protein Structure
• 6.3 Secondary Structure of Proteins
• 6.4 Protein Folding and Tertiary Structure
• 6.5 Subunit Interactions and Quaternary
Structure

6.1 The Weak Forces

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: <40 kJ/mol


6.2 The Role of the Sequence

in Protein 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.

How do proteins recognize and

interpret the folding information?
• Certain loci along the chain may act as
nucleation points
• Protein chain must avoid local energy
minima
• Chaperones may help

6.3 Secondary Structure

The atoms of the peptide bond lie in a plane
• The resonance stabilization energy of the
planar structure is 88 kJ/mol
• A twist about the C-N bond involves a twist
energy of 88 kJ/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
• 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

Steric Constraints on phi & psi

Unfavorable orbital overlap precludes
some combinations of phi and psi
• phi = 0, psi = 180 is unfavorable



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


Classes of Secondary Structure

All these are local structures that are
stabilized 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 167
• First proposed by Linus Pauling and
Robert Corey in 1951
• Identified in keratin by Max Perutz
• A ubiquitous component of proteins
• Stabilized by H-bonds


The Alpha Helix

Know these numbers
• Residues per turn: 3.6
• Rise per residue: 1.5 Angstroms
• Rise per turn (pitch): 3.6 x 1.5A = 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




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 turn



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



Tertiary Structure
Several important principles:
• Secondary structures form wherever

possible (due to formation of large
numbers of H-bonds)
• Helices and sheets often pack close
together

Tertiary Structure
Several important principles:
• The backbone links between elements

of secondary structure are usually short
and direct
• Proteins fold to make the most stable
structures (make H-bonds and minimize
solvent contact

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

Alpha Keratin
• Found in hair, fingernails, claws, horns and
beaks
• Sequence consists of 311-314 residue alpha
helical rod segments capped with non-helical N-
and C-termini
• Primary structure of helical rods consists of 7-
residue repeats: (a-b-c-d-e-f-g)n, where a and d
are nonpolar. Promotes association of helices!


Beta Keratin
Proteins that form extensive beta sheets
• Found in silk fibers
• Alternating sequence: Gly-Ala/Ser-Gly-
Ala/Ser....
• Since residues of a beta sheet extend alternately
above and below the plane of the sheet, this
places all glycines on one side and all alanines
and serines on other side!
• This allows Glys on one sheet to mesh with Glys
on an adjacent sheet (same for Ala/Sers)



Collagen - A Triple Helix

Principal component of connective tissue
(tendons, cartilage, bones, teeth)
• basic unit is tropocollagen:
– three intertwined polypeptide chains (1000
residues each
– MW = 285,000
– 300 nm long, 1.4 nm diameter
– unique amino acid composition

Collagen
The secrets of its a.a. composition...
• Nearly one residue out of three is Gly
• Proline content is unusually high
• Unusual amino acids found:
– 4-hydroxyproline
– 3-hydroxyproline
– 5-hydroxylysine
– Pro and HyPro together make 30% of res.



The Collagen Triple Helix

A case of structure following composition
• The unusual amino acid composition of collagen
is unsuited for alpha helices OR beta sheets
• But it is ideally suited for the collagen triple helix:
three intertwined helical strands
• Much more extended than alpha helix, with a
rise per residue of 2.9 Angstroms
• 3.3 residues per turn
• Long stretches of Gly-Pro-Pro/HyP


Collagen Fibers
Staggered arrays of tropocollagens
• Banding pattern in EMs with 68 nm repeat
• Since tropocollagens are 300 nm long,
there must be 40 nm gaps between
adjacent tropocollagens (5x68 = 340
Angstroms)
• 40 nm gaps are called "hole regions" -
they contain carbohydrate and are thought
to be nucleation sites for bone formation


Structural basis of the

collagen triple helix
• Every third residue faces the crowded center of
the helix - only Gly fits here
• Pro and HyP suit the constraints of phi and psi
• Interchain H-bonds involving HyP stabilize helix
• Fibrils are further strengthened by intrachain
lysine-lysine and interchain hydroxypyridinium
crosslinks



Globular Proteins
Some design principles
• 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
• However, ratio of vdw volume to total volume is
only 0.72 to 0.77, so empty space exists
• The empty space is in the form of small cavities


An amphiphilic helix
in flavodoxin:
A nonpolar helix in
citrate synthase:
A polar helix in
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 to different degrees
• Some segments of proteins are very
flexible and disordered
• Know the kinds and rates of protein motion

Globular Proteins
The Forces That Drive Folding
• Peptide chain must satisfy the constraints
inherent in its own structure
• Peptide chain must fold so as to "bury"
the hydrophobic side chains, minimizing
their contact with water
• Peptide chains, composed of L-amino
acids, have a tendency to undergo a
"right-handed twist"


A New Way to Look at

Globular Proteins
Look for "layer structures"
• 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


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

Antiparallel Alpha Helical

Proteins
See Figure 6.29 for some examples
• Simplest way to pack helices - short
connecting loops and antiparallel packing
• The helix bundle often involves a slight (15
degree) left-handed twist
• The globin proteins - myoglobin and
hemoglobin - are antiparallel alpha
proteins



Parallel or Mixed Beta Sheet

Proteins
See Figure 6.30, 6.31
• Parallel beta sheets distribute nonpolar
residues on both sides of the beta sheet
• This means that both faces of the sheet must
be protected from solvent
• Thus parallel beta sheets are core structures
• Parallel beta barrels are in this class
• Doubly wound parallel beta sheets also



Antiparallel Beta Sheets

See Figures 6.32, 6.33, 6.34
• Antiparallel beta sheets place nonpolar
residues on only one face of the sheet
• Only one face must be protected from solvent
• Thus antiparallel beta sheet proteins may
contain as few as two layers
• Possibilities: barrels, beta sandwiches and
sheets covered by helices on one face only




Metal-Rich and Disulfide-rich

Proteins
See Figure 6.35
• Usually less than 100 residues
• Conformations usually heavily influenced
by metals and/or disulfide bridges
• These proteins are usually unstable if the
metals are removed or the disulfides are
reduced


Thermodynamics of Folding
• Separate the enthalpy and entropy terms for
the peptide chain and the solvent
• Further distinguish polar and nonpolar
groups
• The largest favorable contribution to folding is
the entropy term for the interaction of
nonpolar residues with the solvent


Molecular Chaperones
• Why are chaperones needed if the
information for folding is inherent in the
sequence?
– to protect nascent proteins from the
concentrated protein matrix in the cell and
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 or more "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 same protein
• There is a genetic basis for the use of
modules in nature



Predictive Algorithms
If the sequence holds the secrets of folding, can
we figure it out?
• 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)

Ken Dill’s folding
funnel.
Unfolded structures lie

around the top. As the
protein folds, it falls
down the wall of the
energy funnel to more
stable conformations.
The native, folded

structure is at the
bottom.
Nature Structural Biol.

4, 10-19 (1997).

6.5 Quaternary Structure

What are the forces driving quaternary
association?
• 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!








What are the structural and functional

advantages driving quaternary association?
Know these!
• Stability: reduction of surface to volume
ratio
• Genetic economy and efficiency
• Bringing catalytic sites together
• Cooperativity

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http://biomodel.uah.es/tecnicas/carga/calculo_pI.

Copyright © 1999 by Harcourt Brace & Company

5.1 Proteins are Linear Polymers of Amino Acids

Copyright © 1999 by Harcourt Brace & Company

The Peptide Bond

Copyright © 1999 by Harcourt Brace & Company

Copyright © 1999 by Harcourt Brace & Company

The Coplanar Nature of the Peptide Bond

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Copyright © 1999 by Harcourt Brace & Company

Proteins - Large and Small

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The Sequence of Amino Acids

The sequence of ribonuclease A

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5.2 Architecture of Proteins

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What forces determine the

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How to view a protein?

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5.3 Biological Functions of

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5.4 Other Chemical Groups in

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5.7 Sequence Determination

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Insulin consists of two

The sequence shown is

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Determining the Sequence

• 1. If more than one polypeptide chain,

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Determining the Sequence

• 5. Cleave each chain into smaller

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Determining the Sequence

• 7. Reconstruct the sequence of the

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