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Protein Structure
Student Edition 5/23/13 Version
Pharm. 304
Biochemistry
Fall 2014
Dr. Brad Chazotte
213 Maddox Hall
chazotte@campbell.edu
Web Site:
http://www.campbell.edu/faculty/chazotte
Original material only ©2004-14 B. Chazotte
Goals
•Understand the bases of & differences between primary, secondary, tertiary, &
quaternary protein structure.
•Be familiar with basic protein purification/sequencing methods & how they
depend on the physical & chemical properties of proteins.
•Understand the physical and chemical forces that determine secondary structure,
including the peptide bond. Learn the basic types of secondary structure: -helix,
-sheet, random coil & non-repetitive structures.
•Understand the physical & chemical forces that determine tertiary structure -
protein domains and motifs.
•Be acquainted with techniques like NMR & X-ray crystallography that help
determine protein structure.
•Understand the physical and chemical forces that determine quaternary structure –
protein folding, denaturation, renaturation, hydropathy plots.
•Remember how protein structure and structural changes reflect the influence of
thermodynamic concepts on structural stability.
Levels of Protein Structure
•Primary
•Secondary
•Tertiary
•Quaternary
Refer to the three-
dimensional shape
of folded
polypeptide chains
Protein Diversity
For a protein of n residues there are 20n possible sequences
For 40 residue protein 1.1 x 1052
For 100 residue protein 1.27 x 10130
Definitions
•Peptides – typically less than 40 residues
Dipeptide: 2 amino acids; Tripeptide: 3 amino acids
Oligopeptide: many amino acids
•Proteins – typically polypeptides with 40 or more residues
•Multisubunit proteins - proteins with several identical or
nonidentical subunits.
PRIMARY STRUCTURE
&
ANALYSIS
Primary Structure: the amino acid sequence of a
protein’s polypeptide chain or chains. Sometimes
referred to as the covalent structure.
Conjecture on the Limitations of
Protein Size
Minimum: 40 residues – near the limit for a polypeptide to
be able to fold into a discrete stable shape that permits it to
carry out its basic function.
Maximum: ~1000 residues – near the limit for the protein
synthetic machinery to produce a peptide with minimal errors
in the polypeptide, mRNA template, or gene DNA.
Logic of Amino Acid Sequences in
Proteins
The characteristics of a protein depend more on the
sequence of amino acids rather than its composition.
The presence of an amino acid with its characteristic
physical & chemical properties at a particular place in a
protein influences the protein’s properties. (review
Amino Acids lecture)
The 3-D shape of a protein is a consequence of the
intermolecular forces among its various residues. (review
Chemical Bonding lecture)
Voet, Voet & Pratt 2013 Chap. 5.1
Primary Structure of Bovine
Insulin
Voet, Voet & Pratt 2013 Figure 5.1
Studying Proteins by Isolating
Them
Protein Purification
Crude Extract – 1st step whether protein is from tissue or
microbe, break open the cell and release the proteins into
solution.
Fractionation – step where proteins are separated into
different fractions bases on some chemical or physical
property such as size or charge. May utilize protein
solubility i.e. (pH), salt concentration, temperature, etc.
Proteins Must be Stabilized after Isolation
Care must be taken to preserve protein structure and function after its
is removed from its natural environment were it was stable.
•pH To prevent denaturation (loss of structure) or function proteins are placed in
buffered solutions at or near their native pH.
•Temperature Protein purification is normally carried out at low temperature ~0º C.
While some proteins are thermally stable at high temperatures, others may be
affected by temperature a few degrees higher than the native environment.
•Degradative Enzymes During isolation various nucleases and proteases are
released from their places in the cell and can degrade nucleic acids or proteins unless
temperature, pH or inhibitory agents are added.
•Adsorption to Surfaces Solutions are handled to minimize foaming and are kept
concentrated as interfaces (air-water, glass, plastic) can cause denaturation.
•Storage To maintain protein stability. Cold (-70º C or -196º C liq N2), sometime
under N2(g) to remove oxygen and prevent slow oxidation. Some of the goals are to
minimize microbial growth and/or oxidation. Voet, Voet & Pratt 2013 p.96
Assay of Purified Proteins
To purify a protein it is necessary to measure how much you
have  need a specific assay.
•Easier for enzymes as they produce a product proportional to
the amount of enzyme present.
•Colored or fluorescent products are especially helpful
•Can also use a coupled enzyme reaction, i.e. 2nd enzyme
•Can use immunochemical assays.
ELISA
Enzyme-Linked Immunoabsorbent Assay
Voet, Voet & Pratt 2013 Figure 5.3
Some Separation Techniques
Charge Ion Exchange Chromatography
Electrophoresis
Polarity Hydrophobic Interaction
Chromatography
Size Gel Filtration Chromatography
SDS-Polyacrylamide Electrophoresis
Ultracentrifugation
Binding Specificity Affinity Chromatography
These separation techniques utilize differences in the physical and/or chemical
properties that arise from the differences amino acid composition.
Protein Fractionation by Salting
Out
Voet, Voet & Pratt 2013 Figure 5.5
Protein solubility
depends on:
•Concentration of
dissolved salts
•Solvent polarity
•pH
•Temperature
By careful manipulation of these properties it is possible to selectively
precipitate out certain proteins and leave the other soluble.
Protein Separation by Ion
Exchange Chromatography
Voet, Voet & Pratt 2013 Figure 5.6
Ion exchange chromatography
makes use of the fact that
opposite charges attract
Polyelectrolytes such as proteins
that have both negative and
positive charges will bind to
cation or anion exchangers
depending on the protein’s net
charge
The binding affinity (Strength of
binding) depends on the
presence of other ions that
compete with the protein for
binding sites on the immobile
phase and the pH which in terms
effects the protein’s net charge.
Anion exchanger: e.g., DEAE Matrix–CH2-CH2-NH(CH2CH3)2
+
Cation exchanger: e.g., CM Matrix-CH2COO-
Protein Separation by Gel
Filtration Chromatography
Voet, Voet & Pratt 2013 Figure 5.7
A bead can have different pore
sizes (holes) depending on the
extent of cross-linking in its
component polymer.
The larger proteins that are
excluded from the beads have
a shorter path and leave the
column sooner.
Protein Separation by Affinity
Chromatography
Voet, Voet & Pratt 2013 Figure 5.8
Utilize the ability of certain proteins (via
biochemical properties) that are able to
bind specific molecules non-covalently.
Bind a specific molecule called a ligand to
an inert matrix – immobile phase
Column conditions are then changed, e.g.
pH, ionic strength or high ligand
concentration, to permit the protein to elute
in a highly purified form.
SDS-PAGE of Supernatants &
Membrane Fraction from a Bacterium
Voet, Voet & Pratt 2006 Figure 5.9 & 2013 Fig 5.9 & 5.10
Gel electrophoresis - a
molecular sieving approach
SDS, sodium dodecyl sulfate,
when added to a protein solution
binds 1 molecule of SDS per two
amino acids or 1.4g per g protein.
Separation by Zonal
Centrifugation
Voet, Voet & Pratt 2013 Figure 5.12
Gradient preparation
Berg, Tymoczko, & Stryer 2012 Fig 3.16
Protein Sequencing
1. Sequence is a prerequisite for determining protein’s 3-D
structure and understanding its molecular mechanism.
2. Sequence comparisons among analogous proteins from
different species yield insights into protein function as well as
reveal evolutionary relationships among proteins
3. Many inherited diseases are caused by point mutations in the
amino acid sequence. Sequence analysis can assist with
diagnostic testing and therapy development.
Voet, Voet & Pratt 2006 Chapter 5
A protein must be broken down into fragments small enough to be
individually sequenced. The fragments are used to reconstruct the
protein by analyzing the fragment overlaps.
Sanger Method for Protein Sequencing
Voet, Voet & Pratt 2013 p.108 Box 5.1
Bind to terminal amino
groups to form a yellow
dinitrophenyl derivative
Hydrolyze protein
Identify terminal amino acid
chromatographically
Also today nucleic acids sequencing is frequently used to
determine protein sequences
Basic Logic of Protein Sequencing
Voet, Voet & Pratt 2013 Figure 5.13
1. polypeptide chains that are linked by
disulfide bonds are separated by
reduction of the sulfhydral groups of
cysteine.
2. chemical or enzymatic means are
used to cleave the resultant polypeptide
chains into smaller fragments.
3. Each small fragment is sequenced.
4. Compare overlapping sequences
produced by different enzymes or
chemical degradations to logically
reconstruct original protein sequence
5. Repeat process without cleaving
the disulfide bonds to determine
where those bonds are located
End Group Analysis
Used to determine the number of distinct polypeptide chains in a
protein (if end groups not chemically blocked).
•There are several procedures for the N-terminus.
•No reliable chemical procedure for C-terminus, an enzymatic
approach uses carboxypeptidases.
Protein Sequencing
End Group Analysis with Danzyl
Chloride
Voet, Voet & Pratt 2013 Figure 5.14
Conjugate the
fluorophore to primary
amine(s).
Perform an acid
hydrolysis
Identify via
chromatography the
labeled amino acid.
Disulfide Bond Cleavage
Need to cleave to separate polypeptide chains.
Two methods:
• oxidative cleavage with performic acid
disadvantage: destroys met and Trp indol side
chain
• reductive cleavage with mercaptan
e.g. 2-mercaptoethanol. Usually alkylate product
with iodoacetic acid to prevent disulfide bond
reformation.
Peptide Hydrolysis
Chemical approach
Acid hydrolysis
disadvantages – destroys Ser, Thr, Tyr & Trp
converts Asn & Gln to Asp & Glu, respectively
Base Hydrolysis
disadvantages – destroys Cys, Ser, Thr & Arg
Biochemical Approach
Enzymatic Hydrolysis
disadvantages – often incomplete some autodigestion
Amino Acid Analysis
Separation by HPLC
Voet, Voet & Pratt 2006 Figure 5.15
Complete
hydrolysis will
yield the
composition but
not the sequence
Molecular Mass Determination
by Mass Spectrometry
Voet, Voet, & Pratt 2013 Fig 17a,bBerg, Tymoczko, & Stryer 2012 Fig 3.34
Matrix-assisted Laser desorption/ionization –time of flight
MALDI-TOF
• Permits the ionization of proteins that formerly could not be
efficiently ionized due to their high MW and low volatility.
• Laser vaporizes solvent – some protein enter gas phase too.
• Protein ionizes and is separated on a mass/charge ratio.
Electrospray ionization mass spectrometry
• Peptide in solution sprayed from capillary tube at high
voltage to produce highly charged droplets
• Solvent soon evaporates to give peptide ions in gas phase
• – Yield +0.5 to +2.0 charge per kilodalton from, e.g., Arg
& Lys protonation
ESI mass spectrum of horse heart apomyolobin
• Measures mass/charge (m/z) ratio
• Electrospray ionization (ESI) does NOT destroy proteins as earlier
mass spec techniques did.
• Mass spectrum: series
of peaks of ions
differing by a single
charge and mass of 1
proton.
• Each peak corresponds
to an m/z ratio of an
(M + nH)n+ ion
• Can take two adjacent
peaks and solve two
linear equations to get
MW.
Tandem Mass Spectroscopy for
Peptide Sequencing
Berg, Tymoczko, & Stryer 2012 Fig 3.36
Tandem refers to two mass spectrometers in series
Ions of proteins, i.e. precursor ions, from the 1st
mass spec are broken into smaller peptide chains,
i.e. product ions, by bombardment with atoms of an
inert gas. These are in turn passed to a 2nd mass
analyzer.
• Product ions can be formed such that individual
amino acids are cleaved from the precursor ion such
that a family of ions can be produced
• Each ion represents the original peptide minus one
or more amino acids from the end.
• The mass difference between the peaks in the plot
represent the sequence of the amino acids.
“By comparing molecular masses of
successively larger members of a family of
fragments, the molecular masses and therefore
the identities of the corresponding amino acids
can be determined”
Voet, Voet & Pratt 2013, Fig. 5-18; & p. 113
Polypeptide Cleavage
Endopeptidases
Trypsin Rn-1 = pos chg res: Arg, Lys; Rn≠Pro (C-side)
Chymotrypsin Rn-1 = bulky hydroph res Phe, Trp, Tyr; Rn≠Pro (C-side)
Elastase Rn-1 = small neut. Res: Ala, Gly, Ser, Val; Rn≠Pro
Thermolysin Rn = Ile, Met, Phe, Try, Val Rn≠Pro (N-Side)
Pepsin Rn = Leu, Phe, Trp, Typ; Rn≠Pro (N-side)
Endopeptidase V8 Rn-1 = Glu
Cyanogen Bromide (CNbr) Rn = Met (C side)
Endopeptidases hydrolyze internal peptide bonds and are used to fragment
polypeptides but require certain adjacent side chains.
Voet, Voet, & Pratt 2013 Table 5.4
Edman Degradation of Proteins
Voet, Voet & Pratt 2013 Figure 5.16
Use repeated (sequential)
cycles of the Edman
degradation.
Trifluoroacetic acid cleavage
of the N-terminal amino acid
does NOT hydrolyze the
other peptides bonds.
Identify PTH-amino acid
by chromatographic
techniques.
Protein Sequence Determination
using Overlapping Fragments
Voet, Voet & Pratt 2013 Figure 5.119
Determining Disulfide Bond Location(s)
Voet, Voet & Pratt 2013 Figure 5.20
Sites to Find Sequence Data
Voet, Voet & Pratt 2013 Fig 5.21
Voet, Voet & Pratt 2013 Table 5.5
Cytochrome c Sequence Analyses
Voet, Voet & Pratt 2013 Table 5.6
Cytochrome c Phylogenetic Tree
Voet, Voet & Pratt 2013 Figure 5.22
Protein Evolution, Gene
Duplication & Protein Modules
Protein evolution rates
The rate at which mutations are incorporated into a protein are
dependent on the degree to which a change in an amino acid
effects a protein’s function
Gene duplication
Proteins with similar functions tend to have similar sequences.
New related function can arise by gene duplication. An
aberrant genetic recombination in which one chromosome
acquires both copies of a primordial gene.
Protein modules
New proteins (and functions) can also be generated by
incorporation of various 40-100 amino acid module or motifs.
Sample Protein Evolution Rates
Voet, Voet & Pratt 2013 Figure 5.24
Proteins mutate at different rates
over time.
But mutations in the DNA typically
occur at the same rate
Differences due to the rate at which
functionally or structurally
acceptable changes occurs. That is
those changes that are NONLETHAL
SECONDARY STRUCTURE
The local spatial arrangement of a polypeptide’s backbone
atoms without regard to the conformation of its side chains.
Levels of Protein Structure
Voet, Voet & Pratt 2013 Figure 6.1
The (trans) Peptide Bond -
Structure
Voet, Voet & Pratt 2013 Figure 6.2
In most cases in the protein
backbone the peptide bond is in the
trans configuration
Bond angles and lengths effect to
a large extent the freedom of
movement and the configuration
of the protein. (Important!)
Means -carbons of adjacent amino
acids are on opposite sides
So less steric hindrance of
adjacent amino acids side chains
Find ~ 8 kJ greater stability of
the trans vs the cis configuration
Resonance give rise to
40% double bond character
Polypeptide: Extended
Conformation & Torsion Angles
Voet, Voet & Pratt 2013 Figure 6.3; 6.4
“peptide group”
Definition:  and  = 180º when the
polypeptide chain is fully extended. They
increase clockwise when looking from Cα
Peptide Bonds: Steric Interference
Amide
hydrogen
Carbonyl
oxygen
Steric interferenceVoet, Voet & Pratt 2013 Figure 6.5
Ramachandran Diagram
(Allowed Bond Angles )
Sterically allowed
angles for all aa
except Gly & Pro
α-helix
α-helix (left
handed)
↑↑ -pleated sheet
Note: Gly is less
sterically hindered
van der Waals radii,
the attractive and
repulsive forces we
covered in earlier
lectures have a
significance for
protein structure.
Voet, Voet & Pratt 2013 Figure 6.6
Protein α-Helix Structure
Voet, Voet & Pratt 2013 Figure 6.7
Right-handed
helix
rightleft
Lehninger 2000 Box 6.1
5.4Å
H-bond every 4th residue
Amide H
Carbonyl O
The alpha helix structures
is one of the most stable
and is therefore one of the
most abundant biological
structures.
Helix core is tightly
packed such that the
atoms are at or near their
van der Waals radii.
The carbonyl oxygen on residue N is
hydrogen bonded to the amide
hydrogen on residue N+4 an optimum
bond length of 2.8Å. This is a source
of great thermodynamic stability.
-Helix Stability and Amino Acid Sequence
Interactions between amino acids can stabilize or destabilize the
helix.
•e.g. a long block of Glu residues will not form an -helix at pH 7.0 due to the
negatively charged carboxyl groups overpowering H-bonds
•Many adjacent Lys and/or Arg residues with pos. charges will repel each other at
pH 7.0
•The bulk & shape of Asn, Ser, Thr and Leu can also destabilize a helix if close
together in the backbone sequence
•The twist of the helix ensures that critical interactions occur between a side chain
(R-group) and another 3 or 4 residues away.
•Positively charged amino acids are often found three residues away from a
negatively charge amino acid – supports ion-pair formation
•Aromatic residues are often 3 residues apart to support hydrophobic interactions.
•Proline (N in rigid ring structure) causes a kink in -helix. Rarely found in helix
Constraints on -Helix Stability
(Summary)
1. Electrostatic repulsion or attraction between successive amino
acids with charged R groups.
2. Bulkiness of adjacent R groups.
3. Interactions between amino acid side chains spaced 3 (or 4)
residues apart.
4. The occurrence of Pro or Gly residues.
5. Interaction between amino acid residues at the ends of the helical
segment and the inherent electric dipole of the helix.
Electric Dipole
of the Peptide
Bond
&
Interactions
Between -
Helix Residues
Three Apart
Lehninger 2000 Figure 6.6
Arg103
side chain
Asp100
side
chain
Lehninger 2000 Figure 6.5
Troponin c protein segment
amino
carbonyl
The electric dipole of the peptide bond is transmitted along
an -helical segment via the intrachain hydrogen bonds
and this results in an overall helix dipole.
Protein -Sheet Structures
Pleated -Sheet
-Sheets: parallel vs Antiparallel
Voet, Voet & Pratt 2013 Figures 6.9, 6.10, 6.11
Space-filling
Antiparallel
-Sheet
-sheet makes full use
of the hydrogen
bonding capacity of the
polypeptide backbone
H-bonding occurs
between neighboring
polypeptide chains, i.e.
interchain, rather than
intrachain.
Historical Classification of Proteins
Globular polypeptide chains folded
in to spherical or globular shape.
These often contain several types of
secondary structure.
Typically most enzymes and
regulatory proteins.
Fibrous polypeptide chains
arranged in long chains or sheets.
Usually consist of a single type of
secondary structure.
Typically provide support, shape
and external protection to
vertebrates.
Alberts et al 2004 Fig 4.9
Fibrous Proteins
Fibrous proteins share properties that convey strength and/or
flexibility to structures in which they are part.
In each case the fundamental structural unit is a simple repeating
element of secondary structure.
All fibrous proteins are insoluble in water as a result of the high
concentration of hydrophobic residues on the protein surface and
interior.
The hydrophobic residues are largely buried via packing many
similar polypeptides chains together to form elaborate
supramolecular complexes.
-Keratin Structure: coiled coil
Voet, Voet & Pratt 2013 Figure 6.15
A “Permanent Wave”
Lehninger 2000 Box 6.2
Rich in Ala, Val, Leu, Ile, Met and Phe –
hydrophobic residues
A coiled coil - composed of two parallel -
helices that are twisted around each other to
form a left-handed supertwisted coiled coil.
Silk fibroin -Sheets in Side View
Voet, Voet & Pratt 2002 Figure 6.16
Typical repeat:
(Gly-Ser-Gly-Ala-Gly-Ala)n
Gly
Ala or Ser
•Has great strength
•Not very extensible
(would break
polypeptide chain
covalent bonds)
•Very flexible
(Neighboring sheets
associate with weak van
der Waals forces).
Collagen Triple Helix Structure
Voet, Voet & Pratt 2013 Figure 6.17
Composition:
~33% Gly
~15-30% Pro,
Hyp and Hyl
Repeating Sequence:
Gly-X-Y where:
X is often Pro, Y is often
Hyp. Hyl is sometimes at Y
Hyp = 4-hydroxyprolyl
Hyl = 5-hydroxylysyl
•most abundant
vertebrate protein
occurring
•fibers form the major
stress bearing
components of
connective tissues
•Three parallel, left-
handed helical
polypeptide chains with
three residues per turn
twisted together to form
a right-handed
superhelical structure.
Collagen’s Molecular Interactions
Voet, Voet & Pratt 2002 Figure 6.18
Space-filling model
H-bonding in collagen
triple helix
H-Bond
Voet, Voet & Pratt 2013 Figure 6.18a
Ball & stick model
Every third polypeptide residue passes through the very crowded
center of the superhelix, hence the repeated gly every third residue.
Lehninger 2000 Table 6.1
Secondary Structure & Properties
Table
Nonrepetitive Protein Structure
Native, folded proteins can have nonrepetitive
structures that are also ordered like helices or -
sheets but they are irregular and therefore more
difficult to give a clear, simple description
Globular proteins (majority of proteins in nature) can contain a
number of secondary structure types. They may have these irregular
structures in addition to coils and sheets.
The appearance of certain residues outside an α-helix or β-sheet may be
nonrandom.
Helix capping: Asn and Gln often flank the ends of an α-helix since their
side chains can fold back to H-bond with the 4 terminal residues of the
helix.
β-bulge: a distortion in a β-sheet where a polypeptide strand may
have an extra, non H-bonded residue which produces a structural
distortion.
 Loop almost always located on the protein surface. May be involved in recognition
processes.
Turn & Loop Structures in
Polypeptides
Reverse turn types  Loop in space-filling model
Voet, Voet & Pratt 2006 Figure 6.20
found in most proteins with 60 are more
residues and are composed on 6 to 16
residues.Voet, Voet & Pratt 2013 Figure 6.14
Lehninger 2005 Figure 4.8
Structure of -turns
oxygen
hydrogen
Type II always
Gly at 3
•Connecting elements that link successive runs of an alpha helix or a beta sheet.
•A 180° turn of four amino acids
•Most common type of turn
Relative Probability of an AA Being in
These Secondary Structures
Lehninger 2005 Figure 4.10(for illustrative, informational purposes only)
Take home
message:
chemical and
physical
characteristics
of an amino
acid (charge,
bond angles,
etc.) influence
its ability to
participate in
particular
secondary
structures
TERTIARY STRUCTURE
The three-dimensional structure of an entire polypeptide
including its side chains
Tertiary structure describes the folding of the protein’s
secondary structure elements and also specifies the position of
each atom in the protein.
Lehninger 2005 Figure 4.16
Myoglobin Tertiary Structure: View Types
ribbon mesh Surface
contour
Ribbon
w/ side
chains
Space-
filling
w/ side
chains
Myoglobin is composed of
eight relatively straight
alpha helices interrupted by
bends and some of these are
beta turns.
Protein 3-D structure & X-ray
crystallography
Voet, Voet & Pratt 2013 Figure 6.20b
X-ray diffraction pattern of
sperm whale myoglobin
crystal
3-D electron density of
human rhino virus
crystal
Protein crystal: flavodoxin
from Desulfovbrio vulgaris
Voet, Voet & Pratt 2013 Figure 6.21 Voet, Voet & Pratt 2002 Figure 6.23
Lehninger 2005 Box 4.4
3-D Protein Structure Determination
X-Ray Crystallography
•Generate a good protein
crystal (not easy).
•Detector “sees” a pattern of
spots called reflections from
X-ray beam. EACH atom
makes a contribution to
EACH spot.
•Massive calculations to
produce an electron density
map. Nuclei have greatest
density.
•Yields map of structure
Myoglobin, Globular Proteins, & Tertiary
Structure
•Positioning of amino acid side chains reflects a
structure that derives much of its stability from
hydrophobic interactions
•A dense hydrophobic core is typical of globular
proteins.
•In dense, closely packed environment weak
interactions, e.g. van der Waals, strengthen and reinforce
one another.
Lehninger 2005 Box 4.4 Fig 2
NMR in Protein Structure Determination
1-D
2-D
#1
Only certain atoms such as 1H, 13C, 15N,
19F, and 31P give rise to an NMR signal.
NMR used to identify nuclei and their
immediate chemical environment. Also
use NOE signals provide information
about the distance between atoms
Voet, Voet & Pratt 2013 Fig 6.25
NMR in Protein Structure Determination of
Full Structure from 2D Spectrum
Backbone
showing
possible
constraints
Part of reason for the
multiple structures
shown is that
proteins are
dynamic molecules
with molecular
vibrations occurring
in solution.
Src protein SH3 domain – 64 residue polypeptide
20 possible structures shown w/ backbone in
white
Protein Structural Motifs and Domains
In globular proteins the amino acid side chains are distributed
according to their polarities to achieve the most energetically
favorable conditions.
1. Val, Leu, Ile, Met & Phe occur mostly in the protein interior
away from aqueous solvent molecules.
2. Arg, His, Lys, Asp, & Glu are typically located at the proteins
surface where their charges can be solvated
3. Ser, Thr, Asn, Gln, & Tyr (uncharged polar) are found on the
protein surface but also in the protein’s interior where they are
almost always hydrogen bonded.
Side Chain Locations Seen in Space-filling Models
Voet, Voet & Pratt 2008 Figure 6.26
α-Helix -Sheet
Nonpolar
side chains
Polar side
chains
back
bone
-sheet
interior
this side
sperm whale myoglobin concanavalin A
Voet, Voet & Pratt 2013 Figure 6.27
The hydrophobic side chains are in
orange and are closer to the protein’s
interior and near the porphyrin ring.
Horse Heart Cytochrome c Structure
Hydrophillic
side chains
Hydrophobic side
chains
Fe atom
& heme
Hydrophilic sides chains are shown in
green and can be seen to be at the
protein’s surface.
Protein Motifs
(Supersecondary Structures)
There are grouping of certain secondary structural elements
that occur in many unrelated globular proteins.
•Most common motif is an -helix connecting two parallel strands
of a -sheet.  motif
•Antiparallel strands connected by relatively tight reverse turns
 hairpin
•Two successive antiparallel helices pack against each other with
their axis inclined  motif.
•Extended  sheet can role up to form three different types of
barrels.  barrel
Protein Structural Motifs
α   hairpin α α
 barrels
Voet, Voet & Pratt 2008 Figure 6.28
Different proteins combine these structures in various ways to achieve
their function. Certain successful and energetically favorable designs are
preserved in many diverse proteins.
The Rossman Fold
Nucleotide binding site
Voet, Voet & Pratt 2002 Figure 6.29
A prime example of a
structure function-
relationship
Binds dinucleotides such as
NAD+.
Utilizes  strands which
form a parallel sheet with
-helical connections
Two such  units are
shown.
Voet, Voet & Pratt 2013 Figure 6.31
Protein Domains
Polypeptide chains > ~200 residues usually fold into two or
more globular clusters.
• Typical domain 100 – 200 residues with an 25 Å avg.
diameter.
• Neighboring domains are usually connected by one or two
polypeptide segments. Many domains are structurally
independent units with characteristics of a small globular
protein
• Domains generally consist of two or more layers of secondary
structures – seals off a domain’s hydrophobic core from
aqueous environment.
• Domains often have a specific function, e.g. nucleotide
binding.
Domains in Evolution
1. Form stable folding patterns.
2. Tolerate amino acid deletions, substitutions, & insertions
which makes them better able to survive evolutionary
changes.
3. Support essential biological functions.
Common protein structures likely arose and persisted
because of their ability to:
Studies of proteins support the concept that essential structural
and functional elements of proteins rather than their amino
acid residues are conserved during evolution, e.g. changes in
like residues that do not appreciably change structure are not
dysfunctional.
QUATERNARY STRUCTURE
The spatial arrangement of the subunits of a multisubunit protein
•Subunits typically associate via noncovalent interactions.
•Contact regions between subunits resemble the interior of single
subunit proteins.
Definitions: Oligomer – proteins with more than one subunit
Protomer – repeating structural subunits of a protein
Design Benefits:
•Easier to synthesize multiple smaller subunits than one large
polypeptide chain free from error.
•A subunit with an error can more easily replaced than a single
large polypeptide – more efficient.
Quaternary Structure of
Hemoglobin
Voet, Voet & Pratt 2008 Figure 6.33
•An oligomeric
protein
•Each of the four
subunits 1212 is
shown in a different
color. (Heme is red)
Oligomeric Protein Symmetry
Examples
Voet, Voet & Pratt 2013 Figure 6.34
Proteins can
only have
rotational
symmetry
Related by
single axis of
rotation
When n-fold
rotation axis
intersects a 2-
fold rotation
axis at 90°
Other types of
symmetry
based on
geometrical
objects
Protein Folding & Stability
•Normally for biological structures, the molecules exist in
conformations that are at energy minimums, i.e. the most
thermodynamically stable.
•Hydrophobic effects, electrostatic interactions, and hydrogen
bonding, the noncovalent interactions, each provide energies of
thousands of kJ/mol over an entire protein.
•Thermodynamic studies of native proteins revealed that
native protein are only marginally stable under physiological
conditions as the free energy required to denature them is
approximately 0.4 kcal/mol per amino acid residue. For 100
residue protein ONLY 40kJ/mol more stable than unfolded
Conclude: a protein’s structure is in fact a delicate balance
of counteracting forces.
Stabilizing Forces
Hydrophobic effect - Nonpolar molecules seek
thermodynamically (entropically) to minimize their contact
with water.
This is the major determinant of native protein structure –
greatest contribution to stability
Hydropathy Plot
Voet, Voet & Pratt 2008 Figure 6.35
Protein
interior
Protein
exterior
Plot combines hydrophobic and hydrophilic
tendencies of individual amino acids.
The greater a side chain’s hydropathy the more
like it is to be in a protein’s interior.
Electrostatic Interactions
The association of two ionic protein groups of opposite
charge is called an ion pair or salt bridge.
Approximately 75% of charged residues are involved in
ion pairs and are mostly on the protein surface.
Chemical Cross-Linking
Disulfide Bonds
Thought that disulfide bonds are not so much a
stabilizing force, but they may function to lock in a
particular conformation of a polypeptide backbone.
Since the cell cytoplasm is a reducing environment
most intracellular proteins do not have disulfide
bridges.
Metal Ions
can also internally link proteins
Electrostatic Contributions to
Protein Stability
•Van der Waals interactions are an important stabilizing force in
the closely packed protein interior where they act over short
distances and are lost when the protein is unfolded.
•Hydrogen bonds make a minor contribution because those
groups can H-bond with water as a protein unfolds.
H-bonds do “select” the unique native structure of a protein
from a small number of hydrophobically stabilized
conformations.
•Ions pairs also make minor contributions because the free
energy of the pair does not compensate for the loss of side
chain entropy and the loss of the free energy of solvation.
Protein
Denaturation/Renaturation
Proteins can be denatured by a variety of conditions & substances.
Heating (adding energy) Most proteins exhibit a sharp transition
over a narrow temperature range indicative of the protein unfolding
in a cooperative manner.
pH variation Alters the ionization of amino acid side chains which
results in changes in charge distributions and H-bonding needs.
Detergents (amphipathic molecules) Associated with nonpolar
residues and disrupt the hydrophobic interactions
Chaotropic agents (urea, guanidinium chloride) Ions or small
organic molecules that at high 5- 10M concentration disrupt
hydrophobic interactions
Lehninger 2005 Figure 4.26
Protein Denaturation Curves
Protein Renaturation
Anfinsen classic experiment with RNAse A (4 disulfide bonds
must reform):
Proteins can spontaneously fold into their native
conformation under physiological conditions – protein’s
primary structure dictates its three dimensional structure.
Protein
Denaturation/Renaturation
Voet, Voet & Pratt 2008 Figure 6.39
Thermodynamics of Protein Folding
Lehninger 2000 Figure 6.27
Number of possible conformations
is large and therefore the
conformation energy is large.
At this point only a small
percentage of the intramolecular
interactions found in the native
state are present
Decreasing number of
possible states, i.e. the entropy
Amount of protein in native state
increasing
Free energy is decreasing as we
head to a free energy minimum
at the bottom, i.e., native state.
Energy
max
Energy
min
native
min
Protein
Misfolding:
Prions &
Disease
Lehninger 2000 Box 6.4
•Brain becomes
riddled with holes
•Caused by a
single 28 kd
protein called a
prion protein
•Normal Prp is
mostly alpha
helical.
•Illness occurs
when an altered
form of Prp called
PrpScr is present
which has mixed
alpha helix and
beta sheets
Prion: proteinaceous
infectious particle
Alzheimers and -Amyloid Protein
-Amyloid protein is normally present in the human brain but
its function is unknown
In Alzheimers a 40 residue (shorter) segment of this protein
forms fibrous deposits or plaques. This cleaved protein tends to
aggregate only with itself. This protein does not fold properly!
Amyloid plaques w/ Aβ
protein in brain tissue of
human Alzheimers patient
Native (N) <=> Unfolded (U)
Keq = [U]/[N] = e-∆G˚’/RT
As the ∆G˚’ for unfolding
decreases the portion unfolded
proteins increases.
Proteins that Help Native
Protein Folding
Molecular chaperones – proteins that bind to unfolded and
partially folded polypeptide chains to prevent improper
folding by prevent improper association of hydrophobic
regions that could lead to polypeptide aggregation,
precipitation, or non-native folding.
Important for multisubunit and multidomain
proteins.
First described as heat shock proteins (Hsp)
Most are ATPases
Voet, Voet & Pratt 2012 Figure 6.45
Proteins are Dynamic Structures
Myoglobin
Voet, Voet & Pratt 2013 Figure 6.39
•Remember that proteins are
in fact dynamic molecules
with normal structural
fluctuations
•These fluctuations are
important for function
particularly in enzymes.
•Snapshots are for the
structure of myoglobin seen
over 4 x 10-12 seconds
End of Lectures

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Protein structure

  • 1. Protein Structure Student Edition 5/23/13 Version Pharm. 304 Biochemistry Fall 2014 Dr. Brad Chazotte 213 Maddox Hall chazotte@campbell.edu Web Site: http://www.campbell.edu/faculty/chazotte Original material only ©2004-14 B. Chazotte
  • 2. Goals •Understand the bases of & differences between primary, secondary, tertiary, & quaternary protein structure. •Be familiar with basic protein purification/sequencing methods & how they depend on the physical & chemical properties of proteins. •Understand the physical and chemical forces that determine secondary structure, including the peptide bond. Learn the basic types of secondary structure: -helix, -sheet, random coil & non-repetitive structures. •Understand the physical & chemical forces that determine tertiary structure - protein domains and motifs. •Be acquainted with techniques like NMR & X-ray crystallography that help determine protein structure. •Understand the physical and chemical forces that determine quaternary structure – protein folding, denaturation, renaturation, hydropathy plots. •Remember how protein structure and structural changes reflect the influence of thermodynamic concepts on structural stability.
  • 3. Levels of Protein Structure •Primary •Secondary •Tertiary •Quaternary Refer to the three- dimensional shape of folded polypeptide chains
  • 4. Protein Diversity For a protein of n residues there are 20n possible sequences For 40 residue protein 1.1 x 1052 For 100 residue protein 1.27 x 10130 Definitions •Peptides – typically less than 40 residues Dipeptide: 2 amino acids; Tripeptide: 3 amino acids Oligopeptide: many amino acids •Proteins – typically polypeptides with 40 or more residues •Multisubunit proteins - proteins with several identical or nonidentical subunits.
  • 5. PRIMARY STRUCTURE & ANALYSIS Primary Structure: the amino acid sequence of a protein’s polypeptide chain or chains. Sometimes referred to as the covalent structure.
  • 6. Conjecture on the Limitations of Protein Size Minimum: 40 residues – near the limit for a polypeptide to be able to fold into a discrete stable shape that permits it to carry out its basic function. Maximum: ~1000 residues – near the limit for the protein synthetic machinery to produce a peptide with minimal errors in the polypeptide, mRNA template, or gene DNA.
  • 7. Logic of Amino Acid Sequences in Proteins The characteristics of a protein depend more on the sequence of amino acids rather than its composition. The presence of an amino acid with its characteristic physical & chemical properties at a particular place in a protein influences the protein’s properties. (review Amino Acids lecture) The 3-D shape of a protein is a consequence of the intermolecular forces among its various residues. (review Chemical Bonding lecture) Voet, Voet & Pratt 2013 Chap. 5.1
  • 8. Primary Structure of Bovine Insulin Voet, Voet & Pratt 2013 Figure 5.1
  • 9. Studying Proteins by Isolating Them
  • 10. Protein Purification Crude Extract – 1st step whether protein is from tissue or microbe, break open the cell and release the proteins into solution. Fractionation – step where proteins are separated into different fractions bases on some chemical or physical property such as size or charge. May utilize protein solubility i.e. (pH), salt concentration, temperature, etc.
  • 11. Proteins Must be Stabilized after Isolation Care must be taken to preserve protein structure and function after its is removed from its natural environment were it was stable. •pH To prevent denaturation (loss of structure) or function proteins are placed in buffered solutions at or near their native pH. •Temperature Protein purification is normally carried out at low temperature ~0º C. While some proteins are thermally stable at high temperatures, others may be affected by temperature a few degrees higher than the native environment. •Degradative Enzymes During isolation various nucleases and proteases are released from their places in the cell and can degrade nucleic acids or proteins unless temperature, pH or inhibitory agents are added. •Adsorption to Surfaces Solutions are handled to minimize foaming and are kept concentrated as interfaces (air-water, glass, plastic) can cause denaturation. •Storage To maintain protein stability. Cold (-70º C or -196º C liq N2), sometime under N2(g) to remove oxygen and prevent slow oxidation. Some of the goals are to minimize microbial growth and/or oxidation. Voet, Voet & Pratt 2013 p.96
  • 12. Assay of Purified Proteins To purify a protein it is necessary to measure how much you have  need a specific assay. •Easier for enzymes as they produce a product proportional to the amount of enzyme present. •Colored or fluorescent products are especially helpful •Can also use a coupled enzyme reaction, i.e. 2nd enzyme •Can use immunochemical assays.
  • 13. ELISA Enzyme-Linked Immunoabsorbent Assay Voet, Voet & Pratt 2013 Figure 5.3
  • 14. Some Separation Techniques Charge Ion Exchange Chromatography Electrophoresis Polarity Hydrophobic Interaction Chromatography Size Gel Filtration Chromatography SDS-Polyacrylamide Electrophoresis Ultracentrifugation Binding Specificity Affinity Chromatography These separation techniques utilize differences in the physical and/or chemical properties that arise from the differences amino acid composition.
  • 15. Protein Fractionation by Salting Out Voet, Voet & Pratt 2013 Figure 5.5 Protein solubility depends on: •Concentration of dissolved salts •Solvent polarity •pH •Temperature By careful manipulation of these properties it is possible to selectively precipitate out certain proteins and leave the other soluble.
  • 16. Protein Separation by Ion Exchange Chromatography Voet, Voet & Pratt 2013 Figure 5.6 Ion exchange chromatography makes use of the fact that opposite charges attract Polyelectrolytes such as proteins that have both negative and positive charges will bind to cation or anion exchangers depending on the protein’s net charge The binding affinity (Strength of binding) depends on the presence of other ions that compete with the protein for binding sites on the immobile phase and the pH which in terms effects the protein’s net charge. Anion exchanger: e.g., DEAE Matrix–CH2-CH2-NH(CH2CH3)2 + Cation exchanger: e.g., CM Matrix-CH2COO-
  • 17. Protein Separation by Gel Filtration Chromatography Voet, Voet & Pratt 2013 Figure 5.7 A bead can have different pore sizes (holes) depending on the extent of cross-linking in its component polymer. The larger proteins that are excluded from the beads have a shorter path and leave the column sooner.
  • 18. Protein Separation by Affinity Chromatography Voet, Voet & Pratt 2013 Figure 5.8 Utilize the ability of certain proteins (via biochemical properties) that are able to bind specific molecules non-covalently. Bind a specific molecule called a ligand to an inert matrix – immobile phase Column conditions are then changed, e.g. pH, ionic strength or high ligand concentration, to permit the protein to elute in a highly purified form.
  • 19. SDS-PAGE of Supernatants & Membrane Fraction from a Bacterium Voet, Voet & Pratt 2006 Figure 5.9 & 2013 Fig 5.9 & 5.10 Gel electrophoresis - a molecular sieving approach SDS, sodium dodecyl sulfate, when added to a protein solution binds 1 molecule of SDS per two amino acids or 1.4g per g protein.
  • 20. Separation by Zonal Centrifugation Voet, Voet & Pratt 2013 Figure 5.12 Gradient preparation Berg, Tymoczko, & Stryer 2012 Fig 3.16
  • 21. Protein Sequencing 1. Sequence is a prerequisite for determining protein’s 3-D structure and understanding its molecular mechanism. 2. Sequence comparisons among analogous proteins from different species yield insights into protein function as well as reveal evolutionary relationships among proteins 3. Many inherited diseases are caused by point mutations in the amino acid sequence. Sequence analysis can assist with diagnostic testing and therapy development. Voet, Voet & Pratt 2006 Chapter 5 A protein must be broken down into fragments small enough to be individually sequenced. The fragments are used to reconstruct the protein by analyzing the fragment overlaps.
  • 22. Sanger Method for Protein Sequencing Voet, Voet & Pratt 2013 p.108 Box 5.1 Bind to terminal amino groups to form a yellow dinitrophenyl derivative Hydrolyze protein Identify terminal amino acid chromatographically Also today nucleic acids sequencing is frequently used to determine protein sequences
  • 23. Basic Logic of Protein Sequencing Voet, Voet & Pratt 2013 Figure 5.13 1. polypeptide chains that are linked by disulfide bonds are separated by reduction of the sulfhydral groups of cysteine. 2. chemical or enzymatic means are used to cleave the resultant polypeptide chains into smaller fragments. 3. Each small fragment is sequenced. 4. Compare overlapping sequences produced by different enzymes or chemical degradations to logically reconstruct original protein sequence 5. Repeat process without cleaving the disulfide bonds to determine where those bonds are located
  • 24. End Group Analysis Used to determine the number of distinct polypeptide chains in a protein (if end groups not chemically blocked). •There are several procedures for the N-terminus. •No reliable chemical procedure for C-terminus, an enzymatic approach uses carboxypeptidases.
  • 25. Protein Sequencing End Group Analysis with Danzyl Chloride Voet, Voet & Pratt 2013 Figure 5.14 Conjugate the fluorophore to primary amine(s). Perform an acid hydrolysis Identify via chromatography the labeled amino acid.
  • 26. Disulfide Bond Cleavage Need to cleave to separate polypeptide chains. Two methods: • oxidative cleavage with performic acid disadvantage: destroys met and Trp indol side chain • reductive cleavage with mercaptan e.g. 2-mercaptoethanol. Usually alkylate product with iodoacetic acid to prevent disulfide bond reformation.
  • 27. Peptide Hydrolysis Chemical approach Acid hydrolysis disadvantages – destroys Ser, Thr, Tyr & Trp converts Asn & Gln to Asp & Glu, respectively Base Hydrolysis disadvantages – destroys Cys, Ser, Thr & Arg Biochemical Approach Enzymatic Hydrolysis disadvantages – often incomplete some autodigestion
  • 28. Amino Acid Analysis Separation by HPLC Voet, Voet & Pratt 2006 Figure 5.15 Complete hydrolysis will yield the composition but not the sequence
  • 29. Molecular Mass Determination by Mass Spectrometry Voet, Voet, & Pratt 2013 Fig 17a,bBerg, Tymoczko, & Stryer 2012 Fig 3.34 Matrix-assisted Laser desorption/ionization –time of flight MALDI-TOF • Permits the ionization of proteins that formerly could not be efficiently ionized due to their high MW and low volatility. • Laser vaporizes solvent – some protein enter gas phase too. • Protein ionizes and is separated on a mass/charge ratio. Electrospray ionization mass spectrometry • Peptide in solution sprayed from capillary tube at high voltage to produce highly charged droplets • Solvent soon evaporates to give peptide ions in gas phase • – Yield +0.5 to +2.0 charge per kilodalton from, e.g., Arg & Lys protonation ESI mass spectrum of horse heart apomyolobin • Measures mass/charge (m/z) ratio • Electrospray ionization (ESI) does NOT destroy proteins as earlier mass spec techniques did. • Mass spectrum: series of peaks of ions differing by a single charge and mass of 1 proton. • Each peak corresponds to an m/z ratio of an (M + nH)n+ ion • Can take two adjacent peaks and solve two linear equations to get MW.
  • 30. Tandem Mass Spectroscopy for Peptide Sequencing Berg, Tymoczko, & Stryer 2012 Fig 3.36 Tandem refers to two mass spectrometers in series Ions of proteins, i.e. precursor ions, from the 1st mass spec are broken into smaller peptide chains, i.e. product ions, by bombardment with atoms of an inert gas. These are in turn passed to a 2nd mass analyzer. • Product ions can be formed such that individual amino acids are cleaved from the precursor ion such that a family of ions can be produced • Each ion represents the original peptide minus one or more amino acids from the end. • The mass difference between the peaks in the plot represent the sequence of the amino acids. “By comparing molecular masses of successively larger members of a family of fragments, the molecular masses and therefore the identities of the corresponding amino acids can be determined” Voet, Voet & Pratt 2013, Fig. 5-18; & p. 113
  • 31. Polypeptide Cleavage Endopeptidases Trypsin Rn-1 = pos chg res: Arg, Lys; Rn≠Pro (C-side) Chymotrypsin Rn-1 = bulky hydroph res Phe, Trp, Tyr; Rn≠Pro (C-side) Elastase Rn-1 = small neut. Res: Ala, Gly, Ser, Val; Rn≠Pro Thermolysin Rn = Ile, Met, Phe, Try, Val Rn≠Pro (N-Side) Pepsin Rn = Leu, Phe, Trp, Typ; Rn≠Pro (N-side) Endopeptidase V8 Rn-1 = Glu Cyanogen Bromide (CNbr) Rn = Met (C side) Endopeptidases hydrolyze internal peptide bonds and are used to fragment polypeptides but require certain adjacent side chains. Voet, Voet, & Pratt 2013 Table 5.4
  • 32. Edman Degradation of Proteins Voet, Voet & Pratt 2013 Figure 5.16 Use repeated (sequential) cycles of the Edman degradation. Trifluoroacetic acid cleavage of the N-terminal amino acid does NOT hydrolyze the other peptides bonds. Identify PTH-amino acid by chromatographic techniques.
  • 33. Protein Sequence Determination using Overlapping Fragments Voet, Voet & Pratt 2013 Figure 5.119
  • 34. Determining Disulfide Bond Location(s) Voet, Voet & Pratt 2013 Figure 5.20
  • 35. Sites to Find Sequence Data Voet, Voet & Pratt 2013 Fig 5.21 Voet, Voet & Pratt 2013 Table 5.5
  • 36. Cytochrome c Sequence Analyses Voet, Voet & Pratt 2013 Table 5.6
  • 37. Cytochrome c Phylogenetic Tree Voet, Voet & Pratt 2013 Figure 5.22
  • 38. Protein Evolution, Gene Duplication & Protein Modules Protein evolution rates The rate at which mutations are incorporated into a protein are dependent on the degree to which a change in an amino acid effects a protein’s function Gene duplication Proteins with similar functions tend to have similar sequences. New related function can arise by gene duplication. An aberrant genetic recombination in which one chromosome acquires both copies of a primordial gene. Protein modules New proteins (and functions) can also be generated by incorporation of various 40-100 amino acid module or motifs.
  • 39. Sample Protein Evolution Rates Voet, Voet & Pratt 2013 Figure 5.24 Proteins mutate at different rates over time. But mutations in the DNA typically occur at the same rate Differences due to the rate at which functionally or structurally acceptable changes occurs. That is those changes that are NONLETHAL
  • 40. SECONDARY STRUCTURE The local spatial arrangement of a polypeptide’s backbone atoms without regard to the conformation of its side chains.
  • 41. Levels of Protein Structure Voet, Voet & Pratt 2013 Figure 6.1
  • 42. The (trans) Peptide Bond - Structure Voet, Voet & Pratt 2013 Figure 6.2 In most cases in the protein backbone the peptide bond is in the trans configuration Bond angles and lengths effect to a large extent the freedom of movement and the configuration of the protein. (Important!) Means -carbons of adjacent amino acids are on opposite sides So less steric hindrance of adjacent amino acids side chains Find ~ 8 kJ greater stability of the trans vs the cis configuration Resonance give rise to 40% double bond character
  • 43. Polypeptide: Extended Conformation & Torsion Angles Voet, Voet & Pratt 2013 Figure 6.3; 6.4 “peptide group” Definition:  and  = 180º when the polypeptide chain is fully extended. They increase clockwise when looking from Cα
  • 44. Peptide Bonds: Steric Interference Amide hydrogen Carbonyl oxygen Steric interferenceVoet, Voet & Pratt 2013 Figure 6.5
  • 45. Ramachandran Diagram (Allowed Bond Angles ) Sterically allowed angles for all aa except Gly & Pro α-helix α-helix (left handed) ↑↑ -pleated sheet Note: Gly is less sterically hindered van der Waals radii, the attractive and repulsive forces we covered in earlier lectures have a significance for protein structure. Voet, Voet & Pratt 2013 Figure 6.6
  • 46. Protein α-Helix Structure Voet, Voet & Pratt 2013 Figure 6.7 Right-handed helix rightleft Lehninger 2000 Box 6.1 5.4Å H-bond every 4th residue Amide H Carbonyl O The alpha helix structures is one of the most stable and is therefore one of the most abundant biological structures. Helix core is tightly packed such that the atoms are at or near their van der Waals radii. The carbonyl oxygen on residue N is hydrogen bonded to the amide hydrogen on residue N+4 an optimum bond length of 2.8Å. This is a source of great thermodynamic stability.
  • 47. -Helix Stability and Amino Acid Sequence Interactions between amino acids can stabilize or destabilize the helix. •e.g. a long block of Glu residues will not form an -helix at pH 7.0 due to the negatively charged carboxyl groups overpowering H-bonds •Many adjacent Lys and/or Arg residues with pos. charges will repel each other at pH 7.0 •The bulk & shape of Asn, Ser, Thr and Leu can also destabilize a helix if close together in the backbone sequence •The twist of the helix ensures that critical interactions occur between a side chain (R-group) and another 3 or 4 residues away. •Positively charged amino acids are often found three residues away from a negatively charge amino acid – supports ion-pair formation •Aromatic residues are often 3 residues apart to support hydrophobic interactions. •Proline (N in rigid ring structure) causes a kink in -helix. Rarely found in helix
  • 48. Constraints on -Helix Stability (Summary) 1. Electrostatic repulsion or attraction between successive amino acids with charged R groups. 2. Bulkiness of adjacent R groups. 3. Interactions between amino acid side chains spaced 3 (or 4) residues apart. 4. The occurrence of Pro or Gly residues. 5. Interaction between amino acid residues at the ends of the helical segment and the inherent electric dipole of the helix.
  • 49. Electric Dipole of the Peptide Bond & Interactions Between - Helix Residues Three Apart Lehninger 2000 Figure 6.6 Arg103 side chain Asp100 side chain Lehninger 2000 Figure 6.5 Troponin c protein segment amino carbonyl The electric dipole of the peptide bond is transmitted along an -helical segment via the intrachain hydrogen bonds and this results in an overall helix dipole.
  • 50. Protein -Sheet Structures Pleated -Sheet -Sheets: parallel vs Antiparallel Voet, Voet & Pratt 2013 Figures 6.9, 6.10, 6.11 Space-filling Antiparallel -Sheet -sheet makes full use of the hydrogen bonding capacity of the polypeptide backbone H-bonding occurs between neighboring polypeptide chains, i.e. interchain, rather than intrachain.
  • 51. Historical Classification of Proteins Globular polypeptide chains folded in to spherical or globular shape. These often contain several types of secondary structure. Typically most enzymes and regulatory proteins. Fibrous polypeptide chains arranged in long chains or sheets. Usually consist of a single type of secondary structure. Typically provide support, shape and external protection to vertebrates. Alberts et al 2004 Fig 4.9
  • 52. Fibrous Proteins Fibrous proteins share properties that convey strength and/or flexibility to structures in which they are part. In each case the fundamental structural unit is a simple repeating element of secondary structure. All fibrous proteins are insoluble in water as a result of the high concentration of hydrophobic residues on the protein surface and interior. The hydrophobic residues are largely buried via packing many similar polypeptides chains together to form elaborate supramolecular complexes.
  • 53. -Keratin Structure: coiled coil Voet, Voet & Pratt 2013 Figure 6.15 A “Permanent Wave” Lehninger 2000 Box 6.2 Rich in Ala, Val, Leu, Ile, Met and Phe – hydrophobic residues A coiled coil - composed of two parallel - helices that are twisted around each other to form a left-handed supertwisted coiled coil.
  • 54. Silk fibroin -Sheets in Side View Voet, Voet & Pratt 2002 Figure 6.16 Typical repeat: (Gly-Ser-Gly-Ala-Gly-Ala)n Gly Ala or Ser •Has great strength •Not very extensible (would break polypeptide chain covalent bonds) •Very flexible (Neighboring sheets associate with weak van der Waals forces).
  • 55. Collagen Triple Helix Structure Voet, Voet & Pratt 2013 Figure 6.17 Composition: ~33% Gly ~15-30% Pro, Hyp and Hyl Repeating Sequence: Gly-X-Y where: X is often Pro, Y is often Hyp. Hyl is sometimes at Y Hyp = 4-hydroxyprolyl Hyl = 5-hydroxylysyl •most abundant vertebrate protein occurring •fibers form the major stress bearing components of connective tissues •Three parallel, left- handed helical polypeptide chains with three residues per turn twisted together to form a right-handed superhelical structure.
  • 56. Collagen’s Molecular Interactions Voet, Voet & Pratt 2002 Figure 6.18 Space-filling model H-bonding in collagen triple helix H-Bond Voet, Voet & Pratt 2013 Figure 6.18a Ball & stick model Every third polypeptide residue passes through the very crowded center of the superhelix, hence the repeated gly every third residue.
  • 57. Lehninger 2000 Table 6.1 Secondary Structure & Properties Table
  • 58. Nonrepetitive Protein Structure Native, folded proteins can have nonrepetitive structures that are also ordered like helices or - sheets but they are irregular and therefore more difficult to give a clear, simple description Globular proteins (majority of proteins in nature) can contain a number of secondary structure types. They may have these irregular structures in addition to coils and sheets. The appearance of certain residues outside an α-helix or β-sheet may be nonrandom. Helix capping: Asn and Gln often flank the ends of an α-helix since their side chains can fold back to H-bond with the 4 terminal residues of the helix. β-bulge: a distortion in a β-sheet where a polypeptide strand may have an extra, non H-bonded residue which produces a structural distortion.
  • 59.  Loop almost always located on the protein surface. May be involved in recognition processes. Turn & Loop Structures in Polypeptides Reverse turn types  Loop in space-filling model Voet, Voet & Pratt 2006 Figure 6.20 found in most proteins with 60 are more residues and are composed on 6 to 16 residues.Voet, Voet & Pratt 2013 Figure 6.14
  • 60. Lehninger 2005 Figure 4.8 Structure of -turns oxygen hydrogen Type II always Gly at 3 •Connecting elements that link successive runs of an alpha helix or a beta sheet. •A 180° turn of four amino acids •Most common type of turn
  • 61. Relative Probability of an AA Being in These Secondary Structures Lehninger 2005 Figure 4.10(for illustrative, informational purposes only) Take home message: chemical and physical characteristics of an amino acid (charge, bond angles, etc.) influence its ability to participate in particular secondary structures
  • 62. TERTIARY STRUCTURE The three-dimensional structure of an entire polypeptide including its side chains Tertiary structure describes the folding of the protein’s secondary structure elements and also specifies the position of each atom in the protein.
  • 63. Lehninger 2005 Figure 4.16 Myoglobin Tertiary Structure: View Types ribbon mesh Surface contour Ribbon w/ side chains Space- filling w/ side chains Myoglobin is composed of eight relatively straight alpha helices interrupted by bends and some of these are beta turns.
  • 64. Protein 3-D structure & X-ray crystallography Voet, Voet & Pratt 2013 Figure 6.20b X-ray diffraction pattern of sperm whale myoglobin crystal 3-D electron density of human rhino virus crystal Protein crystal: flavodoxin from Desulfovbrio vulgaris Voet, Voet & Pratt 2013 Figure 6.21 Voet, Voet & Pratt 2002 Figure 6.23
  • 65. Lehninger 2005 Box 4.4 3-D Protein Structure Determination X-Ray Crystallography •Generate a good protein crystal (not easy). •Detector “sees” a pattern of spots called reflections from X-ray beam. EACH atom makes a contribution to EACH spot. •Massive calculations to produce an electron density map. Nuclei have greatest density. •Yields map of structure
  • 66. Myoglobin, Globular Proteins, & Tertiary Structure •Positioning of amino acid side chains reflects a structure that derives much of its stability from hydrophobic interactions •A dense hydrophobic core is typical of globular proteins. •In dense, closely packed environment weak interactions, e.g. van der Waals, strengthen and reinforce one another.
  • 67. Lehninger 2005 Box 4.4 Fig 2 NMR in Protein Structure Determination 1-D 2-D #1 Only certain atoms such as 1H, 13C, 15N, 19F, and 31P give rise to an NMR signal. NMR used to identify nuclei and their immediate chemical environment. Also use NOE signals provide information about the distance between atoms
  • 68. Voet, Voet & Pratt 2013 Fig 6.25 NMR in Protein Structure Determination of Full Structure from 2D Spectrum Backbone showing possible constraints Part of reason for the multiple structures shown is that proteins are dynamic molecules with molecular vibrations occurring in solution. Src protein SH3 domain – 64 residue polypeptide 20 possible structures shown w/ backbone in white
  • 69. Protein Structural Motifs and Domains In globular proteins the amino acid side chains are distributed according to their polarities to achieve the most energetically favorable conditions. 1. Val, Leu, Ile, Met & Phe occur mostly in the protein interior away from aqueous solvent molecules. 2. Arg, His, Lys, Asp, & Glu are typically located at the proteins surface where their charges can be solvated 3. Ser, Thr, Asn, Gln, & Tyr (uncharged polar) are found on the protein surface but also in the protein’s interior where they are almost always hydrogen bonded.
  • 70. Side Chain Locations Seen in Space-filling Models Voet, Voet & Pratt 2008 Figure 6.26 α-Helix -Sheet Nonpolar side chains Polar side chains back bone -sheet interior this side sperm whale myoglobin concanavalin A
  • 71. Voet, Voet & Pratt 2013 Figure 6.27 The hydrophobic side chains are in orange and are closer to the protein’s interior and near the porphyrin ring. Horse Heart Cytochrome c Structure Hydrophillic side chains Hydrophobic side chains Fe atom & heme Hydrophilic sides chains are shown in green and can be seen to be at the protein’s surface.
  • 72. Protein Motifs (Supersecondary Structures) There are grouping of certain secondary structural elements that occur in many unrelated globular proteins. •Most common motif is an -helix connecting two parallel strands of a -sheet.  motif •Antiparallel strands connected by relatively tight reverse turns  hairpin •Two successive antiparallel helices pack against each other with their axis inclined  motif. •Extended  sheet can role up to form three different types of barrels.  barrel
  • 73. Protein Structural Motifs α   hairpin α α  barrels Voet, Voet & Pratt 2008 Figure 6.28 Different proteins combine these structures in various ways to achieve their function. Certain successful and energetically favorable designs are preserved in many diverse proteins.
  • 74. The Rossman Fold Nucleotide binding site Voet, Voet & Pratt 2002 Figure 6.29 A prime example of a structure function- relationship Binds dinucleotides such as NAD+. Utilizes  strands which form a parallel sheet with -helical connections Two such  units are shown. Voet, Voet & Pratt 2013 Figure 6.31
  • 75. Protein Domains Polypeptide chains > ~200 residues usually fold into two or more globular clusters. • Typical domain 100 – 200 residues with an 25 Å avg. diameter. • Neighboring domains are usually connected by one or two polypeptide segments. Many domains are structurally independent units with characteristics of a small globular protein • Domains generally consist of two or more layers of secondary structures – seals off a domain’s hydrophobic core from aqueous environment. • Domains often have a specific function, e.g. nucleotide binding.
  • 76. Domains in Evolution 1. Form stable folding patterns. 2. Tolerate amino acid deletions, substitutions, & insertions which makes them better able to survive evolutionary changes. 3. Support essential biological functions. Common protein structures likely arose and persisted because of their ability to: Studies of proteins support the concept that essential structural and functional elements of proteins rather than their amino acid residues are conserved during evolution, e.g. changes in like residues that do not appreciably change structure are not dysfunctional.
  • 77. QUATERNARY STRUCTURE The spatial arrangement of the subunits of a multisubunit protein •Subunits typically associate via noncovalent interactions. •Contact regions between subunits resemble the interior of single subunit proteins. Definitions: Oligomer – proteins with more than one subunit Protomer – repeating structural subunits of a protein Design Benefits: •Easier to synthesize multiple smaller subunits than one large polypeptide chain free from error. •A subunit with an error can more easily replaced than a single large polypeptide – more efficient.
  • 78. Quaternary Structure of Hemoglobin Voet, Voet & Pratt 2008 Figure 6.33 •An oligomeric protein •Each of the four subunits 1212 is shown in a different color. (Heme is red)
  • 79. Oligomeric Protein Symmetry Examples Voet, Voet & Pratt 2013 Figure 6.34 Proteins can only have rotational symmetry Related by single axis of rotation When n-fold rotation axis intersects a 2- fold rotation axis at 90° Other types of symmetry based on geometrical objects
  • 80. Protein Folding & Stability •Normally for biological structures, the molecules exist in conformations that are at energy minimums, i.e. the most thermodynamically stable. •Hydrophobic effects, electrostatic interactions, and hydrogen bonding, the noncovalent interactions, each provide energies of thousands of kJ/mol over an entire protein. •Thermodynamic studies of native proteins revealed that native protein are only marginally stable under physiological conditions as the free energy required to denature them is approximately 0.4 kcal/mol per amino acid residue. For 100 residue protein ONLY 40kJ/mol more stable than unfolded Conclude: a protein’s structure is in fact a delicate balance of counteracting forces.
  • 81. Stabilizing Forces Hydrophobic effect - Nonpolar molecules seek thermodynamically (entropically) to minimize their contact with water. This is the major determinant of native protein structure – greatest contribution to stability
  • 82. Hydropathy Plot Voet, Voet & Pratt 2008 Figure 6.35 Protein interior Protein exterior Plot combines hydrophobic and hydrophilic tendencies of individual amino acids. The greater a side chain’s hydropathy the more like it is to be in a protein’s interior.
  • 83. Electrostatic Interactions The association of two ionic protein groups of opposite charge is called an ion pair or salt bridge. Approximately 75% of charged residues are involved in ion pairs and are mostly on the protein surface.
  • 84. Chemical Cross-Linking Disulfide Bonds Thought that disulfide bonds are not so much a stabilizing force, but they may function to lock in a particular conformation of a polypeptide backbone. Since the cell cytoplasm is a reducing environment most intracellular proteins do not have disulfide bridges. Metal Ions can also internally link proteins
  • 85. Electrostatic Contributions to Protein Stability •Van der Waals interactions are an important stabilizing force in the closely packed protein interior where they act over short distances and are lost when the protein is unfolded. •Hydrogen bonds make a minor contribution because those groups can H-bond with water as a protein unfolds. H-bonds do “select” the unique native structure of a protein from a small number of hydrophobically stabilized conformations. •Ions pairs also make minor contributions because the free energy of the pair does not compensate for the loss of side chain entropy and the loss of the free energy of solvation.
  • 86. Protein Denaturation/Renaturation Proteins can be denatured by a variety of conditions & substances. Heating (adding energy) Most proteins exhibit a sharp transition over a narrow temperature range indicative of the protein unfolding in a cooperative manner. pH variation Alters the ionization of amino acid side chains which results in changes in charge distributions and H-bonding needs. Detergents (amphipathic molecules) Associated with nonpolar residues and disrupt the hydrophobic interactions Chaotropic agents (urea, guanidinium chloride) Ions or small organic molecules that at high 5- 10M concentration disrupt hydrophobic interactions
  • 87. Lehninger 2005 Figure 4.26 Protein Denaturation Curves
  • 88. Protein Renaturation Anfinsen classic experiment with RNAse A (4 disulfide bonds must reform): Proteins can spontaneously fold into their native conformation under physiological conditions – protein’s primary structure dictates its three dimensional structure.
  • 90. Thermodynamics of Protein Folding Lehninger 2000 Figure 6.27 Number of possible conformations is large and therefore the conformation energy is large. At this point only a small percentage of the intramolecular interactions found in the native state are present Decreasing number of possible states, i.e. the entropy Amount of protein in native state increasing Free energy is decreasing as we head to a free energy minimum at the bottom, i.e., native state. Energy max Energy min native min
  • 91. Protein Misfolding: Prions & Disease Lehninger 2000 Box 6.4 •Brain becomes riddled with holes •Caused by a single 28 kd protein called a prion protein •Normal Prp is mostly alpha helical. •Illness occurs when an altered form of Prp called PrpScr is present which has mixed alpha helix and beta sheets Prion: proteinaceous infectious particle
  • 92. Alzheimers and -Amyloid Protein -Amyloid protein is normally present in the human brain but its function is unknown In Alzheimers a 40 residue (shorter) segment of this protein forms fibrous deposits or plaques. This cleaved protein tends to aggregate only with itself. This protein does not fold properly! Amyloid plaques w/ Aβ protein in brain tissue of human Alzheimers patient Native (N) <=> Unfolded (U) Keq = [U]/[N] = e-∆G˚’/RT As the ∆G˚’ for unfolding decreases the portion unfolded proteins increases.
  • 93. Proteins that Help Native Protein Folding Molecular chaperones – proteins that bind to unfolded and partially folded polypeptide chains to prevent improper folding by prevent improper association of hydrophobic regions that could lead to polypeptide aggregation, precipitation, or non-native folding. Important for multisubunit and multidomain proteins. First described as heat shock proteins (Hsp) Most are ATPases Voet, Voet & Pratt 2012 Figure 6.45
  • 94. Proteins are Dynamic Structures Myoglobin Voet, Voet & Pratt 2013 Figure 6.39 •Remember that proteins are in fact dynamic molecules with normal structural fluctuations •These fluctuations are important for function particularly in enzymes. •Snapshots are for the structure of myoglobin seen over 4 x 10-12 seconds