•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
Refer to the three-
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
•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
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
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
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
Crude Extract – 1st step whether protein is from tissue or
microbe, break open the cell and release the proteins into
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.
Some Separation Techniques
Charge Ion Exchange Chromatography
Polarity Hydrophobic Interaction
Size Gel Filtration Chromatography
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
Voet, Voet & Pratt 2013 Figure 5.5
By careful manipulation of these properties it is possible to selectively
precipitate out certain proteins and leave the other soluble.
Protein Separation by Ion
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
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
Voet, Voet & Pratt 2013 Figure 5.7
A bead can have different pore
sizes (holes) depending on the
extent of cross-linking in its
The larger proteins that are
excluded from the beads have
a shorter path and leave the
Protein Separation by Affinity
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.
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
Identify terminal amino acid
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
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.
End Group Analysis with Danzyl
Voet, Voet & Pratt 2013 Figure 5.14
fluorophore to primary
Perform an acid
labeled amino acid.
Disulfide Bond Cleavage
Need to cleave to separate polypeptide chains.
• oxidative cleavage with performic acid
disadvantage: destroys met and Trp indol side
• reductive cleavage with mercaptan
e.g. 2-mercaptoethanol. Usually alkylate product
with iodoacetic acid to prevent disulfide bond
disadvantages – destroys Ser, Thr, Tyr & Trp
converts Asn & Gln to Asp & Glu, respectively
disadvantages – destroys Cys, Ser, Thr & Arg
disadvantages – often incomplete some autodigestion
Amino Acid Analysis
Separation by HPLC
Voet, Voet & Pratt 2006 Figure 5.15
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
• 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
• 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
Tandem Mass Spectroscopy for
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
• 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
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
Trifluoroacetic acid cleavage
of the N-terminal amino acid
does NOT hydrolyze the
other peptides bonds.
Identify PTH-amino acid
Protein Sequence Determination
using Overlapping Fragments
Voet, Voet & Pratt 2013 Figure 5.119
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
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.
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
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
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 -
Voet, Voet & Pratt 2013 Figure 6.2
In most cases in the protein
backbone the peptide bond is in the
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
Conformation & Torsion Angles
Voet, Voet & Pratt 2013 Figure 6.3; 6.4
Definition: and = 180º when the
polypeptide chain is fully extended. They
increase clockwise when looking from Cα
(Allowed Bond Angles )
angles for all aa
except Gly & Pro
↑↑ -pleated sheet
Note: Gly is less
van der Waals radii,
the attractive and
repulsive forces we
covered in earlier
lectures have a
Voet, Voet & Pratt 2013 Figure 6.6
Protein α-Helix Structure
Voet, Voet & Pratt 2013 Figure 6.7
Lehninger 2000 Box 6.1
H-bond every 4th residue
The alpha helix structures
is one of the most stable
and is therefore one of the
most abundant biological
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
•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
•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
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)
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.
of the Peptide
Lehninger 2000 Figure 6.6
Lehninger 2000 Figure 6.5
Troponin c protein segment
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
-Sheets: parallel vs Antiparallel
Voet, Voet & Pratt 2013 Figures 6.9, 6.10, 6.11
-sheet makes full use
of the hydrogen
bonding capacity of the
polypeptide chains, i.e.
interchain, rather than
Historical Classification of Proteins
Globular polypeptide chains folded
in to spherical or globular shape.
These often contain several types of
Typically most enzymes and
Fibrous polypeptide chains
arranged in long chains or sheets.
Usually consist of a single type of
Typically provide support, shape
and external protection to
Alberts et al 2004 Fig 4.9
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
The hydrophobic residues are largely buried via packing many
similar polypeptides chains together to form elaborate
-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 –
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
Ala or Ser
•Has great strength
•Not very extensible
associate with weak van
der Waals forces).
Collagen Triple Helix Structure
Voet, Voet & Pratt 2013 Figure 6.17
Hyp and Hyl
X is often Pro, Y is often
Hyp. Hyl is sometimes at Y
Hyp = 4-hydroxyprolyl
Hyl = 5-hydroxylysyl
•fibers form the major
•Three parallel, left-
polypeptide chains with
three residues per turn
twisted together to form
Collagen’s Molecular Interactions
Voet, Voet & Pratt 2002 Figure 6.18
H-bonding in collagen
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.
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
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
β-bulge: a distortion in a β-sheet where a polypeptide strand may
have an extra, non H-bonded residue which produces a structural
Loop almost always located on the protein surface. May be involved in recognition
Turn & Loop Structures in
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
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)
of an amino
its ability to
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
Myoglobin is composed of
eight relatively straight
alpha helices interrupted by
bends and some of these are
Protein 3-D structure & X-ray
Voet, Voet & Pratt 2013 Figure 6.20b
X-ray diffraction pattern of
sperm whale myoglobin
3-D electron density of
human rhino virus
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
•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
•Massive calculations to
produce an electron density
map. Nuclei have greatest
•Yields map of structure
Myoglobin, Globular Proteins, & Tertiary
•Positioning of amino acid side chains reflects a
structure that derives much of its stability from
•A dense hydrophobic core is typical of globular
•In dense, closely packed environment weak
interactions, e.g. van der Waals, strengthen and reinforce
Lehninger 2005 Box 4.4 Fig 2
NMR in Protein Structure Determination
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
Part of reason for the
shown is that
Src protein SH3 domain – 64 residue polypeptide
20 possible structures shown w/ backbone in
Protein Structural Motifs and Domains
In globular proteins the amino acid side chains are distributed
according to their polarities to achieve the most energetically
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
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
Hydrophilic sides chains are shown in
green and can be seen to be at the
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
•Two successive antiparallel helices pack against each other with
their axis inclined motif.
•Extended sheet can role up to form three different types of
Protein Structural Motifs
α hairpin α α
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
Binds dinucleotides such as
Utilizes strands which
form a parallel sheet with
Two such units are
Voet, Voet & Pratt 2013 Figure 6.31
Polypeptide chains > ~200 residues usually fold into two or
more globular clusters.
• Typical domain 100 – 200 residues with an 25 Å avg.
• Neighboring domains are usually connected by one or two
polypeptide segments. Many domains are structurally
independent units with characteristics of a small globular
• Domains generally consist of two or more layers of secondary
structures – seals off a domain’s hydrophobic core from
• Domains often have a specific function, e.g. nucleotide
Domains in Evolution
1. Form stable folding patterns.
2. Tolerate amino acid deletions, substitutions, & insertions
which makes them better able to survive evolutionary
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
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
Definitions: Oligomer – proteins with more than one subunit
Protomer – repeating structural subunits of a protein
•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
Voet, Voet & Pratt 2008 Figure 6.33
•Each of the four
subunits 1212 is
shown in a different
color. (Heme is red)
Oligomeric Protein Symmetry
Voet, Voet & Pratt 2013 Figure 6.34
single axis of
intersects a 2-
axis at 90°
Other types of
Protein Folding & Stability
•Normally for biological structures, the molecules exist in
conformations that are at energy minimums, i.e. the most
•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.
Hydrophobic effect - Nonpolar molecules seek
thermodynamically (entropically) to minimize their contact
This is the major determinant of native protein structure –
greatest contribution to stability
Voet, Voet & Pratt 2008 Figure 6.35
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.
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.
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
can also internally link proteins
Electrostatic Contributions to
•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
•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.
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
Lehninger 2005 Figure 4.26
Protein Denaturation Curves
Anfinsen classic experiment with RNAse A (4 disulfide bonds
Proteins can spontaneously fold into their native
conformation under physiological conditions – protein’s
primary structure dictates its three dimensional structure.
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
Free energy is decreasing as we
head to a free energy minimum
at the bottom, i.e., native state.
Lehninger 2000 Box 6.4
riddled with holes
•Caused by a
single 28 kd
protein called a
•Normal Prp is
when an altered
form of Prp called
PrpScr is present
which has mixed
alpha helix and
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 that Help Native
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
First described as heat shock proteins (Hsp)
Most are ATPases
Voet, Voet & Pratt 2012 Figure 6.45
Proteins are Dynamic Structures
Voet, Voet & Pratt 2013 Figure 6.39
•Remember that proteins are
in fact dynamic molecules
with normal structural
•These fluctuations are
important for function
particularly in enzymes.
•Snapshots are for the
structure of myoglobin seen
over 4 x 10-12 seconds