Each chapter summary lists the main points that the reader should have understood from
analysis of each chapter.
Chapter 1.
1. Proteins are incredibly diverse occupying a wide range of biological functions.
2. Proteins are based around the elements of organic chemistry namely carbon,
hydrogen, oxygen and nitrogen.
3. Other elements of the periodic table are directly involved with protein structure
and/or function. This includes particularly metal ions of Group I and II and the
first row transition state elements.
4. Proteins often operate in conjunction with co-factors.
5. Proteins vary in size and complexity.
6. Defective proteins underpin many diseases and the wish to combat many of these
diseases represents a major reason for acquiring structural and functional
information.
Chapter 2.
1. Twenty different amino acids act as the building blocks of proteins.
2. In an isolated state the molecules are found as dipolar ions in solutions.
3. The charged properties result from the presence of amino and carboxyl groups
and lead to solubility in water, an ability to act as electrolytes, a crystalline
appearance and high melting points.
4. Of the twenty amino acids found in proteins 19 have a common structure based
around a central carbon, the C carbon, in which the amino, carboxyl, hydrogen
and R group are arranged tetrahedrally.
5. An exception to this arrangement of atoms is proline.
6. The C carbon is asymmetric with the exception of glycine and leads to at least
two stereoisomeric forms and leads to a chiral centre.
7. Amino acids form peptide bonds via a condensation reaction and the elimination
of water in a process that normally occurs on the ribosomes found in cells.
8. The formation of one peptide bond covalently links two amino acids forming a
dipeptide.
9. Polypeptides or proteins are built up by the repetitive formation of peptide bond.
An average sized protein may contain 1000 peptide bonds.
10. The peptide bond possesses hybrid characteristics with properties between that of
a C-N single bond and those of a C=N double bond. These properties result in
decreased peptide bond lengths compared to a C-N single bond, a lack of rotation
about the peptide bond and a preferred orientation of atoms in a trans
configuration for most peptide bonds.
11. One exception to this rule is the peptide bond preceding proline residues where
the cis configuration is increased in stability relative to the trans configuration.
12. The side chains dictate the chemical and physical properties of proteins. Side
chain properties include charge, hydrophobicity and polarity.
13. Many of these reactions are exploited in understanding the structure and function
of proteins.
Chapter 3.
1. Proteins fold into a precise structure that reflects biological role.
2. All proteins have three levels of organization called the primary, secondary and
tertiary structures.
3. Proteins with more than one polypeptide chain exhibit quaternary levels of
organization.
4. Primary structure is simply the linear order of amino acid residues along the
polypeptide chain from the N to C terminals.
5. Long polymers cannot fold into any shape because of restrictions placed on
conformational flexibility by the planar peptide bond and interactions between
non-bonded atoms.
6. Conformational flexibility along the polypeptide backbone is dictated by  and 
torsion angles.
7. Repetitive values for  and  lead to regular structures known as the  helix and
 strand. These are elements of secondary structure.
8. Secondary structure is defined as the spatial arrangement of residues that are close
together in the primary sequence.
9. The  helix is the most common element of secondary structure found in proteins.
10. The  helix is characterized by dimensions such as pitch (5.4Å), the translation
distance (1.5Å), and the number of residues per turn (3.6). It is stabilized by
hydrogen bonds orientated parallel to the helix axis and formed between the CO
and NH groups of residues separated by four intervening residues.
11. In contrast the  strand represents an extended structure (the pitch distance is
~6.8-7Å, the translation distance is 3.5Å) with fewer residues per turn (2).
12. Strands have the ability to hydrogen bond with other strands to form sheets.
13. Numerous variations on the basic helical and strand structures are found in
proteins.
14. The ideal or model conformation is rare in proteins.
15. Tertiary structure is formed by the organization of secondary structure into more
complex topology or folds by interaction between residues (side chain and
backbone) that are widely separated in the primary sequence.
16. Several identifiable folds or motifs exist within proteins.
17. These units are often seen as ‘sub-structures’ within a protein.
18. Examples include the four-helix bundle, the  barrel, the  helix, the HTH motif
and the  propeller.
19. Proteins are classified according to tertiary structure and this has led to the
description of proteins as all , + and /.
20. Proteins with similar tertiary structures leads to the concept of structural
homology and grouping together in related families.
21. Favourable interactions between residues maintain tertiary structure.
22. These interactions include covalent and non-covalent interactions.
23. A covalent bond formed between two thiol side chains results in a disulfide bridge
but more common stabilizing forces include charged interactions, hydrophobic
forces, van der Waals interactions and hydrogen bonding.
24. These interactions differ significantly in their strength and number.
25. Quaternary structure is a property shared by proteins with more than one
polypeptide chain.
26. DNA binding proteins function as dimers whilst haemoglobin is the classic
example of a tetrameric protein (2,2 subunits).
27. Proteins with more than one chain may exhibit allostery; a modulation of activity
by smaller effector molecules.
28. The oxygen binding properties of haemoglobin and myoglobin differ with the
former exhibiting allostery and shown by sigmoidal as opposed to hyberbolic
binding curves. This curve is described as cooperative and differs from that
shown by myoglobin.
29. Oxygen binding changes the structure of one subunit facilitating the transition
from deoxy to oxy in the remaining subunits. Historically the study of the
structure of hemoglobin provided a platform with which to study larger, more
complex, proteins structures together with their respective functions.
30. Immunoglobulins are proteins that form part of the body’s arsenal of defence
mechanisms in response to foreign macromolecules. Collectively they are called
antibodies.
31. All antibodies are based around a Y shaped molecule composed of 2 heavy and 2
light chains held together by covalent and non-covalent interactions.
32. Antigen binding sites are formed from the hypervariable regions at the end of the
heavy and light chains. These hypervariable regions allow the production of a vast
array of different antibodies within five major classes and allow the host to
combat many different potential antigens.
Chapter 4.
1. Fibrous proteins represent a contrast to the normal topology of globular domains.
2. Fibrous proteins lack true tertiary structure showing elongated structures and
interactions confined to those between local residues.
3. The amino acid composition of fibrous proteins differs considerably from
globular proteins but also varies widely within this group.
4. Variation in composition reflects the different roles peformed by each group of
fibrous proteins.
5. Three prominent groups of fibrous proteins are collagens, silk fibroin and
keratins- all occupy pivotal roles within cells.
6. Collagen is very abundant in vertebrates and invertebrates
7. A triple helix provides a platform for a wide range of structural roles in the
extracellular matrix delivering strength and rigidity to a wide range of tissues.
8. The triple helix is a repetitive structure containing the motif (Gly-Xaa-Yaa) in
high frequency with Xaa and Yaa often found as proline and lysine residues.
9. Repeating sequences of amino acids are a feature of many fibrous proteins and
help to establish the topology of each protein.
10. In collagen the presence of glycine at every third residue is critical because its
small side chain allows it to fit precisely into a region that forms from the close
contact of three polypeptide chains.
11. Although based around a helical design the helix differs considerably in
dimensions to the typical  helix.
12. The triple helix of collagen undergoes post-translational modification to increase
strength and rigidity.
13. Keratins make up hair and nails and contain polypeptide chains arranged in
helical conformations. The helices interact by supercoiling to form “coiled-coils”.
14. Specific non-polar interactions between residues in different helices confer
stability with the basis of this interaction being a heptad of repeating residues
along the primary sequence.
15. A heptad repeat possesses leucine or other residues with hydrophobic side chains
arranged periodically to favour inter-helix interactions.
16. In view of their widespread distribution in all animal cells mutations in fibrous
proteins such as keratins or collagens leads to serious medical conditions.
17. Many disease states arise from inherited disorders that lead to impaired structural
integrity in these groups of proteins.
Chapter 5.
1. The major theme of this section is to understand how membrane proteins fold
into structures allowing functional activity.
2. The determination of the structure of the bacterial photosynthetic reaction
centre was a key step in the pathway towards understanding membrane
proteins.
3. There has been a progressive increase in the number of ‘refined’ structures for
this class of proteins over the last 20 years.
4. However, compared with the large number of structures existing for globular
domains in the protein databank the number of well-defined (rmsd<5Å)
structures available for membrane proteins is much smaller.
5. Crystallization of membrane proteins requires controlled and ordered
association of subunits via the interaction of exposed polar groups on the
surfaces of proteins. The process is assisted by the introduction of amphiphiles
that balance conflicting solvent requirements of membrane proteins
6. The red blood cell membrane has proved a useful paradigm for probing
membrane structure and function.
7. Peripheral or extrinsic proteins are associated with the membrane but are
generally released by mild treatments. In contrast integral membrane proteins
remain firmly embedded within the hydrophobic bilayer and removal from
this environment frequently results in a loss of structure and function.
8. In the red blood cell membrane some integral proteins have the majority of
their mass in large extracellular or cytoplasmic domains. One example is
glycophorin.
9. From studies of bacteriorhodopsin and the bacterial reaction center the
majority of membrane proteins are based on folding primary sequences into
transmembrane helical structures.
10. The structure of bacteriorhodopsin revealed seven transmembrane helices ~
23 residues in length correlating with seven “blocks” in the primary sequence
composed predominantly of residues with non-polar side chains.
11. The helices have comparable geometry to those occurring in soluble proteins
exhibiting similar bond lengths and torsion angles.
12. Major differences with soluble proteins lie in the relative distribution of
hydrophobic amino acid residues.
13. Sequencing proteins assists in the definition of transmembrane domains via
identification of hydrophobic side chains. Proteins containing high
proportions of residues with non-polar side chains such as Leu, Val, Ile, Phe,
Met and Trp in blocks of ~ 20-30 residues are probably membrane proteins.
14. The seven transmembrane helix structure is a common architecture in
membrane proteins with G protein coupled receptors based on these motifs.
15. Membrane proteins such as toxins or pore proteins are based on  strands that
assemble into compact, barrel structures of great stability via inter-strand
hydrogen bonding.
16. Methods developed for crystallization of the bacterial reaction centre proved
applicable to other classes of membrane proteins including respiratory
complexes found in mitochondrial membranes and in aerobic bacteria as well
as members of the ATPase superfamily
Chapter 6.
1. The major theme of this chapter is molecular evolution and how evolution of
the basic twenty amino acids allowed formation of a variety of proteins
making up cells. Some of these proteins are related by homology or
evolutionary lineage. After reading this chapter an appreciation of the
following facts should be gained by the reader.
2. Despite the incredible diversity of living cells all organisms are made up of
the same twenty amino acid residues linked together to form proteins.
3. The origin of the amino acid alphabet occurred early in evolution before the
first true cells. All subsequent forms of life evolved using this basic alphabet.
4. Pre-biotic synthesis is the term applied to non-cellular based methods of
amino acid synthesis that existed over 3.6 x 109 years ago.
5. The famous experiment of Urey and Miller demonstrated that the formation of
organic molecules such as adenine, alanine and glycine - precursors of nucleic
acids and protein systems - was possible by simulating the conditions thought
to be present on early Earth.
6. A major development in molecular evolution was the origin of self-replicating
systems. This role is reserved today for DNA but the first replicating systems
were based on RNA a molecule now known to have catalytic function.
7. The fossil record shows primitive prokaryotic cells resembling the blue green
cyanobacteria evolved over 3.6 billion years ago. Evolution of these cells
through compartmentalisation, symbiosis and specialization yielded single cell
and metazoan eukaryotes.
8. Protein sequencing is performed using the Edman procedure. Labelling and
identification of the N terminal residue with phenylisothiocyanate in a cyclic
process can be repeated ~50-80 times before cumulative errors restrict the
accuracy of sequencing.
9. Nucleic acid sequencing is based on dideoxy chain termination procedures
and PCR methods. The result of efficient DNA sequencing methods is the
completion of genome sequencing projects and the prevalence of enormous
amounts of bio-information within databases.
10. Databases represent the ‘core’ of the new area of bioinformatics.
11. One of the first uses of sequence data was to establish homology between
proteins. Sequence homology arises from a link between proteins as a result of
evolution from a common ancestor.
12. Serine proteases show extensive sequence homology and this is accompanied
by structural homology. Chymotrysin, trypsin and elastase share homologous
sequences and structure.
13. Structural homology will also result when sequences show low levels of
sequence identity. The c type cytochromes from bacteria and mitochondria
exhibit remarkably similar folds achieved with low overall sequence identity.
14. The bioinformatics revolution allows analysis of protein sequences at many
different levels. Common applications include secondary structure prediction,
conserved motif recognition, identification of signal sequences and
transmembrane regions, determination of sequence homology, and structural
prediction ab initio.
15. In the future bioinformatics is likely to guide the directions pursued by
biochemical research by allowing the formation of new hypotheses to be
tested via experimental methods.
Chapter 7.
1. The major idea of this chapter is to understand enzyme catalysis and the
methods used in biological systems to control activity.
2. Enzymes, with the exception of ribozymes, are catalytic proteins.
3. Enzymes accelerate reactions by factors up to 1017 when compared with the
corresponding rate in the uncatalysed reaction.
4. All enzymes are grouped into one of six functional classes; each represents a
generic catalytic reaction. These classes include oxidoreductases, transferases,
hydrolases, lyases, isomerase and lyases.
5. Many enzymes require the presence of accessory cofactors to perform
effective catalysis. Co-factors can be tightly bound to the enzyme including
covalent linkage or more loosely associated with the enzyme. Metal ions as
well as organic components such as pyridoxal phosphate or nicotinamide
adenine dinucleotide (NAD) are co-factors. Many co-factors or co-enzymes
are derived from vitamins.
6. All elementary chemical reactions are described by a rate equation that
summarizes the progress with time. Reactions are generally described by first
or second order rate equations.
7. Enzymes bind substrate forming enzyme-substrate complexes that decompose
to yield product.
8. A plot of the velocity of an enzyme catalysed reaction as a function of
substrate concentration exhibits a hyperbolic profile rising steeply at low
substrate concentrations before reaching a plateau above which further
increases in substrate have little effect on overall rates.
9. The kinetics of enzyme activity are described via the Michaelis-Menton
equation that relates initial velocity to substrate concentration.
10. Analysis yields several important parameters; Vmax the maximal velocity that
occurs when all of the enzyme is found as ES complex, Km the substrate
concentration at which the reaction velocity is half maximal and kcat/Km a
second order rate constant that represents the catalytic efficiency of enzymes.
11. Enzymes catalyse reactions by decreasing the activation free energy (G‡)
associated with the transition state.
12. Enzymes use a wide range of catalytic mechanisms to convert substrate into
product. These mechanisms include acid-base catalysis, covalent catalysis,
metal ion catalysis, proximity and orientation effects together with preferential
binding of the transition state complex. The latter effect is responsible for the
greatest enhancement of catalytic activity in enzymes.
13. Catalytic mechanisms of numerous enzymes have been determined from a
combination of structural, chemical modification and kinetic analysis. Enzyme
mechanisms for lysozyme, the serine protease family including trypsin and
chymotrypsin, triose phosphate isomerase and tyrosinyl tRNA synthetase are
now understood at a molecular level.
14. Enzyme inhibition represents a vital mechanism to controlling catalytic
function. In vivo inhibitors bind tightly to enzymes causing a loss of activity
and regulation of activity.
15. A common form of enzyme inhibition involves the competition between
substrate and inhibitor for an active site. Such inhibition is classically
recognized from an increase in Km whilst Vmax remains unaltered.
16. Other forms of inhibition include uncompetitive inhibition where inhibitor
binds to the ES complex and mixed (non-competitive) inhibition were binding
to both E and ES occurs.
17. Different modes of inhibition are identified from Lineweaver-Burk or EadieHofstee plots.
18. Irreversible inhibition involves the inactivation of an enzyme by an inhibitor
through covalent modification frequently at the active site. Irreversible
inactivation is the basis of many forms of poisoning but is also used
beneficially in therapeutic interventions with, for example, the modification of
prostaglandin H2 synthase by aspirin alleviating inflammatory responses.
19. Allosteric enzymes are widely distributed throughout many different cell
types and play important roles as regulatory units within major metabolic
pathways such as glycolysis or biosynthetic reactions.
20. Allosteric enzymes contain at least two subunits.
21. Changes in quaternary structure arise as a result of ligand (effector) binding
leading to alterations in enzyme activity. Conformational changes frequently
involve rotation of subunits relative to one another and may produce large
overall movement.
22. Such movements are the basis for the transitions between high (R state) and
low (T state) affinity states of the enzyme with conformational changes
leading to large differences in substrate binding.
23. The ability to modify enzymes via allosteric effectors allows “fine-tuning” of
catalytic activity to match fluctuating or dynamic conditions.
24. Alternative mechanisms of modifying enzyme activity exist within all cells.
Most important are covalent modifications that result in phosphorylation or
remove parts of the protein, normally the N terminus region, that limit
functional activity.
25. Covalent modification is to secrete proteolytic enzymes as inactive precursors
with limited proteolysis revealing the fully active protein. Caspases are one
group of enzymes using this mechanism of modification and play a vital role
in apoptosis.
26. Apoptosis is the programmed destruction of cell and is vital to normal growth
and development.
Chapter 8.
1. Fifty years have passed since the discovery of the structure of DNA. This
event marking the beginning of molecular biology increased our
understanding of all events in the pathway from DNA to protein. This
includes structural and functional description of proteins involved in the cell
cycle, DNA replication, transcription, translation, post-translational events
and protein turnover. In many instances determining structure has uncovered
intricate details of their biological function.
2. A cell cycle is characterized by four distinct stages; a mitotic or M phase is
followed by a G1 (gap) phase representing most of the cell cycle, then a
period of intense synthetic activity called the S (synthesis) phase and finally a
short G2 phase as the cell prepares for mitosis.
3. Genetic studies of yeast identified mutants in which cell division events were
inhibited or slowed. Control of the cell cycle is mediated by protein kinases
known as Cdks
4. Activity of Cdks is dependent on cyclin binding with optimal activity
occurring for Cdk2 in a complex with cyclin A and phosphorylation of
Thr160.
5. The structure of this complex results in the critical movement of the T loop, a
flexible region of Cdk2, governing accessibility to the catalytic cleft and the
active site threonine.
6. Transcription is DNA directed synthesis of RNA catalyzed by RNA
polymerase. Transcription proceeds from a specific sequence (promoter) in a
5’-3’ direction until a second site known as the transcriptional terminator is
reached.
7. In eukaryotes three nuclear RNA polymerases exist with clearly defined
functions. RNA polymerase II is concerned with the synthesis of mRNA
encoding structural genes.
8. Sequences associated with transcriptional elements have been identified in
prokaryotes and eukaryotes. In eukaryotes the TATA box is located upstream
of the transcriptional start site and governs formation of a pre-transcriptional
initiation complex.
9. Specific TATA binding proteins have been identified and structural studies
reveal that basal transcription requires in addition to RNA polymerase the
formation of pre-initiation complex of TBP, TFIIB, TFIIE, TFIIF and TFIIH.
10. In eukaryotes transcription is followed by further mRNA processing that
involves addition of 5’ G caps and 3’ polyA tails that influence translation
rates and mRNA stability.
11. Non-coding regions of mRNA known as introns are removed creating a
coherent translation-effective mRNA. Introns are removed by splicing in
esterification reactions catalysed by the spliceosome.
12. The spliceosome contains snRNA complexed with specific proteins.
Processes initial mRNA transcripts involves cutting at specific pyrimidine rich
recognition sites splicing them together to create mRNA that is exported from
the nucleus for translation at the ribosome.
13. Translation converts mRNA into protein and occurs in ribonucleoprotein
components known as the ribosomes.
14. The ribosomes convert the genetic code, a series of three non-overlapping
bases known as the codon, into a series of amino acids covalently linked
together in a polypeptide chain.
15. All ribosomes are composed of two subunits -large and small subunits- based
predominantly on highly conserved rRNA molecules together with over 50
different proteins.
16. Biochemical studies identified two major sites known as the A and P sites
along with an E site in ribosome. The P site (peptidyl) contains a growing
polypeptide chain attached to tRNA whilst a second site the A site
(aminoacyl) contains charged tRNA species with a single amino acid that will
be added to the extending chain.
17. Protein synthesis is divided into initiation, elongation and termination. All
stages involve accessory proteins such as IF-1, IF-2 and IF-3 together with
elongation and release factors such as EF-1, EF-2 and EF-3 as well as RF-1,
RF-2 and RF-3.
18. Elongation is the most extensive aspect of protein synthesis and can again be
divided into three steps. These processes involving amino acyl tRNA binding
at the A site, peptidyl transferase activity linking the P site tRNA bound chain
to the new incoming amino acid followed by translocation of the ribosome to
the next codon.
19. Structures for 50 and 30S subunits revolutionized understanding of ribosome
function. The structure of the large subunit confirmed conclusively that the
peptidyl transferase reaction is catalysed entirely by RNA; the ribosome is a
ribozyme.
20. Initial protein translation products undergo post-translational modification that
vary dramatically in type from oxidation of thiol groups to the addition of new
covalent groups such as GPI anchors, oligosaccharides, myristic acid ‘tails’,
inorganic groups such as phosphate or sulfate and larger organic skeletons
such as heme.
21. The removal of peptide ‘leader’ sequences in the activation of zymogens is
another important post translational modification.
22. Many enzymes such as proteolytic digestive enzymes,caspases and
components of the blood clotting cascade are activated in this type of
pathway.
23. N terminal signal sequences share physicochemical properties and are
recognised by a signal recognition particle.
24. The SRP directs nascent chains to the ER membrane or cell membrane of
prokaryotes. Signal sequences from different proteins do not exhibit sequence
homology but have a basic N terminal region followed by a hydrophobic core
and a polar C terminal region proximal to the cleavage site.
25. SRP’s are found in the three major kingdoms (archae, eubacteria and
eukaryotes) with the mammalian SRP the most extensively characterized
system and shown to consist of rRNA and six distinct polypeptides.
26. The SRP directs polypeptide chains to the ER membrane and the translocon, a
membrane bound protein-conducting channel.
27. Other forms of intracellular protein sorting exist within eukaryotes.
28. Proteins destined for the mitochondria, chloroplast and nucleus all possess
signals within their polypeptide chains.
29. For the nucleus protein import requires the presence of a highly basic stretch
of residues arranged either as a single block or as a bipartite structure
anywhere within the primary sequence.
30. Nuclear localization signals (NLS) are recognized by specific proteins
(importins). Importins shuttle ‘cargo’ towards the nuclear pore complex.
31. Proteins are not immortal –they are degraded and turnover rates of proteins
vary from minutes to weeks. Turnover is controlled by a complex pathway
involving labelling of proteins marked for degradation with a highly
conserved protein called ubiquitin.
32. Ubiquitin is a signal for destruction by the proteasome.
33. The proteasome has multiple catalytic activities in a core unit based around 4
heptameric rings. The 20S proteasome from T. acidophilum has an 7777
assembly forming a central channel guarded by two ante-chambers located on
opposite sides.
34. The central chamber catalyses proteolysis based around the N terminal
threonine residue (Thr1) where the side chain acts as a nucleophile attacking
the carbonyl carbon of peptide bonds.
35. In prokaryotes the proteasome degrades proteins in ubiquitin independent
pathways. In eukaryotes the 20S proteasome catalyses ubiquitin-dependent
proteolysis in the presence of a cap or 19S assembly bound to the cylindrical
core at either end of the channel.
36. Further pathways of protein degradation exist in the lysosome where defects
are responsible for known metabolic disorders such as Tay-Sachs disease and
by caspases that promote programmed cell death known as apoptosis.
Chapter 9.
1. This chapter emphasizes the methods and principles underlying protein
purification. After reading this chapter you should be able to list, describe and
give examples of the different methods used today in biochemistry.
2. Purification is the isolation of a protein from a complex mixture. The aim of
purification strategies is the isolation of a single protein retaining most, if not
all, biological activity and the absence of contaminating proteins.
3. Purification methods are helped enormously by advances in cloning and
recombinant DNA technology. This allows proteins to be over-expressed in
foreign host cells.
4. Methods of purification rely on the biophysical properties of proteins with the
properties of mass, charge, hydrophobicity, and hydrodynamic radius being
frequently used as the basis of separation techniques.
5. Chromatographic methods form the most common group of preparative
techniques for use in protein purification.
6. Chromatography involves the a mobile, usually aqueous, phase containing
proteins that interacts with a stationary inert phase. The stationary phase is
usually an inert support (resin) containing functional groups that enhance
interactions with some proteins.
7. In ion exchange chromatography the supporting matrix contains negatively or
positively charged groups. Similar methods allow protein separation on the
basis of hydrodynamic radius (size exclusion), ligand binding (affinity), and
non-polar interactions (hydrophobic interaction and reverse phase).
8. Alongside preparative techniques are analytical methods that establish the
purity and mass of the product.
9. SDS-polyacrylamide gel electrophoresis involves the separation of
polypeptides under the influence of an electric field solely on the basis of
mass. This technique is widely used in ascertaining subunit molecular mass
as well as overall protein purity.
10. An extension of the basic SDS-PAGE technique is Western Blotting. This
method allows the identification of an antigenic polypeptide within a mixture
of size-separated components by its reaction with a specific antibody.
11. 2D electrophoresis allows the separation of proteins according to mass and
overall charge. It is proving possible to identify large numbers of different
proteins within proteomes of single celled organisms or individual cells.
12. Of all analytical methods mass spectrometry has expanded greatly in
importance as a result of technical advances permitting accurate identification
of the mass/charge ratio of molecular ions.
13. The most popular methods of analysis of proteins are MALDI-TOF and
electrospray spectrometry. Using modern instrumentation protein mass is
determinable to within 1 a.m.u. This precision supports the identity of a
purified protein or permits identification of mutant or altered forms of proteins
from respective changes in molecular mass.
14. In combination with 1 and 2 dimensional gel methods mass spectrometry is
proving immensely valuable in characterization of the proteome of individual
organisms.
15. The expansion of proteomics in the post-genomic revolution has placed
greater importance on preparative and analytical techniques.
16. When the methods described here are combined with the techniques such as
NMR spectroscopy, X-ray crystallography or cryo-EM it is possible to go
from gene identification to protein structure within a comparatively short
space of time.
Chapter 10.
1. After reading chapter 10 an impression of the methods used to determine
structure should be gained along with some knowledge of the physical
principles that underpin these experimental methods.
2. Using biophysical techniques it is possible to determine the structure of
proteins at incredible levels of resolution. These methods allow details of
catalytic or functional roles to be uncovered and are playing a major part in
the battle to design drugs that combat disease states.
3. By exploiting different regions of the electromagnetic spectrum and the
interaction of radiation with atoms considerable information can be acquired
on the structure and dynamics of proteins.
4. A plethora of techniques are available for studying proteins so that if one
technique is unsuitable there is almost certainly another method that can be
applied.
5. X-ray crystallography and multi-dimensional NMR spectroscopy yield the
most detailed pictures of protein structure at an atomic level.
6. X-ray crystallography relies on the diffraction of X-rays by electron dense
atoms constrained within a crystal. Some proteins fail to crystallize and this
represents one of the major limitations of the technique. Another limitation is
the ‘phase problem’.
7. The ‘phase problem’ is overcome through the use of isomorphous replacement
-a basic requirement is that addition of heavy metal atoms does not alter
protein structure. The structures derived for the proteasome, the ribosome and
viral capsids serve to emphasize the success of the technique.
8. Membrane proteins or proteins with extensive hydrophobic domains are
notoriously difficult to crystallize although several structures now exist within
the protein databank.
9. NMR spectroscopy measures nuclear spin reorientation in an applied
magnetic field most frequently for spin ½ nuclei. In proteins this involves
mainly the 1H but with isotopic labelling includes 15N and 13C nuclei.
10. A major hurdle in NMR spectroscopy is the assignment problem or
identifying which resonances belongs to a given residue. Sophisticated multidimensional heteronuclear NMR methods developed to facilitate this process
are based around the interaction of nuclei ‘through bonds’ and ‘through
space’.
11. Solution structure is defined from the use of a combination of torsion angle
and distance restraints. The former are obtained from J coupling experiments
whilst the r-6 dependence of the NOE is the basis of distance constraints.
12. NMR structures are usually presented as a family or ensemble of closely
related protein topologies. Although most structures of proteins determined by
NMR spectroscopy have masses below ~20kD the size limit is increasing
steadily with the introduction of new methods.
13. The vast majority of structures deposited in the protein databank (>95%) have
been determined using crystallography or NMR spectroscopy but slowly a
third technique of cryo electron microscopy is gaining prominence.
14. Cryo-EM methods have the advantage of requiring little sample preparation
and are particularly suitable to very large protein complexes. The technique
relies on electron diffraction by particles immersed in a frozen lattice. Single
particle analysis is the current ‘hot’ area and involves trapping
macromolecular complexes in random orientations within vitreous ice and
combining thousands of images to provide an enhanced picture of the system.
15. Optical techniques based around electronic transitions provided information
on the absorbance and fluorescence of chromophores found in proteins.
16. Tryptophan exhibits a significantly greater molar extinction coefficient when
compared to either tyrosine or phenylalanine.
17. Time resolved measurements allow changes in fluorescence or absorbance to
be followed over very short time scales sometimes in the sub nanosecond
range. This allows measurements of protein mobility such as the motion of
aromatic side chains.
18. Circular dichroism involves the measurement of differential absorption of
right- and left circularly polarized light as a function of wavelength. In
proteins the far UV region from 260-180nm is dominated by the CD signals
due to elements of secondary structure such as helix, turns and strands.
19. Proteins composed extensively of helical structure have very different CD
spectra to those containing proportionally more  strands. CD spectra allow
the secondary structure content of unknown proteins to be predicted with
reasonable accuracy.
20. Infrared spectroscopy of proteins has been used to measure changes in the
vibrational states associated with the amide bond. Amide bonds in helices,
turns and strands show characteristic transitions in a region known as the
amide I band between 1600 and 1700 cm-1. Experimental spectra are fitted as
combinations of helices and strands and can be used to estimate secondary
structure content in a comparable manner to CD spectroscopy.
Chapter 11.
1. Protein folding is central to effective biological activity and after reading chapter
11 you should be able to describe the folding of proteins in vitro and in vivo as
well as the parameters that influence rates of folding and overall protein stability.
2. The folding of individual polypeptide chains from less structured states to highly
organised topologies is vital to biological function.
3. All of the information directing protein folding resides within the primary
sequence.
4. Thermodynamically the folded state is the global energy minimum with the free
energy decreasing in the transition from unfolded to native protein.
5. Native proteins are marginally more stable than unfolded states with estimates of
conformational stability ranging from ~10-70 kJ mol-1.
6. Several factors influence stability in globular proteins: conformational entropy,
enthalpic contributions from non-covalent interactions and the hydrophobic
interaction.
7. Protein denaturation is the loss of ordered structure and occurs in response to
elevated temperature, extremes of pH or the addition of reagents such as urea or
guanidine hydrochloride.
8. Denaturation involves the disruption of interactions such as hydrophobic
interactions, salt bridges and hydrogen bonds that normally stabilize tertiary
structure.
9. Experimental measurements of protein stability include differential scanning
calorimetry, absorbance and fluorescence optical methods, NMR spectroscopy
and circular dichroism.
10. Kinetic methods allow protein folding to be followed with time. Small soluble
proteins (<100 residues) usually fold within 1s.
11. The observation of slower folding rates is normally associated with disulfide bond
formation or cis-trans proline isomerization. To overcome slow folding reactions
in vivo cells possess specific enzymes such as prolyl peptide isomerases and
protein disulfide isomerase.
12. Critical events in the formation of the native fold are the acquisition of ordered
stable secondary structure, the formation of hydrophobic cores, and the exclusion
of water from the protein interior.
13. For proteins such as lysozyme, barnase and chymotrypsin inhibitor 2 the reaction
pathway has been exquisitely defined to identify properties of intermediates and
transition states.
14. Chaperones are multimeric systems found in all cells that prevent incorrect
protein folding. They are universally based around a toroidal structure of seven,
eight or nine subunits that is capped at either end to form a central cavity that
binds unfolded polypeptide.
15. Binding occurs via hydrophobic surfaces with ATP driven conformational
changes directing the peptide towards the native state.
16. Kinetics associated with membrane protein folding in vitro are much slower than
those observe for globular domains but the principles governing formation of the
native state remain similar.
17. Incorrect folding leads to a loss of activity that is the basis of many disease states.
In some cases misfolding leads to incorrect protein trafficking within the cell mutant proteins of CFTR are one example.
18. Incorrect protein unfolding is also a key event in amyloidosis –the accumulation
of irreversibly aggregated protein within fibrils.
19. Amyloidogenic diseases include many neurodegenerative disorders such as
Alzheimer’s, TSEs and hereditary forms of systemic amyloidosis.
20. Fibrils have a common structure based on collections of  strands aligned in
parallel sheets that twist into helical conformations known as  helices.
21. Neurodegenerative disorders such as CJD, BSE and scrapie are linked via the
spongiform appearance of neuronal tissue and the accumulation of amyloid
deposits. The disease arises from changes in the prion protein generating
conformations with different secondary and tertiary structure.
22. The abnormal form promotes amyloidosis by inducing other prion proteins to
change conformation. These events occur spontaneously at very low frequency
leading to sporadic occurrences of disease but are facilitated by mutations within
the PNRP gene.
Chapter 12.
1. This chapter provides the link between proteins and molecular medicine –a
subject area of growing importance- through a series of example diseases such
as influenza, HIV, prion based diseases, emphysema, neurodegenerative
conditions like Alzheimer’s and cancer.
2. Molecular medicine represents a multi-disciplinary approach to understanding
human disease through the use of structural analysis, pharmacology, gene
technology and even gene therapy.
3. In 1955 sickle cell anemia was identified as arising from a single amino acid
change within hemoglobin. Many other diseases arise from similar genetic
mutation.
4. Unprecedented progress has been made in understanding the molecular basis
of many diseases including new, emerging, ones such as HIV and vCJD
alongside familiar conditions such as influenza.
5. Understanding at a molecular level offers the prospect of better disease
diagnosis and in some cases improved therapeutic intervention.
6. Viruses pose a persistent problem to human health and are responsible for
many diseases.
7. Vaccination using attenuated viruses or purified proteins effectively
eradicated infections such as smallpox and polio but threats from rapidly
mutating viruses such as influenza and the human immunodeficiency virus
continue.
8. HIV will continue to contribute to significant mortality despite structural
characterisation of all proteins encoded by the virus.
9. Despite considerable differences in organization HIV and influenza are
responsible for the biggest pandemics of the 20th century and will continue to
trouble us in the 21st century.
10. The unique ability of virus to mutate rapidly changes the primary sequence of
many antigens on the surfaces. These antigens are the normal targets for drug
action or the basis of antibody directed immune response. In each case
variation severely compromises our ability to fight infection.
11. Identification of the molecular basis of many neurodegenerative disorders
provides a structural platform with which to fight these diseases.
12. Alzheimer’s disease is a major problem in elderly populations.
13. The underlying problem is the production of amyloid protein from
endoproteolysis of membrane bound APP.
14. Cleavage by enzymes known as  and  secretases releases a small 40-42
residue A from a much larger protein and aggregates to form the amyloid
deposits characteristic of neuritic plaques found in the brains of patients.
15. Genetic analysis of Alzheimer’s disease reveals a small percentage of all
forms of the disease is inherited. This is attributed to either mutations in the
amyloid precursor protein or to mutations in two additional genes called
presenilin 1 and 2.
16. Slowly the structures of some of the enzymes contributing to the A peptide
insight are being determined and this insight into the properties of the active
site may allow the development of drugs that inhibit catalytic properties and
by implication limit amyloid formation.
17. The protein p53 has been called the “Guardian of the genome”.
18. Its major role is to prevent the perpetuation of damaged DNA by causing cell
cycle arrest or apoptosis.
19. The protein is a conformationally flexible molecule consisting of an N
terminal transcriptional activation domain, a central or core DNA binding
domain and a third C terminal domain that contains multiple functions
including the ability to promote tetramerization.
20. The core DNA binding domain is based on a  sheet scaffold that binds to
major and minor groove of the double helix through the presence of charged
side chains arranged precisely in conserved loops that link elements of 
strands.
21. Cancer and p53 are inextricably linked through the demonstration that many
tumours have mutations in p53.
22. Most, but not all, are located in the central DNA binding domain and
mutational hot spots involve residues involved in DNA binding.
23. The cell requires exquisite control pathways to modulate the activity of p53
and corruption of these pathways can often lead to the development of cancer.
24. 1-antitrypsin is a serpin that acts to limit the unregulated activity of
neutrophil elastase.
25. The protein inhibits elastase by forming a tightly bound complex. It is found
at high concentrations in the globulin fraction of serum where its abundance
helped identify individuals with deficiencies in serum 1-antitrypsin.
26. Deficiency arises from a gene mutation and is an inherited condition. This
may lead to emphysema as a result of damage cause to the alveolar lining of
the lungs by unregulated elastase activity.
27. Mutated forms of 1-antitrypsin are unable to inhibit elastase effectively and
also exhibit a tendency to polymerization – a process that causes the
irreversible association of serpins within the cell.