Lecture/s Title: AMINO ACIDS & PEPTIDES

Subject: BIOCHEMISTRY Module # 1 Lecture # 4

Lecturer: MelitonBato,MD Date: August 16.2017
Transcribers: Angcianco D., Bayawa S., Dilag A.

(SCID) the Immunoglobulin (IgG).

OUTLINE o 6 month old male infant
I. Clinical Cases o recurrent fever, otitis media,
a. SCID
b. Progeria sorethroat, upper
c. Myasthenia Gravis respiratory infection.
d. Muscular Dystrophy o “Bubble Boy”
e. Prion Disease
o died due to Epstein-Barr
f. Acute Myocardial Infarction
II. Proteomics & Proteome virus
III. Amino Acids  Tx: Bone Marrow transplant
a. Properties Progeria o Lamin A defect.
b. Side Chains
c. Hydropathy o Involves nuclear membrane
d. pKa or cytoskeleton defect.
e. pI o Irreversible aging
f. Peptide Bond
IV. Protein associated with fascie of an
a. Properties & Functions old man.
b. Protein Translation o Fatal.
c. Post translational Modification
d. Protein Folding
Myasthenia Gravis  B-cells produces Abs
e. How does body destroy unwanted or damaged against Acetylcholine
proteins? receptors.
i. Ubiquitin-Proteasome Proteolytic
o Autoimmune disease
Pathway
ii. LysosomalDegradative Pathway o Ptosis with profound
muscle weakness in the
V. Protein Structure afternoon
VI. Protein Purification Muscular Dystrophy o Dystrophin defect (protein
a. Types of Chromatography
b. Isoelectric Focusing located between
c. Sanger Amino Acid Sequencing sarcolemma and outermost
d. Edman Amino Acid Sequencing layer of myofilaments
e. Hybrid Approach
f. Genomics Elucidates Proteins o Gower sign, hypertrophic
g. Mass Spectrometry calf muscle.
h. X ray Crystallography  Becker / Duchenne –
i. Nuclear Magnetic Resonance Microscopy
j. Molecular Modelling.
defects are located both on
the same gene but differs at
the exon site.
o Exon – part of DNA that are
REFERENCES (CITE ALL REFERENCES!!!) converted into mature
messenger RNA via
Legend: transcription.
Remember  Tx: Exon skipping
Lecturer Book
(Exams) Prion disease o Infectious proteins
  o Bovine Spongiform
encephalophaty,CJD,
scrapie, kuru
I. Clinical Cases o Associated with
Neurodegenerative
 Mutations
syndrome.
o Any changes in the base or sequences of the
Acute Myocardial o Luminal obstruction
genes or DNA.
Infarction (AMI) o Associated with
o Either due to mistakes when the DNA is copied
Diapheresis
or as the result of environmental factors.
o Lab exams: Troponin I,T
Severe Combined o Adenine deaminase
and CK-MB
Immunodeficiency enzyme defect / mutation in

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 AMINO ACIDS & PEPTIDES
 At physiologic pH (approximately 7.4), the
carboxyl group is dissociated, forming the
negatively charged carboxylate ion (COO–),
II. Proteomics & Proteome
and the amino group is protonated (NH3+).
 Proteomics
o Study of all proteins in the body which are  Most simple amino acid: Glycine (has a
mostly with unknown function but exists. symmetrical alpha-carbon)
o Study of proteome  The α-carbon is ASYMMETRIC –meaning to
Protein + Genome say that the central α-carbon atom is bounded
by 4 different groups of molecules or atoms
coined by Mark Wilkins in 1994.
that are molecularly oriented in a tetrahedral
o Analogy to Genomics.
configuration.
o Coined in 1997.
 19 out of the 20 amino acids exhibits chirality
o Interdisciplinary domain benefitting from
except GLYCINE because there are 2
the humangenome project
hydrogen atoms are covalently bonded to the
o Experimental analysis: protein purification
á-carbon atom.
& massspectrophotometry
 Amino acids have chirality
o Genomics, transcriptomics, metabolomics
o Chiral aa can exist as stereoisomer
o Aim to ID the entire complement of
 Compound with identical
proteins elaborated by a cell under diverse
molecular formula but different
conditions.
configuration or atomic
o Many proteins undergo posttranslational
arrangement.
modifications during maturation into
functionally competent forms as a means
 Stereoisomers are mirror image
that has identical molecular
of regulating their properties.
formulas or molecular weights
o Protein appearance or disappearance is
but their spatial configurations
associated with a specific physiologic
are different.
condition or disease.
o Determination of proteomes characteristic
 Most common stereoisomer: L-
amino acid.
of each cell type requires utmost efficiency
o Same as enantiomerism
in the isolation and ID of individual
proteins.  Enantiomers: 2 stereoisomers
that are nonsuperimposable
III. Amino Acid mirror images.
o Isoleucine and Threonine have 2
A. Properties chiral carbons
 Organic acid compound that contains both an o Racemase enzyme converts L and D-
amino (-NH) group and a carboxylic acid (- amino acid to each other.
COOH) functional group. B. Side Chains
 Exhibits amphoteric property, allowing it to  It is the nature of the side chains that
function aseither base or acid. Excellent for ultimately dictates the role an amino acid
buffering mechanism. plays in a protein. It is, therefore, useful to
 Basic structural and functional monomer classify the amino acids according to the
subunit ofproteins and polypeptides properties of their side chains: nonpolar
 300 known amino acids in nature. (have an even distribution of electrons) or
 Only 20 amino acids are commonly found as polar (have an uneven distribution of
constituents of mammalian proteins. electrons, such as acids and bases).
o 21st Amino acid: Selenocysteine
 Modification of aa Serine.
 Occurs when serine is bounded to
tRNA then translated into polypeptide
production.
 Each amino acid has a carboxyl group, a
primary amino group (except for proline, which
has a secondary amino group), and a
distinctive side chain (“R group”) bonded to the
á-carbon atom.

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 AMINO ACIDS & PEPTIDES
C. Hydropathy Index
 Relative hydrophilicity or hydrophobicity of
each aa.
 Important determinant in protein folding.
 High (+) values : highly hydrophobic
 High (-) values : hydrophilic
 Most hydrophilic: Arginine
 Most hydrophobic: Isoleucine
Molecular interactions between aa side chains
1. Hydrophobic Interactions
2. Hydrogen bonds
3. Electrostatic Interactions
Transcribed by: Angcianco D., Bayawa S., Dilag A.
D. pKa
 measurement of acid strength
 Amino acids are known weak acids
 pKa is affected by environment
 polar environment favors charged
forms
 non-polarenvironment favors
uncharged forms
 Net charge: algebraic sum of all the (+) and (-)
charged groups present depends on the pKa
values
 Altering the charge on the aa by varying the pH
fascilitates the physical separation
 Net charge of zero found in Zwitterions.
 Larger Ka : Stronger the Acid
(dissociation of HA into H+ and A-)
 Smaller Ka : weaker acid (less acid
dissociation)
 It determines the ideal amino acid form in a
certain environment with a particular pH.
E. pI
 pH midway between pKa values of an
Isoelectric species
o Isoelectric: molecules has an equal
number of (+) and (-) charges
o Neutral
o 0
 Zwitterions
 at pH 2.3 the carboxylic group is deproteinated
(gives off H+)
 beyond 9.1 the amine group is deproteinated.
 the sum of 2 pKa divided by 2 = the pH of
Isoelectric point (pI).
F. Peptide bond
 Amino acids are covalently bonded together
bymeans of peptide bond (amide linkage) that
formsmolecular polypeptide chains.
 A process called dehydration synthesis
willchemically combine one amino acid to
another aminoacid via reaction between the
carboxylic acid group ofthe preceding (first)
amino acid and the amino group of the
succeeding (second) amino acid.
 Thiscondensation reaction will lead to the
formation ofpeptide bond and eventually
release water molecule.
How do Amino acids bind together?
 Special with the peptide bond
1. Partial double bond character
2. Rigid and planar (they cannot move)
3. Trans Configuration
4. Uncharged but polar
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 AMINO ACIDS & PEPTIDES
IV. Proteins

A. Properties& Functions
 Characteristic
o Polypeptides
o One of the most important
biomolecule
o Physically and functionally complex
o Made from amino acid
 Functions
o Enzymes
o Motor proteins
o Receptor proteins
o Structural proteins
o Storage proteins
o Gene regulatory proteins
o Transport proteins
o Signal proteins
o Special purpose proteins

B. Protein Translation
 Process whereby biological cells generate
newproteins; it is balanced by the loss of
cellular proteins via degradation or export
 TRANSLATION  assembly of amino
acids byribosomes

a. Transcription
 mRNA chain is generated, with one strand
of theDNA double helix in the genome as
a template

b. Translation E. How does body destroy unwanted / damaged

 synthesis of proteins from RNA
In eukaryotes, translation occurs in the
cytoplasm,where the ribosomes are
located
 In activation, the correct amino acid (AA)
is joined tothe correct transfer RNA
(tRNA). While this is not, inthe technical
sense, a step in translation, it is
requiredfor translation to proceed.
C. Post Translational Modification
 Covalent and generally enzymatic
modification of proteinsduring or after
protein biosynthesis
 Occurred on the amino acid side chains or
at the protein's CorN- termini.
D. Protein Folding
 process by which a protein structure
assumes its functionalshape or
conformation
 physical process by which a protein chain
acquires its native3-dimensional structure,
a conformation that is usuallybiologically
functional, in an expeditious and
reproduciblemanner
proteins?
i. Ubiquitin-Proteasome Proteolytic Pathway
 ATP-dependent
 Cytosol
 Uses “ubiquitin” (evolutionarily
conserved 76-amino acid proteinthat
is central to multiple cellular function)
 Endogenous peptides
 Ub molecule contains seven lysine
residues, andfunctional selectivity is
provided by diverse patterns
ofprotein linkage to these amino
acids
 Linkage to some lysines leads to
passage of the taggedprotein to the
proteasome for degradation
 fate of Ub-conjugated proteins
among the severalpathways is
determined by the number of Ub
moietiesconjugated and the site of
the conjugation linkages onthe Ub
molecule
 AUb-activating enzyme, E1, binds to
Ub and then transfers itto one of
dozens of Ub-conjugating enzymes
(E2).
 These act together with one of about
800 different Ubligatingenzymes (E3)
to add Ub to a lysine on the doomed
protein

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 AMINO ACIDS & PEPTIDES
 Additional Ub moieties are added to
the original Ub, forming
apolyubiquitin chain (atleast four
Ubs)
 Proteasomes are highly conserved
organelles in thecytoplasm and
possibly the nucleus:
o barrel-shaped complexes whose
main (but not only)function is to
digest polyubiquitinated proteins
o two types of proteasomes: 20S
and 26S
o The degradative unit of both is a
20S destructionchamber, to
which, in the 26S proteasome,
two 19S“caps” are attached
o The capsat the entrance to the
proteolytic coreregulate entryThe
20S proteasomeslack these caps
o The 20S proteasomes are
important in degradation
ofoxidized proteins
o In 26S proteasomes,
polyubiquitinated proteins
aredegraded.
 Mutations that interfere with normal
proteasomal function are lethal
ii. LysosomalDegradative Pathway
 ATP dependent
 Lysosomes
 Exogenous peptides
Transcribed by: Angcianco D., Bayawa S., Dilag A.
V. Protein Structure
 Four Orders of Protein Structure
1. Primary – Amino Acid sequence
2. Secondary – alpha helix & beta pleated sheets
3. Tertiary – 3D conformation (with domain)
4. Quaternary – Spatial arrangements
Primary structure
 Defined linear sequence of amino acids in
a one dimensionalplane.
 Amino Acid Sequence (Gly-X-Y)
o X-Proline
o Y-HydroxyProline
Secondary structures
 Primary polypeptide structure folds 3 to 30-
amino acid residues forming geometrically
ordered units via intramolecular hydrogen
bonding.
 The way in which the primary structure of a
polypeptide chain folds (Left Handed Manner)
 Free rotation possible on a-carbon (Ca) to
carbonyl carbon (Co) and Ca to N.
 Partial double bond character of Co to a-N
requires carbonyl carbon, carbonyl oxygen and
a-nitrogen remain coplanar = prevents rotation.
 Phi : angle about the Ca – N
 Psi: angle about Ca-Co
 Proline is retricted due to absence of Ca – N
 Regions of ordered secondary structures arise
when aa residue adopt similar Phi and Psi
angles.
 3 types;
 Alpha- helix
o Polypeptide backbone twisted by an
equal aount of Phi at -57 degrees and
Psi at -47 degrees.
o 1 complete turn = 3.6 aa residues
o Pitch per turn = 0.54nm
o R group face downward
o Right handed a-helix are more stable
due yo L-amino acids.
o Represented as Cylinders
o Stability due to H bonds between
oxygen of the peptide bond carbonyl
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 AMINO ACIDS & PEPTIDES
and H atom of the peptide bond
nitrogen
o Maximum number of H bonds coupled
with Van der Waals forces provides
the thermodynamic driving force.
o Proline lacks a hydrogen atom and
could only be stable accommodated
within the first turn.
o Glycine induces a bend
o Amphipathic – compounds with both
hydrophilic and hydrophobic
properties, these amphipathic helices
form interfaces between polar and
non-polar regions creating channel
pores.
 Beta- sheet
o aa residues form a zigzag or
pleated pattern
o R-groups of adjacent residues
point on opposite directions
o Peptide backbone highly
extended
o Stability formed from H bond
between carbonyl oxygen and
amide hydrogen
o Parallel: adjacent segments
proceed in the same amino to
carboxyl
 Parallel has extended loops
o Antiparallel: opposite directions
 Anti-parallel has unextended
loops
 Arrow head represents the
carboxylic group.
 Arrow tail represents the amino
group.
o Many globular proteins.
Transcribed by: Angcianco D., Bayawa S., Dilag A.
 Loops and Bends
o Short aa segments joining 2 units
of secondary structures
o B turn: 4 aa residues, in which
the first residue is H-bonded to
the 4th resulting in a 180 degree
turn (Proline and Glycine)
o Loops are regions that contain
residues beyond the minimum
number necessary to connect
adjacent regions of secondary
stuructures.
Irregular conformation
Serve key biologic
functions
Can serve as the active site
of the enzyme
Lack apparent structural
regularity
Stabilized via H bonding, salt
bridges and hydrophobic forces
o Helix-loop-helix motif provides
aligonucleotide binding portion of DNA-
binding proteins such as repressors and
transcription factors.
o Supersecondary structures: intermediate
between secondary and tertiary structures
o Epitope is the accessible site for Abs
recognition and binding
 Loops and bends reside on
protein surfaces.
o Not all protein portion are ordered,
Disordered regions often at extreme
amino or carboxyl terminal.
 High conformational flexibility
 Assume an ordered
conformation upon ligand
binding
 Enables regions to act as ligand
controlled switches.
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 AMINO ACIDS & PEPTIDES
Tertiary structure
 Refers to the entire 3-D conformation of the
polypeptide
 3-D Spatial behavior: helices, sheets, bends,
turns and loops
 Domain: protein structure sufficient to perform
a particular chemical or physical task such as
substrate or ligand binding
o Anchor protein to a membrane
o Interact with a regulatory molecule
modulating its function
o Single domains: triose phosphate
isomerase, myoglobin
 Two domains : Protein kinase
o Catalyze the transfer of a phosphoryl VI. Protein Purification
group from ATP to a peptide or
protein Chromatography
o Amino terminus is rich in B-sheets
and binds ATP  Discovered by Mikhail Tsvetin 1900
o Carboxyl terminus is rich in a a-
helices and binds peptide/protein  Based on thin-layer chromatography of
substrate separating plant extracts
o Group that catalyze phosphoryl
transfer reside in a loop positioned at  Depends on the relative affinity of different
the interface of the 2 domains proteins for a given stationary phase and for
Quaternary structure the mobile phase
 Defines the polypeptide composition of a
 Association between each protein and the
protein and the spatial relationships between
matrix is weak and transient
subunits or protomers
 Proteins are assembled from more than one
 Proteins interacting more strongly with the
polypeptide stationary phase are retained longer
 Monomer: single polypeptide chain
 Dimer: two polypeptide chains  Length of time that a protein is associated with
 Homodimer: 2 copies of the same polypeptide the stationary phase is a function of the
 Heterodimer: 2 copies of different polypepide composition of both stationary and mobile
 Protomers are polypeptide chains with phases
domains that are similar in functions.
 Multimers are polypeptide chains with domains  Optimal separation achieved by manipulation
of the composition of the 2 phases
that are different in functions and amino acid
sequences.
A. Types of Chromatography
 Greek letters are used to describe the subunits
of a heterooligomeric protein
 Techniques by chromatographic bed shape
 Ribbon diagrams trace conformation o Column
 Cylinders are helices while arrow are sheets o Planar
 Line for the polypeptide backbone  Techniques using separation mechanism
o Size exclusion
o Ion exchange
o Hydrophobic interaction
o Affinity
 Techniques based on mobile phase type
o High-pressure liquid
o GasTypes of Chromatography

a. Column Chromatography

 Stationary phase: column containing

small spherical beads of modified

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 AMINO ACIDS & PEPTIDES
cellulose, acrylamide, or silica whose
surface has been coated with
chemical functional groups
 The matrix interacts with proteins
based on 1) charge 2) hydrophobicity
3) ligand-binding properties
 A protein mixture is applied to the
column
 Liquid mobile phase is percolated
through it
 Small portions of the mobile phase or
e.
eluent are collected
b. Planar Chromatography
 Separation of mixtures in a filter paper
based on partition of a solute between
2 solvents one of which is
immobilized by the substance paper
c. Size exclusion Chromatography
 Also known as gel filtration or gel
permeation
 Separates proteins based on their
Stokes radius (sphere diameter)
o Stoke radius is a function of
molecular mass and shape
o A tumbling elongated protein
occupies a larger volume
than a spherical protein of
the same mass
f.
 Employs porous beads
 Proteins with large Stokes radii
remain in the eluent and emerge
before proteins that have a smaller
Stokes radii and are able to enter the
porous beads
 Proteins emerged via descending
order of their Stokes radii
d. Ion Exchange Chromatography
 Proteins interact with the stationary
phase via charge-charge interactions
 Proteins with net (+) charge at a given
pH adhere to beads with negatively
charged functional groups such as
carboxylates or sulfates
(cationexchangers)
 Negatively charged proteins adhere to
tertiary or quaternary amines (anion
exchangers)
 Proteins compete with
monovalentions for binding hence “ion
exchange”
 Example: negatively charged proteins
bind to diethylaminoethyl(DEAE)
Transcribed by: Angcianco D., Bayawa S., Dilag A.
cellulose via replacing the Cl-or
CH3COO-
 Bound proteins are selectively
displaced by gradually raising the
concentration of the monovalent ions
in the mobile phase
 Proteins elute in inverse order of
strength of their interactions with
stationary phase
 Sequential elution achieved via pH
manipulation
Hydrophobic Interaction Chromatography
 Separates via tendency to associate
with a stationary phase matrix coated
with hydrophobic groups (phenyl
Sepharose, octylSepharose)
 Proteins with exposed hydrophobic
surfaces adhere to the matrix via
hydrophobic interactions enhanced by
a mobile phase of high ionic strength
 Non-adherent proteins are washed
first
 Polarity of the mobile phase is then
decreased by gradually lowering salt
concentration
 If interaction between protein and
stationary phase is particularly strong,
ethanol or glycerol is added to
decrease polarity and weaken
hydrophobic interactions
Affinity Chromatography
 Exploits the high selectivity of most
proteins for their ligands
 Enzymes may be purified using
immobilized substrates, products,
coenzymes, or inhibitors
 In theory, only proteins interacting
with immobilized ligands adhere
 Bound proteins eluted either by
competition with soluble ligand or less
selectively by disrupting protein-ligand
interactions using urea, guanidine
HCl, mildly acidic pH, high salt
concentration
 Stationary phase matrices available
commercially contain ligands such as
NAD+or ATP analogs
 Most powerful and widely applicable
affinity matrices are used for
recombinant protein purifications
o Uses a Ni2+ matrix that
binds proteins with an
attached polyhistidinetagand
a glutathione matrix that
binds a recombinant protein
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 AMINO ACIDS & PEPTIDES
linked to glutathione S-
transferase
g. High-Pressure Liquid Chromatography
 Employs incompressible silica or
alumina micro-beadsas stationary
phase and pressures of up to a few
thousand psi
 Incompressible matrices permit both
high flow rates and enhanced
resolution
 Resolve complex mixtures of lipids or
peptides whose properties differ only
slightly
 Reversed phase HPLC exploits a
hydrophobic stationary phase of
aliphatic polymers 3 -18 carbon atoms
in length
 Peptide mixtures are eluted using a
gradient of a water-miscible organic
solvent such as acetonitrile or
methanol
h. PolyacrylamideGel Electrophoresis (PAGE)
 Most widely used method for
determining protein purity
 Uses sodium dodecyl sulfate
 Electrophoresis separates charged
biomolecules based on the rates at
which they migrate in an applied
electrical field
 Acrylamide is polymerized and cross-
linked to form a porous matrix
 SDS denatures and binds to proteins
at a ratio of one molecule of SDS per
2 peptide bonds
 2-mercaptoethanol or
dithiothreitolused to reduce or break
disulfide bonds
 Large number of anionic SDS
molecules overwhelms the charge
contributions of the aafunctional
groups
 Charge to mass ratio of each SDS-
polypeptide complex is equal
 The physical resistance encountered
by the polypeptide determines the
rate of migration
 Large complexes encounter greater
resistance
 Polypeptides separate based on their
relative molecular mass (Mr)
 Individual polypeptides trapped in the
gel are stained using Coomassieblue
Transcribed by: Angcianco D., Bayawa S., Dilag A.
B. Isoelectric Focusing
 Ionic buffers called ampholytesand an
applied electric field are used to
generate a pH gradient within a
polyacrylamide matrix
 Applied proteins migrate until they
reach the region of the matrix where
the pH at which a molecule’s net
charge is 0
 Used in conjunction with SDS-PAGE
 Separates polypeptides based on pIin
one dimension and based on Mrin the
second
C. Sanger Amino Acid Sequencing
 Frederick Sanger won the Nobel Prize
in 1958 for determining the amino
acid sequence of insulin
 Insulin: 21 –aaresidue A chain and 30
–aaresidue B chain linked by disulfide
bonds
 Separated both chains by reducing
the disulfide bonds and cleaved with
trypsin, chymotrypsin, and pepsin
 Resulting peptides isolated and
treated with acid to hydrolyze peptide
bonds and generated 2 –3 aa
 Each peptide was reacted with 1-
fluoro-2,4 -dinitrobenzene (Sanger’s
reagent)
 While the ε-amino group of lysine
reacts with Sanger’s reagent, amino-
terminal lysinescan be distinguished
from other positions because they
react to 2 mol of the Sanger’s reagent
D. EdmanAmino Acid Sequencing
 PehrEdman introduced
phenylisothiocyanate(Edman’sreaget)
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 AMINO ACIDS & PEPTIDES
 Phenylthiohydantoin(PTH) derivative
can be removed under mild conditions
to generate a new amino terminal
residue
 Successive rounds of
derivatizationwith Edman’sreagent
can be used to sequence the first 20
–30 residues
 Most polypeptides must be cleaved
into smaller peptides prior to
sequencing
 Cleavage is necessary to circumvent
posttranslational modifications
rendering a proteins á-amino group
unreactive to Edman’sreagent
 Following cleavage, the peptides are
purified by reversed-phase HPLC and
sequenced.
E. Hybrid Approach
 Edmansequencing provides a partial
amino acid sequence
 Oligonucleotide primers modeled on
this partial sequence are used to ID
the gene and amplify it using PCR
 Once the gene is ID, oligonucleotide
sequence is determined to infer the
primary structure of the encoded
polypeptide
 Enhances the speed and efficiency of
primary structure analysis
 Circumvents obstacles brought about
by amino-terminal blocking group or
the lack of key overlap peptide
 Only a few segments of the primary
structure should be determined using
Edman’sapproach
 DNA sequencing reveals the order in
which amino acids are added but
does not provide posttranslational
modification
o Proteolyticprocessing
o Methylation
o Glycosylation
o Phosphorylation
o Prolineand lysine
hydroxylation
o Disulfide bond formation
F. Genomics Elucidates Proteins
 Primary structure analysis
revolutionized by genomics using the
hybrid approach
Transcribed by: Angcianco D., Bayawa S., Dilag A.
 Automated oligonucleotide
sequencing and computerized data
retrieval and analysis
 Identification of the open reading
frame (ORF) that encodes the protein
can be accessed from genome
databases
 A segment of amino acid around 4-5
residues in length is sufficient to
identify the correct ORF
 Computerized search algorithms
assist the identification of the gene
encoding the protein
 Peptide mass profiling: a peptide is
digested and introduced into the mass
spec
 A computer program is used to find
an ORF whose predicted protein
product would produce a set of
peptides whose masses match the
ones in the mass spec
G. Mass Spectrometry
 Replaced Edman’ssequencing
 Posttranslational modification of proteins
identified via mass increments
 Discriminates molecules based solely on
mass
 Can detect subtle physical changes in
proteins that occur during the life cycle of
a cell or organism
 A sample in a vacuum is vaporized under
conditions where protonation can occur,
imparting a positive charge
 An electrical field propels cationsthrough a
magnetic field which deflects them at right
angles to their original direction of flight
and focuses them onto the detector
 The magnetic force required to deflect the
path of each ionic species onto the
detector measured as the current applied
to the electromagnet is recorded
 The force of ions with identical net charge
is proportionate to their mass
 In a time-of-flight mass spectrometer, a
briefly applied electric field accelerates the
ions towards a detector that records the
time at which each ion arrives
Molecules with identical charge have
velocities inversely proportional to their
mass
 Conventional mass specs are used to
determine the masses of molecules of
1000 Da or less
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 AMINO ACIDS & PEPTIDES
 Time-of-flight mass specs are suited for
large protein masses
 Previously, analysis of peptides and
proteins by mass spec was hindered
bydifficulties in volatilizing large organic
molecules
 Matrix-assisted laser-desorption (MALDI)
and electro-spraydispersion (e.g.
nanospray) permitted determination of
large polypeptide masses (Ĩ100,000 Da)
at +/-1 Da
 Using electrospray dispersion, peptides
eluting from a reversed-phase HPLC
column are introduced directly into the
mass spec
 Peptides inside the mass spec are broken
down into smaller units by collision with
neutral helium (collision-induced
dissociation)
 Peptide bond are more labile than carbon
–carbon bonds
 Molecular mass of each amino acid is
unique except for leucineand isoleucine
 Tandem mass spec:
o Complex peptide mixtures can be
analyzed without purification
o Employs the equivalent of 2
mass specs linked in series
o First mass spec: separates
individual peptides based on
mass
o Second mass spec: fragments
single peptide
o Used to screen blood samples
from newborns (newborn
screening)
o Abnormalities in metabolite
(phenylketonuria,
ethylmalonicencephalopathy and
glutaricacidemiatype I)
monochromatic x rays of 0.15 nm to
confirm protein nature
 Crystals are frozen in liquid nitrogen
 Diffraction patterns recorded
 Data analyzed using Fourier synthesis
which summates wave functions
 Laue Xraycrystallography:
o Diffraction of polychromatic X
rays
o Crystal rotation is avoided
I. 3-D Structures: Nuclear Magnetic
Resonance Spectroscopy
 Measures the absorbance of radio
frequency electromagnetic energy by
certain atomic nuclei
 The frequency or chemical shift at which a
particular nucleus absorbs energy is a
function of both the functional group within
which it resides and the proximity of other
NMR-active nuclei2-D NMR permits 3-D
representation by determining the
proximity of the nuclei from one another
 Analyzes proteins in aqueous solutions
therefore conformation accompanying
ligand binding or catalysis is possible
 Less than or equal to 30 kDain size is
analysable.

H. 3-D Structures: XrayCrystallography

 Protein is first precipitated to form crystals

 Crystals are mounted into quartz
capillaries and irradiated with

Transcribed by: Angcianco D., Bayawa S., Dilag A. 11

 AMINO ACIDS & PEPTIDES
J. Molecular Modelling

 Computer technology determining the 3-D

protein structure
 Molecular dynamics programs can be
used to simulate conformational dynamics
of a protein and the manner in which
factors such as temperature, pH, ionic
strength, amino acid substitutions
influence motion
 Molecular docking programs simulate
interactions between a ligand-enzyme,
etc.
 Homology modelling: known 3-D structure
of a protein is used as a template to build
a model of the probable structure of a
related protein

Transcribed by: Angcianco D., Bayawa S., Dilag A. 12