Lecture 4: Protein Sequence and Structure Determination

Question 1

Uses of determining amino acid sequence in a protein?

Answer 1

• compare with all other known sequences (including DNA) to determine whether similarities exist
• sequence comparison of the same protein in different species can yield cluse about evolutionary pathways
• sequence comparison of the same protein in the same species (ie humans) can reveal the molecular mechanisms of (genetic) disease

* conserved amino acids (between very different species) suggest that a pathway is very significant

Question 2

HCl - Determination of sequencing amino acid composition

Answer 2

• hydrolyze polypeptide in HCl at 110 c for 24 hours
• amino acids can then be separated by ion exchange chromatography
• ninhydrin can be used to ID amino acids
• modifies asparagines and glutamines to aspartic acid and glutamic acid

Question 3

What does ninhydrin do?

Answer 3

• chemically modifies asparagines and glutamines to aspartic acid and glutamic acid

Question 4

Elution profile of amino acids

Answer 4

• Peaks are in a ratio
• double height means double of that amino acid compared to another
• if asparagine is present is degrades it into aspartic acid and NH3 (see those peaks)

Question 5

Purpose of cleavage and reduction of polypeptides

Answer 5

• accuracy of amino acid sequencing generally declines as the length of the polypeptide increases
• must be enzymaticaly (proteases) or chemically fragmented to be sequenced efficiently
• if disulfide bridges are present, they must be broken (reduced) and the resulting cysteine-sulfhydryl groups prevented from reformation of disulfide bonds (modified) [add reductant and then modify sulfhydryl groups]

Question 6

What is Edman degradation?

Answer 6

• sequentially removing one residue at a time from the amino end of a peptide (fragment)

Question 7

Steps of Edman degradation

Answer 7

1. phenyl isothiocyanate reacts with the uncharged terminal amino group of the peptide to form a phenylthiocarbamoyl derivative
2. the cyclic form of the derivative is liberated and can be identified by chromatographic methods
3. in multiple labeling-release rounds, the amino acid sequence of a peptide can be determined

* keeps attackign N-terminus over and over to remove successive amino acids

Question 8

Overlapping and Edman degradation

Answer 8

• divide the same polypeptide chain by different segments
• arrange the segments so the two kinds overlap
• this way you can tel how to connect the segments

Question 9

What is mass spectrometry?

Answer 9

• analytical technique that measures the mass to charge ratio (m/z) of charged particles in a gas phase

Question 10

Steps of mass spectrometry

Answer 10

1. molecules to be analyzed (anylate) are first ionized in a vacuum
2. when the newly charged molecuels are introduced into an electric and or magnetic field, their paths through the firls are a function of theis m/z ration (mass/charge)
3. measure property of the ionized species can be used to deduce the mass (M) of the anylate with high precision

Question 11

Three essential components of mass spectrometer

Answer 11

• ion source
• mass analyzer
• detector

Question 12

Conversion of macromolecules into gas phase ions required?

Answer 12

conversion of macromolecular anylates such as proteins and nucleic acids into gas-phase ions (ionization) could not be achieved efficiently until the development of

–> electrospray ionization mass spectrometry (ESI MS)

–> matrix assisted laser desorption/ionization mass spectrometry (MALDI MS)

Question 13

Mass to charge ration of ions and molecular mass of proteins

Answer 13

• gas phase macromolecules acquire a variable number of protons and thus positive charges, from the solvent, which creates a spectrum of species with different mass to charge ratios
• each successive peak corresponds to a species that differs from that of its neighboring peak by a charge difference of 1 and a mass difference of 1.
• the molecular mass M can be determined from any two neighboring peaks

Question 14

Mass Spectrometry Equations

Answer 14

compare two peaks

p1 = (M + z1) / Z1

p2 = (M + z1 - 1) / (z1 - 1)

• just subtract everything by 1
• adjacent peaks have difference on 1 in protons and in mass

can solve for M and z1

1. M = z1 (p1 - 1)
2. z1 = (p2 - 1) /(p2 - p1)

Question 15

ESI MS process

Answer 15

• analyte solution is passed through an electricaly charged nozzle into a chamber of low pressure, evaporating the solvent and ultimately yielding the ionized analyte

Question 16

How fast do different sized particles move in ESI MS

Answer 16

more charge/more mass –> slower

less charge/less mass –> faster, detected first

Question 17

MALDI MS process

Answer 17

• analyte solution is evaporated to dryness in the presence of a volatile, aromatic compound (the matrix) that can absorb light at specific wavelengths
• laser pulse tuned to one of these wavelengths excites and vaporates the matrix, converting some of the analyte into gas phase
• subsequent gaseous collisions enable the intermolecular transfer of charge, ionizing the analyte

* matrix is important to keep proteins from getting burned, makes sure they end up in gas form

Question 18

MALDI TOF analyzer

Answer 18

1. protein sample is ionized
2. electrical field accelerates ions
3. lightest ions arrive at the detector first
4. laser triggers a clock

Question 19

TOF and mass to charge ratio

Answer 19

mi/zi = 2eEl/(ti/ld)²

* mass to charge ratio is dependent on time

plug in time and get out the mass/charge ratio

–> first particles to arrive have the largest mass/charge ratio

Question 20

Tandem mass spectrometry

Answer 20

• alternative to edman degradation as a means of sequencing proteins
• ionized proteins are analyzed by a first mass spectrometer and then broken down into smaller peptide chains

Question 21

Steps of tandem MS

Answer 21

ion source –> MS1 –> collision cell –> MS 2 –> detector

1. catch protein as it arrives after a certain time
2. analyze it further

* pretty difficult to do

Question 22

From protein mixtures to peptide mass identification

Answer 22

protein mixture (separation/2D electrophoresis) –> individual proteins (fragmentation/spot excision, site specific cleavage) –>

peptide fragments (fingerprinting/ mass spec) –>

peptide mass spectrum (database search/MSfit, MASCOT, Proetinlynx) –>

ID

Question 23

Unique peptide sequences

Answer 23

• most of at least 6 amino acids are unique in the proteome of an organism and map to single gene products

Question 24

Effect of sig figs in m/z searches

Answer 24

no decimals could lead to MANY hits (478)

going up to 4 decimals could lead to only 2 hits

Question 25

Determination of fidelity of “known” sequences

Answer 25

• recombinant proteins
• synthetic peptides

Question 26

detection of natural or biosynthetic mutations

Answer 26

isoforms

in vitro mutated proteins - random or site specific

Question 27

identification of endogenous postranslational modifications

Answer 27

phosphorylation - regulatory or catalytic

disulfide bonds

Question 28

identification of experimental chemical modifications

Answer 28

affinity or group secific labels

Question 29

X ray diffraction general steps

Answer 29

crystal –> diffraction pattern –> electron density map –> atomic model

(refinement)

Question 30

Componnts in an X ray crystallographic analysis

Answer 30

• an xray source generates a beam, which is diffracted (scattered) by a crystal
• the resulting diffraction pattern is collected on a detector
• want a lot of crystals for it ro work
• to get crystals get close to the ppt point and crystals will sometimes form

Question 31

Physical principles of x ray crystallography

Answer 31

1. electrons scatter x rays –> the amplitude of the wave sattered by an atom is proportional to its number of electrons
2. the scattered waves recombine
3. the way in which the scattered waves recombine depends only on the atomic arrangement

Question 32

Resolution and electron density

Answer 32

• the better the crystal the better the resolution of th eimage

Question 33

NMR

Answer 33

• carried out on macromolecule sin solution (xray crystalloggraphy is limited to molecules that can be crystallized)
• illuminate the dynamic side of protein structure, including conformational changes, protein folding and interactions with other moelcules
• depends on the fact that certain atomic nuclei are intrinsically magnetic due to a nuclear spin angular momentum, which can take either of two orientations or spin states calle a and b when a magnetic field is applied

Question 34

spin states

Answer 34

• the enrgies of the two orientations of a spin 1/2 (such as 1H) depend on the strength of the applied magnetic field
• absorption of electromagnetic radiation of appropriate frequency induces a transition from the lower to the upper level (and resonance will be obtained)
• change of energy of one H will be transferred only to others in close proximity

* see chemical shifts of Hs on the neighboring C

Question 35

2D NMR - overhauser effect

Answer 35

• identifies pairs of rpoteins that ar ein close proximity
• whatever is on straight line has no interactions
• dots off the line interact
• when proteins fold Hs that are far away int he chain still come into contact and interact

Question 36

homologs

Answer 36

• biochemical entities related by common ancestry
• detectable by significant similarity in nucleotide or amino acid sequence and is (alomst always) manifested in three-dimensional structure

Question 37

paralogs

Answer 37

homologs that are present within one species

• usually a rsult of gene duplication
• often differ in their detailed biological function
• different versions of the same protein for different tissues

* evolution –> develop from existing gene “tamplate” and are altered from there –> every gene begins from another

Question 38

orthologs

Answer 38

• homologs that are present within different sepcies and have very similar or identical functions

Question 39

statistical analysis of sequence alignments

Answer 39

• significant sequence similarity between two DNA, RNA or protein molecules implies that they are homologs and hence have same evolutionary origin
• as proteins are composed of a larger number of building blocks (20 aas) than DNA or RNA (4 nucleotides) random sequence agreements are less likely

Question 40

alignment algorithms to compare sequences

Answer 40

• identities
• gap insertion
• conservative sumstitution

Question 41

conservation of 3d structures

Answer 41

3d structures of proteins or RNA relate directly to their functions and hence are more evolutionarily conserved than primary structures or amino acid sequences

• similarities in structures:

–> can be detected without significant similarities in sequence aliggnments

–> usually indicate common functional mechanisms

Question 42

similar structures but different functions

Answer 42

example:

• in primates a-lactalbumin expression is upregulated in response to the hormone prolactin and increases the production of lactose
• lysozymes are generally enzymes that damage bacterial cell walls by hydrolyzing their petidoglycan component

Question 43

Convergent evolution

Answer 43

some proteins are structureally and funcitonally similar in many important ways but do not have a common ancestor

–> very different evolutionary pathways can elad to the same biochemical solution

chymotrypsin vs subtilisin