Home exam 2011

1.      Protein folding

   Because the protein contains functional groups COOH and NH2 so when pH varies, these groups change their charges.

   Protein can be tolerant to certain pH values. The bonds COO-NH may be broken due to extreme pH (too high or too low), protein then will be denatured, unfolded, or change structures.

 In proteins the isoelectric point (pI) is defined as the pH at which a protein has no net charge (zezo). pI varies for different proteins. When the pH > pI, a protein has a net negative charge (the formation of  COO­^-) and when the pH < pI, a protein has a net positive charge (the formation of NH⁺, NH₂⁺, NH₃⁺).  

1.      Centrifugation

·         Differential centrifugation: The separation is achieved mainly based on the size of particles. The separation can be achieved with certain time and speed of centrifugation. But if we increase the centrifugation time, which does not affect the position of the particles band in the centrifugation tube.

·         Rate zonal centrifugation: for sample containing particles with very similar density, but these particles are different in sizes. The sample is isolated according to the mass and the size of sample particles. The rate zonal centrifugation takes advantages of sizes and mass of particles. So protein is suitable for this centrifugation method.

·         Isopycnic centrifugation: for sample containing particles differ in density but not in size.

The differences between these centrifugation methods are listed below following time for separation, mass, size and density of particles contained in the sample based on different methods, and position of the sample after centrifugation.

Particles	Differential centrifugation	Rate zonal centrifugation	Isopycnic centrifugation
Mass	sililar/different
Size	different	different	Same size
Density	different	Similar density	different
Time	dependent(K factor)	dependent	independent
Position of            the sample	Pellet at the bottom	pallet at the bottom	No pellet

2.      Electrophoresis

a.       The advantage of a stacking gel during denaturing electrophoresis and the principle behind it.

  A stacking gel with low pH and big pore size allows protein moves faster through than separation gel. Stacking gel ions migrate more quickly than protein, and the buffer electrophoresis ions move more slowly. The proteins are trapped in a sharp band between these ions and then enter the separation gel. This makes it possible to analyze and compare many protein samples at once. The proteins are separated in separation gel ( SDS-PAGE) with small pore size, according to their sizes. So the advantages are to increase the resolution, and many proteins can be analyzed at the same time.

b.      The principles of a 2-D gel, staining methods for gels, sensitivity and advantages and disadvantages.

·         The principle of a 2-D gel

·         The protein first is separated by the 1D- dimension electrophoresis, and then it is separated following 90 degree direction of the first. Many molecules are in the same band in the first separation dimension, but they are different in properties and structures and they will be continuously separated in the second dimension. The methods applied to 2-D gel include isoelectric focusing, protein complex mass in the native state and protein mass

·         Isoelectric focusing, the proteins are separated according to their charges in the first gel applied pH gradient and electric potential. Proteins will accumulate at the positions in the gel where they reach their isoelectric points. SDS-PAGE gel will separate proteins in the second dimension.

Figure 1

·         Protein complex mass in the native state

·         Protein mass.

·         Staining methods

To detect the proteins where their positions are in the separation gel, proteins are bonding to specific substances to make them possible to be detected (color).

·         Coomassie Brilliant Blue R-250: this one can bind to basic amino acids, for complicated proteins this method may not be used. In addition, the sensitivity is 100 ng. The linear relation and easy to use are advantageous.

·         Silver stain: AgNO₃ Silver nitrate becomes reduced to Ag-protein, sensitivity: 1 ng, not linear. The amount of AgNO₃ reduced to Ag-protein and the quality of protein is not linear relation, which is disadvantageous.

·         Western blotting: Antibodies (protein has to be known) we cannot use this method for unknown protein. And it is the most sensitivity that can detect 0.1 ng protein.

3.      Mass spectrometry

a.      The two important special features of ESI (electrospry). ESI is produced by applying the high voltage between the capillary tube carrying the sample and the counter electrode. The space of ESI is limited by a low flow-rate inert gas such as Nitrogen.

·         One feature is that ESI produces multiple charged ions from large molecules. This is advantageous as it improves the sensitivity and high-molecular-weight molecules (proteins) can be analyzed. The multiple charged ions are originated from electrochemistry and electric field.

·         The other is that ESI is sensitive to concentration and not to the total quality of the injected sample.

b.      A simple drawing of MALDI-TOF mass spectrometer

Figure 2

After the sample molecules are embedded through the matrix, all they turn to dried state. And then, the intense laser is conducted in the short pulse to the sample-matrix solid state. The sample molecules are desorbed and ionized by protonatation.

c.       The simple relation that is used to determine molecular masses in MALDI-TOF mass spectrometry.

·         The molecular masses are determined by the ions being protonated. This can be done by following the process that ions corresponding to molecules are detected, and the mass differentces between consecutive peaks. To determine the molecular masses, time of flight-method is applied. Ion mass-to-charge-ratio, m/z is defined by time measurement.

 1.1

Ions are speed up in the known applied electric field (up to 20 kV applied voltage). The time for ions to reach the detector is measured base on m/z following equation 1.1

d.      The lowest detection limit of peptides in MALDI-TOF

The detection limits of peptides are dependent on several factors such as the nature of sample, the preparation, purification factor, handling sample and operation of experiments. The detection limit is in the range of femtomoles to picomoles. The lowest detection limit of peptides is attomole.

e.       The identity of protein present in a mix with other proteins when using peptide mapping can be determined by the following :

The peptide mapping is related to small amounts of a pure protease to “break” a pure protein into peptides of characteristics, reproducible sizes. Each protein is characterized by certain peptides that are peptide fingerprints. A peptide fingerprint is a set of peptides that is generated by proteolysis with a sequence specific protease such as trypsin.

 Peptide mapping can indentify an unknown protein based upon its fingerprint. From peptide mapping, if there are a groups of peptides that match to a certain protein, this protein must be in the sample.

f.       MALDI-TOF mass spectrometry

·         Determine a protein is phosphorylated or not on a serine or threonine amino acid.

After proteolysis a protein is transferred to peptides characterized as fingerprints for this protein. The In the MALDI-TOF is used to detect these peptides. Now if a protein contains serine or threonine, and we need to determine is there phosphorylation of serine or threonine amino acid. The signals from MALDI-TOF differ from signals obtained from this protein without phosphyrylation of serine

Chromatography

4.      We can separate the protein of interest with mass 22kDa from proteins different masses ( 6, 28, 60 kDa) by Size exclusion chromatography, large protein will be diluted first, and smaller proteins will dilute late. The VIP can be collected based on the peak position from the chromatogram.

The advantages of this method: preserving the biological activity of VIP, it can separate small amount of VIP with high sensitivity and various solutions can be applied without interfering with the filtration process. The separation time is short and VIP is saparated in the narrow band. Because the VIP does not interact with stationary phase, No sample loss is noticed.

Disadvantages are the separation time is shorts so if there are a lot of proteins are run by this methods meaning that a lot of bands may overlap each other, which makes difficult to collect the protein of interest. To have a good resolution, there has to be a 10% difference in molecular mass, in this case the purification of VIP is suitable for this method.

5.      Spectroscopy

a.      The principles of fluorescence spectroscopy

After receive the energy (hn) to the excited state (M + hn ® M* ), an electron turns back to its ground state.  This process can occur by non radiative decay, the energy is released to surroundings and converted to heat; fluoresence, energy is released by light emission (M* ® M + hn), this is a very fast process ns timescale; Phosphoresence, light emission that occurs much slower then fluorescence. So the principle of fluorescence is the measurement of intensity light emitted by sample that is received light from xenon lamp.

A typical instrumental set-up

Figure 3

The sample is placed in the sample compartment; the light from xenon lamp directly passes through the sample then to reference detector. The sample emits light passing through emission monochometer and detector functioned by photomultiplier. The signals from reference detector and signal detector are compared to have intensity of light emitted by the sample.

b.      A protein has one single flourophore, an tryptophan. Tryptophan causes the protein to be fluorescent. When a fluorophore is at its excited state induced by photon, its electronic and vibrational energy levels start to relaxe to the ground state. It will release energy in the form of a photon. The maximum emission wave lenghs from the flourophores depend on the state of protein(folded or unfolded), one flourophores can absord light emited from other flourophores. The movement of protein chain also affects the emission light.  If the protein has emission maxima at 310, 330, 350nm, the changing of solvent polarity or protein chain, conformational changes of fluorophor ligand binding due to quenching, changing polarity and solvent accessibility of the fluorophor environment affect the emission of light so the maxima emission wave length of flourescene is changed.

Emission maximum may vary because the environment surrounding the flourophores is variable. This include the movement of protein caused by hydro bonds, flourophores absorb photons from other.

c.       The energy transfer occurs between tryptophan and tyrosines because one of them absorbs light from the other with higher emission energy (hv). The come out of experiment is that the intensity or signal does not appear in sharp. The maximum wavelength and the light emitted from the sample will vary.

d.      Solvent exposure of a flourophore

-          We use Fluorescence spectra of a protein obtain information about the solvent exposure of a flourephore. If a molecule is unfolded a flourophore will be exposed to the solvent, it tends to be easily flourescent. The absorption and emission efficiency are enhanced. So the fluorescence efficieny is very different in folded protein than in unfolded protein, and for the same protein, the intensity of signals obtained from unfolded protein is higher than folded protein.

e.       The principles of CD-Spectroscopy

Circular Dichroism (CD) refers to the differential absorption of left and right circularly polarized light that has constant amplitude and the direction of the electronic vector is modulated. Proteins that are assymetric absorbe right and left handed circularly polarized light to different extents. CD-Spectroscopy measures the difference in left and right circularly polarized light by applying Lambert Beers law

f.       A far-UV CD-spectrum at wave length range: 170-250 nm can give information about:

·         How peptides are bond to each other, the picture of peptide backbone in protein is seen (3D structure). We can predict the secondary structure of proteins in Far-UV by fitting CD spectra with various structures.

A near-UV CD-spectrum at wave length range 250-320nm can give information about:

·         Whether proteins are native or not because the adsorption, dipole orientation and the nature of the surrounding environment oftryptophan, tyrosine, phenylalanine and to a lesser extent disulphide groups give signals in A near-UV CD-spectrum. So it provides the structure information on the nature of these groups in proteins and the tertiary structure of proteins.

6.      DNA-binding protein(DNA-BP) and DNA interaction can be characterized by

- The affinity between DNA and DNA from DNA-BP

- The DNA and protein from DNA-BP

- The DNA with both DNA and protein form DNA-BP

The experiment

·         using A far-UV CD-spectrum obtained from C-spectroscopy to define:

-    Find out which functional groups of DNA exposed to DNA-BP to have affinity

-   Protein can separate from DNA-BP then bonds with other DNA

-   Functional groups of DNA can bond DNA and protein from DNA-BP.

1.       Figure 2. Mass Spectrometry - Principles and Applns 3rd ed - E. de Hoffmann, V. Stroobant [34]

2.       Figure 3 from the lecture.