PORTFOLIO

Portfolio

Table of Content

I) PROTEIN STRUTURE AND SYNTHESIS4

Protein Structure4

Primary, Secondary And Tertiary Structure6

Primary Structure7

Secondary Structure7

Tertiary Structure7

Protein Synthesis8

Ribosomes And Chemical Synthesis9

Protein Modification12

II) CELLULAR FUNTIONS14

Enzymes14

Structural Protein16

Protein Complex19

Protein Interactions21

Model Key-Lock23

Thermodynamics24

Affinity25

Kinetics28

The Kinetic Constants Of Enzymes29

III) THE PROTEOME30

Number of Proteins33

Protein Networks34

Evolutions of Proteins36

IV) PROTEIN ANALYSIS TECHNIQUES40

In Vitro Separation40

Ion Exchange41

Affinity45

Gel Electrophoresis47

Isoelectric Focusing (IEF - Isoelectric Focussing)47

Mass Spectrometry48

Protein Sequencing49

Immunoblotting51

2D Gel52

In Vitro Translation56

Affinity Analysis57

Calorimetry57

Biacore58

In Vivo Yeast 2-Hybrid59

Immunocytochemistry60

Fusion Proteins62

Reporter Proteins63

Problem Excercise64

V) CELL IMAGING65

2D Light Microscopy65

Fluorescence Microscopy65

Deactivation67

Non-Staining Methods69

Phase Contrast Microscope Or Light Background With Zernicke Device69

+ 3D image (Microscopes 4,5,6)70

Polarized light70

5 - Atomic Force Microscope70

6 - Confocal Microscope70

Ultraviolet Microscopy71

Ultraviolet Light Microscope71

Fluorescence Microscopy71

Electron Microscopy71

1 - Transmission Microscope (TEM)72

2 - Micoscopio (SEM)72

Beyond The Resolution Limit Of Light73

Electron Microscopy73

X-ray Defraction74

3D Microscopy75

Stereomicroscopy75

Confocal Microscopy76

Multiphoton Microscopy78

Technology Multiphoton79

Optimized Detectors79

Deconvolution Software80

6) 4D Microscoopy81

7 ) Beyond the 4th Dimension82

Spectral Scans82

CARS Microscopy83

Benefits of CARS microscopy84

References86

PORTFOLIO: PROTEIN ANALYSIS TECHNIQUES

I) PROTEIN STRUTURE AND SYNTHESIS

Protein Structure

Proteins are polymers of amino acids covalently linked through peptide bonds into a chain. Within and outside of cells, proteins serve a myriad of functions, including structural roles (cytoskeleton), as catalysts (enzymes), transporter to ferry ions and molecules across membranes, and hormones to name just a few.

(Source: Wu et al.(2001)

Proteins are polymers of amino acids joined together by peptide bonds. There are 20 different amino acids that make up essentially all proteins on earth. Each of these amino acids has a fundamental design composed of a central carbon (also called the alpha carbon) bonded to:

a hydrogen

a carboxyl group

an amino group

a unique side chain or R-group (Adams, 1992)

Thus, the characteristic that distinguishes one amino acid from another is its unique side chain, and it is the side chain that dictates an amino acids chemical properties. Examples of three amino acids are shown below, and structures of all 20 are available. Note that the amino acids are shown with the amino and carboxyl groups ionized, as they are at physiologic pH.

(Source: RJ Lipshutz et al 1999)

Except for glycine, which has a hydrogen as its R-group, there is asymmetry about the alpha carbon in all amino acids. Because of this, all amino acids except glycine can exist in either of two mirror-image forms. The two forms - called stereoisomers - are referred to as D and L amino acids. With rare exceptions, all of the amino acids in proteins are L amino acids. (Adams, 1992)

The unique side chains confer unique chemical properties on amino acids, and dictate how each amino acid interacts with the others in a protein. Amino acids can thus be classified as being hydrophobic versus hydrophilic, and uncharged versus positively-charged versus negatively-charged. Ultimately, the three dimensional conformation of a protein - and its activity - is determined by complex interactions among side chains. Some aspects of protein structure can be deduced by examining the properties of clusters of amino acids. For example, a computer program that plots the hydrophobicity profile is often used to predict membrane-spanning regions of a protein or regions that are likely to be immunogenic.

Amino acids are covalently bonded together in chains by peptide bonds. If the chain length is short (say less than 30 amino acids) ...