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Kód předmětu: BÍ8980
1  MASARYKOVA UNIVERZITA
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Protein expression and purification
III. The important properties of proteins and how to explore them
Lubomír Janda, Blanka Pekarové and Radka Dopitová
Tento projekt je spolufinancován Evropským sociálním fondem a státním rozpočtem České republiky.
OP Vzdělávání pro konkurenceschopnost
EVROPSKÁ UNIE «
NVESTICE  I    )  ROZVOJE VZDĚLÁVÁNI
Název prezentace v zápatí
1
■xplore them -Lubomír Janda
3.1. Introduction: the context for studies and data analysis
Key concept:
Appreciating the complexity of cellular systems in terms of the numbers of distinct protein species present
Yeast Saccharomyces cerevisiae Nematode worms Caenorhabditis elegans Vertebrate Homo sapiens
6,000 genes 12,000 genes 23,000 genes
Probably over 10 times as many distinct proteins:
• differential RNA processing
• post-translational modification
E-1D-C
E0a
E1
E0
Elf
Eial
Eih[
EllIZ EliL
N-terminal domain
i
Plectin  <j —
2   2a   3    3d 4
h 200 bp
rod
C-terminal domain >-Štr$ÉMIHD$IM±)-
— O
RDs        Linker Module
3.2. The key questions about a protein
■xplorethem- Lubomír Janda
Key concepts:
Being aware of the range of functions of proteins Understanding the levels of protein structure Identifying appropriate methods to explore protein structure and protein interactions
Proteins are composed of amino acids which are linked by peptide bonds; however, it is the sequence of amino acids within a given protein that dictates its unique function and structure.
Biological systems are not static entities; they respond to: Environmental signals Developmental signals Metabolic signals • Proteins almost always occur within complex cellular enenvironments, interacting with other proteins, metabolites and cellular structures.
3.2. The key questions about a protein 3.2.1. What are the functions of proteins?
I   1. Catalytic proteins
II. 2. Transport proteins 3. Signalling proteins
III. 4. Structural proteins
5. Motor proteins
6. Binding proteins
IV. 7.   Storage proteins
■xplorethem- Lubomír Janda
I. Enzymes
II. Proteins binding small molecules
III. Proteins binding macromolecules (protein-protein or proteinDNA interaction)
IV. Storage proteins
■xplorethem- Lubomír Janda
3.2. The key questions about a protein
3.2.1. What are the functions of proteins?
3.2.1.1. Protein classification
Enzymes are classified on the basis of their composition (60% of all proteins are enzymes). Activity of enzymes is also affected by changes in pH, temperature and substrate concentration.
Simple enzymes
Complex enzymes (Holoenzymes)
Apoenzymes
Coenzyme
Prosthetic group
enzymes composed entirely of proteins
protein + small organic molecule (ligand)
protein component
non-protein component
(not covalently bound)
small organic molecule
(covalently bound)
3.2. The key questions about a protein.
3.2.1. What are the functions of proteins?
3.2.1.2. Enzyme classification - EC number
■xplorethem- Lubomír Janda
Number	Classification	Biochemical properties
i.	Oxidoreductases	Act on many chemical groupings to add or remove electron or oxygen
2.	Transferases	Transfer functional groups between donor and acceptor molecules
3.	Hydrolases	Add water across bond, hydrolyzing it
4.	Lyases	Add water, ammonia or carbon dioxide across double bonds, or remove these elements to produce double bonds
5.	Isomerases	Carry out many kinds of isomerization (L/D), mutase reaction
6.	Ligases	Catalyze reactions in which two chemical group are joined with use of energy from ATP
xplorethem- Lubomír Janda
3.2.1. What are the functions of proteins?
3.2.1.2. Enzyme classification - EC number 3.2.1.2.1. Oxidoreductases
They are involved in redox reactions in which hydrogen or oxygen atoms or electrons are transferred between molecules.
Dehydrogenases hydride transfer
Oxidase electron transfer
Oxygenase oxygen transfer
Peroxidase electron transfer to peroxide
EC 1.1.3.4
ho- r y
,0. .oh
ho
ho
oh
oh
ho^^Y o
C00198
o
0=0
C00007
ho-oh
C00027
beta-D-glucose oxidase beta-D-Glucose + Oxygen
D-Glucono-1,5-lactone + H2O2
xplorethem- Lubomír Janda
3.2.1. What are the functions of proteins?
3.2.1.2. Enzyme classification - EC number 3.2.1.2.2. Transferases
They catalyse the transfer of an atom or group of atoms (e.g. Acyl-, alkyl-, and glucosyl-) between two molecules, but excluding such transfers as are classified in the other groups.
0
HO
OH
NH2 O
C00049
2.6.1.1 aspartate aminotransferase
O HO
C00036
L-Aspartate + 2-Oxoglutarate   ~-» Oxaloacetate + L-Glutamate
■xplore them - Lubomír Janda
3.2.1. What are the functions of proteins?
3.2.1.2. Enzyme classification - EC number 3.2.1.2.3. Hydrolases
They are involved in hydrolytic reactions and their reversal. This group of enzymes includes esterases, glycosidases, lipases and proteases.
HO
HO
C00208
HO
o. x1fi* "Y",Jf°H
OH
OH
'*OH
O. .OH
'*OH
C00001
EC 3.2.1.20 alpha-1,4-glucosidase
2 alpha-D-Glucose
'xplorethem- Lubomír Janda
3.2.1. What are the functions of proteins?
3.2.1.2. Enzyme classification - EC number 3.2.1.2.4. Lyases
They are involved in elimination reactions in which a group of atoms is removed from the substrate. Lyases include aldolases, decarboxylases, dehydratases and some pectinases.
h
N
N
C00135
oh
o
C00785
o
EC 4.3.1.3. L-histidine ammonia-lyase
L-Histidine
C00014
Urocanate + NH3
They catalyse molecular isomerizations and include epimerases, racemases and intramolecular transferases.
■xplorethem- Lubomír Janda
HO
C00095
D-Fructose
xplorethem- Lubomír Janda
3.2.1. What are the functions of proteins?
3.2.1.2. Enzyme classification - EC number 3.2.1.2.6. Ligases
They are known as synthetases involved in the formation of a covalent bond joining two molecules together, coupled with the hydrolysis of a nucleoside triphosphate.
H2r\T
HS )=0 HO
NH     +   HO-^ /
o        o S3h
NH?        M O
OH
0 0 0 II      II II
HO-P-O-P-O-P-O
1 I I HO    HO HO
HO OH
HO-P-O-P-O i i HO HO
NH?
HO'VOH OH
6.3.2.3
glutathione synthase
ATP + gamma-L-Glutamyl-L-cysteine + Glycine
ADP + Orthophosphate + Glutathione
I. The molecular pri
Lubomír Janda
Please solve the problem.
Question 1: To which enzyme group (EC number according to enzyme classification) does it belong?
L-Aspartate + 2-Oxoglutarate   "-» Oxaloacetate + L-Glutamate
5 points
3 points
Aspartate aminotransferase catalyses this reaction.
2 points
This enzyme group can transfer functional groups between donor and acceptor molecules.
1 point
Second group EC 2.-.-.-.
I. The molecular principles for understa
Please solve the problem.
Question 2: To which enzyme group (EC number according to enzyme classification) does it belong?
HO
OH
C00031
D-Glucose
D-Fructose
D-glucose aldose-ketose-isomerase catalyses this reaction
5 points
3 points
2 points
This enzyme group catalyses molecular isomerizations and includes epimerases, racemases and intramolecular transferases.
1 point
Fifth group EC 5.-.-.-.
xplorethem- Lubomír Janda
'xplorethem- Lubomír Janda
3.2.1. What are the functions of proteins?
3.2.1.4. Signalling proteins - small molecules
IH ATP ks 7.
MNWALNNHQEEEEEPRRIEISDSESLEJ^^CSSDFYQLGG
KGSTFIQEHRALLPKALILWIIIVGFISSC^YQWMDDANKIRREEVLVSMCDQRARML^
TFAEYTARTAFERPLLSGVAYAEKVVNFEREMI^RQHNWVIKTMDRGEPSPVRDEYAPVIFSQDSVSYLESLDMM
KAVLTSPFRLLETHHLGVVLTFPVYKSSLPENPTVEERIAATAGYLGGAFDVESLVENLLGQLAGNQAIVVHVYDITNASD
EADRSLSHESKLDFGDPFRKHKMICRYHQKAPIPLNVL^
TVSHEIRTPMNGILGMLAMLLDTELSSTQRDYAQTAQVCGKALIALINEVLDRAKIEAGKLELESVPFDIRSILDDVLSLFSEESRNKGIE LAVFVSDKVPEIVKGDSGRFRCT \'™"r TT™- 1_,^11_1^T^T^ T ""^^"^"."^""^"""^SYNTLSGYEAADGRNS
WDSFKHLVSEEQSLSEFDISSJV _\ "'"      \ / MRGQINFISRPHIGST
FWFTAVLEKCDKCSAINHMKKI W FERNGSPLPTKPQLDM
ILVEKDSWISTEDNDSEIRLLJ> *J        J  V                      J^Sf J  VA_ QVLELRKTRQQHPEGS
SPATLKSLLTGKKILVVDDNI\ J /   |         ^K-        \           '\" -7 ^                    \. "■ RQIRMMEKEAKEKTNL
.0.1     ^, J"
NH
>r27S
ir 27Ů
HNJ
r r »
IM
N 1
trans-zeatin
kinetin
Extracelular domain of AHK4/CRE1
'xplorethem- Lubomír Janda
3.2.1. What are the functions of proteins?
3.2.1.4. Signalling proteins - macromolecules
insulin
glycogen
acids
pyruvate
xplorethem- Lubomír Janda
F-actin
G-actin
Structural proteins -most abundant proteins within biological systems and include such proteins as collagen, keratin, actin, tubulin, and spectrin
	65 '■■ ■■■
	
m	■ ■
	
	
	;
1	/ f
	
^^^^	
actin molecule
minus end
50 nrn
B   37 nm
plus end
] how to explore them - Lubomír Janda
Actin
flii <>tairiíilDti h on Inh Q[gflulai mnu *f fikmims thar sappcrta and tfiapB tho (all. Ihť rmnt plw hlul tfpa oF Nrrnanr it mnipoisd oF adin. diawnhmvln but Thfl fylDiiJalai hnnr^ii nnr a -joik Jlmclurp Tinr? it muvraipandId lha ihtfialnp awfcif tbs (HI.
Tho prDlviiiríhftfii Ihdplo růíliQpftltia cflwk&lMDn bp nssumUiriQ pi diMEMmUing atin Flaimnls ai niíiiidiy. h nidHiilo of AT?, which e
bound Irctii ndi actin mnlňailn h impwlant in rin1, prawn. Wh^n it \i hyrirah/ied io^DP tha filaniml bams unartote and falliDparl.
Sdsaln brooks dawn adin Mamini* by a^tpg tha hydrate wt ATf and unking hra sHn dF Inrorndkn wih othěf adin jraMm. hw diFtar Enq-mnnb ol grj^m wishwnti lrih end 1 in bound ta urin. lha crntsn fjipi Fffirc a cap ai Hip a-:dn írbnente úvm n hin which
IrrttianmbJy.
lha fiahinlnrmhiEEE rea^mihlynl nnin bydlpiirgr»a<hn pftfam h rlio popu oilfifTtation which ilarte ih* prams of Filainonr jfawtti. Qno domain pí formin it shewn bound la adin In 1-^
HphIii links nalghhnrinc) ociln FilDmanft ma hlohu udar itructani Tho Dilin-LiniJiiifl domain c ^awn in khS
1,'ji
xplore them-Lubomír Janda
1
Actin filament ■
Fascin c ros&linker
36 nrn
'xplorethem- Lubomír Janda
3.2.1. What are the functions of proteins?
3.2.1.6. Motor proteins
Actin and myosin - muscle contraction (60% efficiency) Microtubules - kinesin
dynein
IF wriicki
IF Kiltie
Nature Reviews I Molocuht C*3l Biology
xplorethem- Lubomír Janda
y V rcflions (variable)
	SO    IDO "20
	residue
	
■ M	
O     20    4^60    80   IDO "20 F n i     fr2     frh fqj
Antibodies play a central role in the adaptive immune response of vertebrates by recognizing and binding antigens present on the surface of viruses, bacteria and other infectious agents.
'xplorethem- Lubomír Janda
3.2.1. What are the functions of proteins?
3.2.1.8. Storage proteins
• The casein content of milk represents about 80% of milk proteins.
• Calcium binding by the individual caseins is proportional to the phosphate content.
• The high number of proline residues in caseins causes particular bending of the protein chain and inhibits the formation of close-packed, ordered secondary structures.
• Caseins contain no disulfide bonds. Furthermore, the lack of tertiary structure accounts for the stability of caseins against heat denaturation as there is very little structure to unfold.
Hydrophobic core
kappa casein surface
Casein or ferritin
sequester ligands (Ca2+ or Fe3+), which may prove toxic within a
biological system.
hydrophobic casein submicelles
cluster
kappa casein submicelles
I. The molecular principles for understanding proteins - Lubomír Janda
Please solve the problem.
Question 3: To which group does this protein belong?
The protein is dynein. 5 points
This protein does not interact with small molecules. 3 points
It is a motor protein.        ^^^^^^^^^1 2 points
This protein interacts with microtubules. 1 point
Third protein group (macromolecules binding proteins).
I. The molecular principli
Lubomír Janda
Please solve the problem.
Question 4: To which group does this protein belong?
The protein is AHK4.
5 points
This protein is a receptor histidine kinase.
3 points
This protein interacts with small molecules and macromolecules and has enzymatic activity.
2 points
This protein contains three domains.
1 point
Complex protein - Group 1, 2 and 3.
44,749 structures in the PDB have a structure
e file.
n
Lubomír Janda
3.2. The key questions about a protein
3.2.2. What is the structure of the protein?
3.2.2.1. Determination of the amino acid sequence
The amino acid sequence of a protein can be used to generate a wealth of structural information:
• theoretical mass,
• potential post-translational modification sites,
• structural and functional motifs, and
• X-ray crystallographic and NMR spec. structures.
3.2.2.2. Experimentally determined mass
The theoretical mass of a protein, calculated on the basis of the amino acid sequence, does not take into account any post-translational modifications:
disulphide bond formation phosphorylation glycosylation proteolysis
Number
2,500  5.000  7,500 10,000 12,500 15,000 17,500 20,000 22,500 25,000 27,500 30,000 32,500
2006 2005 2004 2003 2002 2001 2000 1999 1998 1997 1996
1995 1994 1993 1992 1991 1990 S3 1989 f ^ 19SS 1987
Experiment Method	Proteins	Nucleic Acids	Protein/N ucleic Acid complexes	Other	Total
		1202	2386	17	55361
diffraction	51756				
NMR	7240	895	154	1	8296
Electron microscopy	139	17	73	0	279
Hybrid	IS	1	1	Í	21
Other	120	4	4	13	141
Total:	59323	2119	2618	38 "	64098 " 1
'xplorethem- Lubomír Janda
3.2. The key questions about a protein
3.2.2. What is the structure of the protein?
3.2.2.3. Characterization of secondary and tertiary structure
Proteins adopt three-dimensional structures that are dictated by the amino acid sequence of the protein. The predominant forms of secondary structure are oc-helices and p-sheets. The secondary structure of a protein can be determined theoretically from its amino acid sequence or measured experimentally.
a helix
(a)
p-defensin
ATP synthase
(b)
hlHHHl
p-sheets type I p-turn \ / poly (Pro) II helix
160 180 200 220 240 260
Wavelength (nm)
Far-UV CD spectra of various types of protein secondary structure
'xplorethem- Lubomír Janda
3.2. The key questions about a protein
3.2.2. What is the structure of the protein?
3.2.2.4. Characterization of quaternary structure
The quaternary structure can range in complexity from two identical subunits to multiple non-identical subunits.
• ribulose-biphosphate carboxylase EC 4.1.1.39 (photosynthetic bacteria)      2x55 kDa.
• RUBISCO (algae, plant)      (8 x 55 kDa and 8 x 15 kDa)
High-resolution X-ray crystallography is required to provide the molecular details of subunit arrangements within multi-subunit proteins.
• proteasome
• RNA polymerase II i S
ixplore them - Lubomír Janda
3.2. The key questions about a protein
3.2.3. What factors might affect the function of the protein?
Biological systems are dynamic and are capable of responding to environmental, developmental and metabolic changes.
• altering rate of gene expression
• altering rate of protein degradation altering the activity of the protein
non-covalent binding of other molecules availability of ligands reversible covalent modification irreversible covalent changes
■
■
■
ll*yJUjM|m f proteins and how to explore them - Lubomír Janda
3.2. The key questions about a protein
3.2.4. How does the protein interact with other molecules?
Prokaryotic cell has a protein concentration: > 200 mg/ml.
In erythrocytes the concentration was found to be: > 300 mg/ml. Mitochondrial matrix has a protein concentration:        > 500 mg/ml.
To address how proteins interact with ligands, we should aim to determine:
>the three-dimensional structure of the protein-ligand complex, >the site on the protein which interacts with the ligand and the molecular details of the interaction, >the number of ligands interacting with the protein (stoichiometry), and
>the strength of the interaction and the rate constant involved.
^g^^^M f proteins and how to explore them - Lubomír Janda
3.3. Assays for biological activity
Key concept:        Appreciating the range of assays for biological activity
measurements and their limitations
A specific assay is required for purification of a protein and to address key questions, relating its structure and function.
We must consider the care which must be exercised when interpreting assay data in general:
>ls the activity measurement due solely to the presence of the protein of interest? I
H—O
H H
n / O /O
° O O
enolase
0 H b
\   ,0 PGAM
0-\ H
1 O—H H
>ls the activity measurement proportional to the amount of protein present?
assay component limiting the activity measurement continuous or discontinuous assay
•
•
IMAMJUWm Iproteins and how to explore them - Lubomír Janda
3.3. Assays for biological activity
3.3.1. Catalytic proteins (enzymes)
Direct reaction:
EC 1.1.1.1. alcohol dehydrogenase absorbs at 340 nm
V V ethanol + NAD* _Z—>   acetaldehyde (ethanal) + NADH + H+
Coupled reaction:
EC 2.7.1.1. hexokinase ATP + D-glucose —ADP + D-Glucose 6-phosphate
EC 1.1.1.49. Glucose 6 - phosphate dehydrogenase absorbs at 340 nm
V v
D-glucose 6-phosphate + NADP+ —->>D-glucono-1,5-lactone 6-phosphate + NADPH + H+
'xplorethem- Lubomír Janda
3.3. Assays for biological activity 3.3.2. Binding proteins
Protein binding assays can be used to both identify ligands and to characterize the nature of the protein-ligand interaction. Two categories:
• those based on biophysical changes in protein or ligand upon complex formation
• those that employ direct quantification of free and bound ligands.
❖Spectroscopic method - absorbance, fluorescence, CD spectroscopy, NMR analysis ❖ Equilibrium dialysis and membrane filtration
❖Solid phase techniques - SPR (surface plasmon resonance), overlay assay, ELISA.
'xplorethem- Lubomír Janda
3.3. Assays for biological activity 3.3.3. Transport proteins
Transport protein assays are designed to measure the rate of transport of a ligand from one location to another. >ln a typical assay, a known amount of isotopically labelled ligand is added to the system and incubated for a fixed period of time.
3.3.4. Other types of proteins
How to test the biological function of proteins which cannot be measured using the more conventional assays?
❖GFP - green fluorescent protein, which naturally occurs in the jellyfish Aequorea victoria.
❖ YFP _-. ,.....
♦>/?FP B&P^S^sl     The diversitV of genetic mutations is
*CFP I P!ljJSjjP^     illustrated by this San Diego beach scene
W$£mm     drawn with living bacteria expressing 8
different colors of fluorescent proteins.
'xplorethem- Lubomír Janda
3.4. Purification of proteins
Key concepts:
Knowing the objectives of protein purification
Being aware of the experimental considerations required to
meet these objectives
❖ To retain maximum biological activity.
❖ To ensure the protein is indeed pure. kDa
❖ To maximize the amount of protein recovered. 97.6 -
66.2 —L
3.4.1. Wild-type proteins I The isolation of proteins from their natural source exploits their heterogeneous biochemical properties - molecular mass, pi, hydrophobicity, post-translational modifications, putative ligands.
2     3     4    5     6 7
42.7 —
31.0 —
21.5 —
3.4.2. Recombinant proteins
Protein purification has been simplified greatly by the advent of recombinant DNA technologies that enable high levels of protein expression - E. coli, Aspergillus nidulans, S. cerevisiae, P. pastoris, insect cells, mammalian cells, and transgenic plants and animals.
xplorethem- Lubomír Janda
3.5. Structure determination
Key concepts:
• Understanding the tools required to explore the different levels of protein structure
• Appreciating the importance of an integrated experimental approach to provide a more complete picture of protein structure
The ultimate aim of protein structure determination is to gather and interpret data to reveal the three-dimensional structure of the protein at an atomic level.
> X-ray crystallography
> High-resolution NMR spectroscopy
The nature of the post-translational modification can be determined by SDS-PAGE combined with specific removal of modifications and mass spectrometry to analyze the mass of peptides with modifications.
Purify peptides by chromatography
Peptide mixture applied to first analyser to separate peptides by mass/charge
Edman degradation of peptides to generate amino acid sequence
Individual peptides selected from first analyser are fragmented in a collision cell and mass (sequence) determined in the second analyser
Direct determination of the structure.
3.6. Factor affecting the activity of proteins
Key concepts:
■xplorethem- Lubomír Janda
• Defining the major factors which influence the activity of proteins
• Understanding how to monitor their effects on protein structure and function
3.6.1. pH and temperature
vity	* * s			
• l-H -4—' O o	*s			
	2        4 6	8 10 pH	12	14
10      20      30      40      50      60      70      80 90 Temperature (°C)
pH conditions that elicit the highest level of activity promote side chain side ionization states (pKa values of 4.0 and 10.5 are indicative of glutamic acid and lysine, respectively).
'xplorethem- Lubomír Janda
3.6. Factor affecting the activity of proteins 3.6.2. Inhibitor and activator molecules
Inhibitors and activators are important effectors of protein activity within the cell. W      ^ ^
Biochemists usually quantify the effects of inhibitors in terms of an inhibitor constant, K;, whereas pharmacologists quantify the effects of an inhibitor with the term IC50 (or l0 5), which is the concentration of inhibitor required to reduce the protein activity by
50%. (a)
Asp 29
Asp 25 Asp 25' Gly 27 V
C o''    OH (T'^o
Gly 48      =      Gly 48
= H H = N N =lle 50   He 50'
Gly 48'
Hydrophobic pocket -— ~~
\
Ser
195-CH2-0 I
H
CH,
O I
<s-CH
NH ri
(N-terminus) P3
I n\ i i-as* (Saquinavir)
P2
n-N
His
-N
57 H,
(C-terminus)
o- o
Asp' 102
o
HjN X>
Ro 31-8959
FDA Approved
Norvir (Ritonavir)
OH
Crbdven (Indinavir)
oh
ť
MK-630(L-735,524) H pH
O NH
Competitive inhibition and non-competitive
inhibition
^g^^^M f proteins and how to explore them - Lubomír Janda
3.6. Factor affecting the activity of proteins 3.6.3. Post-translational modifications
Post-translational modification is one of the major effectors of protein activity within the cell.
The complexity of higher organisms is due to the differential RNA processing and post-translational modification of gene products.
Examples of post-translational modifications
Modifications
Cysteine: disulphide bond formation
Lysine biotinylation
Serine phosphorylation
Threonine phosphorylation
Tyrosine phosphorylation
Addition of prosthetic group - thiamine
diphosphate
Proteolytic processing
Protein targetting (signal sequences)
Example Lysozyme
Acetyl CoA carboxylase Glycogen phosphorylase Cyclin-dependent kinase
Cortactin
Pyruvate dehydrogenase
Chymotrypsin Penicillin acylase
'xplorethem- Lubomír Janda
3.7. Interactions with other macromolecules
Key concepts:
• Appreciating the importance of protein-protein interactions in vivo
• Understanding the need to use appropriate experimental conditions to study these interactions
Whilst it is convenient to study isolated proteins in vitro, this does not reflect how they function in vivo.
it
The colocalization of vimentin dots and microtubules at the edges of spreading cells. (a) Confocal immunofluorescence localization of vimentin at the edge of a fixed cell 30-45 minutes after replating. (b) Microtubule localization in the same cell. (c) An overlay of images a and b. Yellow, the regions of colocalization between vimentin dots and microtubules. Bar, 2.5 mm.
IMljLUyiUW Iproteins and how to explore them - Lubomír Janda
3.8. Use of bioinformatics
Key concepts:       • Appreciating the range of databases and bioinformatic tools
available to assist protein characterization • Understanding the theoretical basis of the tools used to calculate properties of a protein from its sequence
The field of bioinformatics has harnessed the exponential growth in the amount of information relating to nucleotide sequences, protein sequences, and biomolecular structures.
3.8.1. Web resources and databases
Nucleotide sequences, amino acid sequences, and protein structures are collected within a number of web-based databases. More recently, there has been an effort to integrate data sets (e.g. linking individual nucleotide/protein sequences to related three-dimensional structures, metabolic pathway databases, enzyme databases, disease databases, organism-specific databases, two-dimensional gel databases, and associated references), allowing researchers to characterize more fully the structural and functional properties of proteins.
ixplore them - Lubomír Janda
3.8. Use of bioinformatics 3.8.2. Sequence analysis
Protein sequences, derived from experimental data or database entries, can generate a wealth of information that can assist protein characterization.
EBP - GAG EBP I AKAj 'EBP ■ EKGAG ^BPf PSH 'EBP/ PSKG L_
5SP
EBR
(a)
BR
LZ
LZ
A number of software packages which calculate the physico-chemical properties of proteins from their sequences are available on the web, e.g. ProtParam, available on the ExPASy server.
br
Tyr285
(b)
N
N
^^^^^^ |proteins and how to explore them - Lubomír Janda
3.8. Use of bioinformatics 3.8.2. Sequence analysis
It is possible to compute the absorption coefficient of a protein knowing the amino acid sequence and the molar absorption coefficients (E) of tyrosine, tryptophan, and disulphide bonds at A280 using the following equation:
EProt = NTyr x ETyr + NTrp x ETrp + NCys x ECys
where Nx is the number of amino acid X per polypeptide chain. The values of ETyr, ETrp and ECys are 1,490, 5,500 and 62.5 M-1cm-1, respectively.
Sample calculations of the absorption	coefficients for a range of			proteins.				
Protein	Molecular	Nyyr	NTrp	Ncys	EProt (calc) (1M)		EProt(calc)	Eprot (exp)
	mass (Da)						(1 mg/ml)	(1 mg/ml)
Insulin (bovine)	5,734	4	0	6	5,960	(6,335)	1.04 (1.10)	0.97
Lysozyme (hen)	14,314	3	6	8	37,470	(37,970)	2.62 (2.65)	2.63
Chymotrypsinogen (bovine)	25,666	4	8	10	49,960	(50,585)	1.95 (1.97)	1.98
Phosphoglycerate kinase (yeast)	44,607	7	2	1	21,430	(21,430)	0.48 (0.48)	0.49
Pyruvate kinase (rabbit muscle)	57,917	9	3	9	29,910	(30,410)	0.52 (0.53)	0.54
Serum albumin (bovine)	66,296	20	2	35	40,800	(42,925)	0.62 (0.65)	0.66
'xplorethem- Lubomír Janda
3.8. Use of bioinformatics
3.8.3. Sequence comparison
Whilst pairwise alignments provide a measure of the similarity of two sequences, the information arising from multiple sequence alignments, using more than two sequences from a protein family, can be used to:
>Suggest the function of unknown proteins: E-scores less than 0.2 are
indicative of homology and values greater than 1.0 indicate that the similarities are just as likely to have arisen by chance.
>Identify functionally and/or structurally important residues.
>Improve structure prediction tools.
>Detect and characterize evolutionary relationships.
Copper A
E. californica
N. madagascariensis
C. salei
P. interruptus
P. leniusculus
210
220
230
240
250 I
EYKLAYFREDIGVNAHHWHWHWYPSTYDPAFFGKVKDRKGELFYYMHQQMCARYDC EYKLAYYREDIGWAHHWHWHWYPSW
EYKLAYFREDVATOAHHWYWHWYPANWDESLTGKVKDRKGELFYYMHQQMSARYDC EQRVAYFGEDIGMNIHHVTWHMD F P FWWEDSY-GYHLDRKGEL F FWVHHQLTARFD F EQRGAYFGEDVGLNSHHVHWHMD F P FWWN----GAKIDRKGEL F FWAHHQLTARYDA
Copper B
californica
madagascariensis
salei
interruptus leniusculus
370 380 390 400 410
GYYGS LHNWGHVMMAYIHD PDGRFRETPGVMTDTATS LRD PIFYRYHRFIDNV Q FYGNLHNWGHVMMAYIHDPDGRFRETPGVMTDTATS LRD PIFYRFHRFIDNV G FYGS LHNWGHVMMARMHD PDARFQENPGVMSDTSTS LRD PIFYRWHRFVDNI QYYGS LHNTAHVMLGRQGD PHGKFNL P PGVMEHFETATRD PS F FRLHKYMDNI AYYGALHNQAHRVLGAQSDPKHKFNMPPGVMEHFETATRDPAFFRLHKYMDGI
xplorethem- Lubomír Janda
3.8. Use of bioinformatics
3.8.4. Structure comparisons
Protein structure comparisons are powerful tools in establishing structure, function, and evolutionary relationships, particularly when comparing distantly related proteins with low-level sequence identity.
>SCOP (Structural Classification of Proteins)
>CATH (Class Architecture Topology Homologous superfamily)
3.8.5. Predicting possible functions of proteins
The classical experimental approach to determining the function of a novel protein requires structure characterization, determination of factors influencing activity, and identification of ligands.
NetPhos http://www.cbs.dtu.dk/services/NetPhos NetNGlyc http://www.cbs.dtu.dk.services/NetNGlyc CASTp http://www.sts.bioengr.uic.edu/castp/ PONDR http://www.pondr.com
I. The molecular principles for understanding proteins - Lubomír Janda
Please solve the problem.
Question 5: What is the name of this method?
I can measure protein-protein interaction, but predominantly I
determine if a protein is folded. 5 points
I work in far UV. 3 points
I am able to determine secondary but not tertiary structure. 2 points
I am a spectroscopic method. 1 point
CD spectroscopy.
I. The molecular principles for understa
Please solve the problem.
Question 6: What is the name of this post-translational modification?
I modify arginine, lysine and five other amino acids.
5 points
I am detectable by MALDI and western blot (but not for all kinds of modified amino acids).
3 points
Very often I need ATP for modification.
2 points
Serine and threonine are the most often used amino acids for this modification.
1 point
Phosphorylation.