Day 1 (3h)Introduction to protein chemistryStrategies used by enzymes to accelerate reaction ratesDay 2 (3h)Protein stability elucidated and enhanced via protein engineeringProtein folding & unfolding probed via protein engineeringDay 3 (2.5h)Protein folding and unfolding probed via protein engineering (continued)
Day 4 (3h)The combined power of in vitro chemical modification and paper-supported chromatography as a probe of structure and functionDay 5 (3h)The combined power of in vitro chemical modification and paper-supported chromatography as a probe of structure and function (continued)In vitro manipulation of protein monomers or their environment to enhance performanceDay 6 (2.5h)In vitro manipulation of protein monomers or their environment to enhance performance (continued)A closer look at optimizing protein function in non-aqueous environmentsProtein purification and related analytical methodsShort examination scheduling
References Relevant to this Material1. Lundblad, R.L., Techniques in protein modification, CRC Press, 1995, 0-8493-2606-02. Wong, S.S., Chemistry of protein conjugation and crosslinking, CRC Press, 1991, 0-8493-5886-83. Nagradova, N.K., Lavrik, O.I., Kurganov, B.I., Chemical Modification of Enzymes, Nova Science, Inc., 1995, 1-5607-2238-X4. Brown, W.E., Howard, G.C., Practical Methods in Advanced Protein Chemistry, CRC Press, 2000, 0-8493-9453-85. J.M.Walker, Ed., Protein protocols on CD-ROM, Humana Press, 1998, 0-89603-514-X6. Darbre, A., Practical Protein Chemistry: A Handbook, John Wiley and Sons, 1986. 7. Eyzaguirre, J., Chemical Modification of Enzymes: Active Site Studies, Prentice Hall, 1987, 0-47020-763-98. Methods in Enzymology Series, Academic Press,Vols.11, 25-27, 47-49, 61, 91, 117, 130, 131, 135-137.9. Glazer, A.N, Delange, R.J., Sigman, D.S., Chemical Modification of Proteins, Elsevier Science, 1975, 0-44410-811-410. Feeney, R.E., Whitaker, J.R., American Chemical Society Advances in Chemistry Ser. (No. 160) - Food Proteins: Improvement Through Chemical & Enzymatic Modification, Books on Demand, 0-31710-649-X11. Feeney, R.E., Whitaker, J.R., Modification of Proteins: Food, Nutritional & Pharmacological Aspects, 1982, 0841206104 Advances in Chemistry Ser. (No. 198) American Chemical Society12. Feeney, R.E., Whitaker, J.R., Protein Tailoring & Reagents for Food & Medical Uses, Marcel Dekker Incorporated, 1986, 0-82477-616-X.13. Bailey, J. L., Techniques in Protein Chemistry, Elsevier Publishing Company, 1962, Lib. Congress 62-19691.14. Lundblad, R. L., Chemical Reagents for Protein Modification, 2nd Ed., CRC Press, 1991, 0-8493-5097-2.15. Walsh, G., Headon, D.R., Protein Biotechnology, John Wiley and Sons, 1994, 0-471-94393-2.16. Mean, G., Feeney, R.E., Chemical Modification of Proteins, Holden Day, Inc., 1971, Lib. Congress 74-140785.17. McGrath, K., Kaplan, D., Protein-Based Materials, Birkhauser, 1996, 0-8176-3848.18. Koskinen, A.M.P. and Klibanov, A.M., Enzymatic Reactions in Organic Media, Blackie Academic and Professional, 1996, 0-7514-0259-1.19. Suckling, C.J., Gibson, C.L., Pitt, A.R., Enzyme Chemistry: Impact and Applications, Blackie Academic and Professional, 1998, 0-7514-0362-8.20. Magdassi, S., Surface Activity of Proteins: Chemical and Physicochemical Modifications, Marcel Dekker, Inc., 1996, 0-8247-9532-6. 21. Rawn, J. D., Proteins, Energy and Metabolism, Neil Patterson Publishers, 1989, 0-89278-404-0.22. Fersht, A.R., Structure and Mechanism in Protein Science, W.H. Freeman and Company, 1999, 0-7167-3268-8.23. Oxender, D.L., Fox, F.C., Protein Engineering, Alan R. Liss, Inc., 1987, 0-8451-4300-X.24. Crieghton, T.E., Protein Structure: A Practical Approach, 2nd Edition, Oxford University Press, 1997, 0-19-963618-4.25. Crieghton, T.E., Proteins: Structures and Molecular Properties, 2nd Edition, W.H. Freeman and Company, 1993, 0-7167-2317-4.26. Wüthrich, K., NMR of Proteins and Nucleic Acids, John Wiley and Sons, 1986, 0-471-82893-9.27. Journals focused on the subject of protein chemistry: Journal of Protein Chemistry; Protein Science; Biochemistry; Journal of Biological Chemistry; Biomacromolecules28. Catalogues! Promega Protein Guide: Tips and Techniques; Pierce Products; Biorad Life Science Research Products
The way to operate -True or false? A student of protein chemistry…
1. buys the best possible instrument and then tries to force the problems of protein chemistry to suit the use of the machine.
2. works in a problem-oriented manner in which experience and knowledge are adopted to accommodate available machines.
3. relies first on imagination, then knowledge, then machines (Consider the contrast between H. Noyrath vrs. B. Hartley). What was one of Einstein's quotations?
4. believes that protein investigation is as simple and amusing as watching Indiana Jones running away from a band of sword-wielding bandits (The Okum's Razor argument).
5. should use all his/her time reading primary references and never use his/her own ideas, intuitions or beliefs
To study proteins is to study diversity! i.e., diversity of structure, function, chemistry, analysis, etc.To emphasize the scope of diversity, let us focus on structural diversity...
• Structure is a shape, sequence, order, orientation, configuration, etc. of an atom or molecule.
• Eg. The electronic structure of carbon is 1s22s22p2.
• Eg. CCl4 has a tetrahedral shape.
• Eg. The primary structure of insulin begins with:
• Eg. The tertiary structure of cytochrome c is globular.
Cl
CCl
ClCl
Diversity of structure: Static vs. dynamic• Structure (an other traits) may be static (fixed)
or dynamic (changing) over time.
• The time frame of structural change may be very long (the half life of 238U is 4.5x109 years) or very brief (a 10 fs chemical interaction)
We must characterize structural diversity to understand proteins
• Question 1: Can you see the 3-D shape of myoglobin with your eyes?
• Question 2: Can you live 4.5 billion years to see ½ of the 238U decay?
• Question 3: Can you react quickly enough to measure a chemical interaction?
The answer to each is NO! >> So we must use machines...Why? Reason one: We are limited by resolving power…If our information carrier is visible light, we are limited to an approximate resolution of 0.2m. Details smaller than 0.2m are lost.
Look Wilma, whata nice smoothsurface!! Light
you see
actual surface
500nm
Reason two: The event is often faster than the speed of the measurement…You obtain a blurred average.
• Eg. Try photographing chicks in a bowl
a.
b.
c.
Nature features an invisible world of details & diversity. Instruments
allow us to see these details…
Balloons pierced with a bullet
Dynamic changes along an aqueous surface: Droplets captured in motion
A Look at Diverse Protein Structures1. Protein structure is not rigid!2. Protein structure bears many aspects
Proteins are generally made from20 types of amino acids, which are:
The peptide bond (like formamide, below & above right) is stabilized by resonance: 60% amide and 40% hydroxyimine character
What else do we observe?All 6 atoms lie in the same plane, i.e., the peptide bond is planar.-electrons are distributed over the C-O and C-N bonds.The C-N bond is 10% shorter than a normal C-N bond.The peptide bond is trans.
The peptide bond has a permanent dipole ( = 3.7D)
H2N
O
OH
R H
O
CH N
H
H
O
CH N
H
H
O
CH N
H
H
O
CH N
H
H
-
+
O
CH N
H
H
Linking the building blocks: Stereoelectronic properties of the peptide bond
Diversity of Protein Structural Elements: Basic Structural Hiearchy
1. Primary structure: The exact specification of atomic composition and the chemical bonds connecting those atoms, including stereochemistry. (i.e., L-amino acid sequence, disulphide bridges, other postsynthetic modifications, e.g., insulin A & B chains; chymotrypsin A, B & C chains)
2. Secondary structure: Regular arrangment of the backbone polypeptide without reference to side-chain types or conformation. The secondary structure is usually held by H-bonds (e.g., helix, sheets, random coils)
3. Tertiary structure: 3-D arrangement of polypeptide backbone and amino acid side-chains (e.g., lysozyme). Domain structure: compactly folded units
4. Quaternary structure: Noncovalent association of folded protein subunits (e.g., haemoglobin)
>> Most enzymes: Globular shape, with hydrophobic interior & hydrophilic exterior
So are protein physical traits diverse?Compare keratin versus collagen versus albumin (all from the same 20 amino acid types)
How do we draw protein 3-D structure?Space filling, stick/skeletal (backbone only, sometimes labeled) and ribbon/ schematic models:Show helices (coils), strands (arrows) & random structure
1. Enzymatic proteins: Proteinases, lipases, epimerases, kinases, polymerases...Proteins, which transduce chemical to chemical signals
Note - Proteins are not just enzymes – antibodies, connective tissue (collagen), fluid media, transportation vehicles (Haemoglobin, serum albumin), buffers (serum albumin), signal transducers (rhodopsin), etc.
2. Cytoskeleton – Actin (muscle), Tubulin (cell motility), Intermediate filaments (mechanical protection near membranes and cells subjected to stresses), Spectrin (cytoskeletal protein, particularly found in erythrocytes)
3. Human Plasma – Albumin (osmotic regulation, buffering, transport), -Globulins (transport),‑Globulins (iron transport {transferrin}, histocompatibility antigen {-Microglobulin}), ‑Globulins Antibodies, Fibrinogen (proteolised by thrombin to form fibrin clot), Complement A (11 different protein types working to complement the immune system)
4. Extracellular Matrix – Glycosaminoglycans (hydrated gels), Proteoglycans (long glycosaminoglycans linked to a core protein), Collagen (extracellular matrix; Type I-III tissue supporting fibrils, Type IV laminar network), Elastin (random coil protein gives elasticity to tissues), Fibronectin (cell adhesion), Integrin (integral membrane proteins, also adhesion of cells to extracellular matrix)
5. Digestive Enzymes of Digestive Tract – Amylase (starch to disaccharides), Pepsin, Trypsin, Chymotrypsin (proteins to large peptides), Peptidases (large peptides to small peptides; small peptides to amino acids), Lipases (lipids to fatty acids and glycerol), Ribonuclease (RNA into oligonucleotides), Disaccharidases (disaccharides to monosaccharides)
6. Cytosol Proteins (300-1000 types) – Synthesis of most small molecules, proteins, carbohydrates & lipids of cell
7. Nuclear Proteins – Histones (5, complex to DNA to make chromosomes), Nucleic Acid polymerising enzymes (5-10, used in DNA and RNA synthesis)
8. Mitochondrial & Chloroplast Proteins (300-1000) – Energy production from metabolites or light
9. Endoplasmic Reticulum & Golgi Apparatus Proteins (50-200) - Protein modification, oligosaccharide and lipid synthesis
Diversity of protein physico-chemical traits:>> Diversity among proteins is high but not “random”>> Structure/construction and function are related>> Some 1˚, 2˚ & 3˚ features are retained among proteins of similar function
• Global shape and morphology: Round, tight, loose, fibrous, skinny, crystalline
• Local function-related structures: Active site, receptor site, allosteric regions, catalytic residues
• Solubility: Highly variable• pI: Highly variable• pH stability: Highly variable• Tolerance to other environmental factors: Highly variable
Understanding protein structure, protein function, and their relationships are the central problems of protein science. The rules that govern structure-function relationships are simple
Nature is presumed to provide simple solutions.The challenge is to ask the right questions.
What is protein chemistry?Area of science related to:1. Obtaining/purifying protein,2. Investigating protein structure & function, and3. Controlling and engineering proteins
Protein chemistry contributes to the following subject areas:1. Biochemistry, Biotechniques & Bioengineering2. Analytical Chemistry and Spectroscopy 3. Surface and Colloid Science4. Clinical Chemistry5. Polymer Science6. Medicinal and Pharmaceutical Chemistry7. Organic Chemistry
Why is protein chemistry highly interdisciplinary? Protein chemistry has developed together with analytical methods such as sequencing, X–ray, NMR structure determination and site–directed mutagenesis.
Protein chemistry is useful to whom?Researchers, professionals and students in various areas of specialization:
Protein chemists, molecular biologists, materials scientists, enzymologists, clinicians, analytical chemists, biophysicists and industrial scientists
E.g.: Protein chemists help X-ray crystallographers & genetic engineers:
Protein chemist X-ray crystallographer•Purifies 1g protein •Attempts crystallization•Chemically modifies to aid crystallization •Obtains diffraction patterns
or to form heavy atom derivatives, •Uses heavy atomwhich aid the phase problem derivatives to solve
structures at 3-4Ǻ
Protein chemist Genetic engineer•Purifies 1mg protein •Synthesizes oligonucleotides•Sequences peptides •Screens the gene-bank•Compares peptides & sequence codes •Sequences DNA of insects•Probes posttrans processing by FabMS •Constructs expression vector•Prepares antibodies •Screens using western blots•Develops protocols to purify•Compares properties of wild-type & mutant
One of the most common and often ambitious experiments in protein chemistry is the structure-function study
I.e., How does structure perturb function? How does function define structure?e.g. Consider the pKa of active-site thiols in cysteine proteases
Structure-function experiments: probe the interdependence of structure & function in proteins; generally reflect elements of both structure & function:
Examples along this continuum:1. One end – X-ray; Emphasizes analysis of structure2. Middle ground - pH titration of protein groups, showing hysteresis; Reflects
elements of structure and function substantially3. 2nd end – Bioassay; Weighted toward functional assessment
How do S-F studies work? How would you learn about a system that you cannot see? You interact with the system & note the consequences.
If you walk into an icicle, your “initial state” becomes altered. Your “final state” indicates something sharp. Thus, any change in you during the interaction can probe structure.
Physical methods:Generally nonintrusive, require more protein, performed in water or water-free state.
Chemical methods: Generally intrusive, may be destructive. Potentially very sensitive, performed in water, organic solvent or dry state.
Some physical methods to assess structure & functionDiffraction: X-ray, neutron diffraction Spectroscopies: Infrared, ultraviolet, Raman, optical rotary dispersion, circular dichroism, NMR, esr (principle is to infer structure by perturbing light)Thermal analysis: MicrocalorimetrySpectrometry: Mass analysisIn silicio: Computer modelingOther: Electrophoresis, hydrodynamic techniques, chromatographies
Typical outputs:Composition and secondary structure, quantification, folding energies (spectroscopies)Identifying/purifying biological materials by exploiting adsorption, isoelectric point, size/mass, affinity, etc. (chromatography & electrophoresis)Unfolding enthalpies of protein (microcalorimetry)3-D "Static" structure (X-ray, neutron diffraction) 3-D dynamic structure, kinetic folding, association constants, etc. (NMR)Local environment of coordinated metal ions (Mossbauer spec.)
Some chemical methods to assess structure & function
Titration studies: nature & number of ionizable groups.
Proteolysis in vitro: Limited proteolysis to elucidate the structural motifs of protein.
Kinetic studies: Applied to any protein, but mainly enzymes.
Classic chemical modification: Used to identify important residues. E.g., acetylation of chymotrypsinogen vs. chymotrypsin showed the role of Ile16.
Competitive labeling: Very sensitive and powerful. Reports on individual residue pKa values, structural information such as accessibility of groups, and stereoelectronic perturbations of a group. E.g., the surface reactivities and pKa values of the 12S subunit of a native 50-protein ribosome complex was characterized.
Site-directed mutagenesis: Reports on the role of specific groups. All groups can be investigated. SDM is complementary to chemical modification. Using SDM, the role of active site groups of barnase on stability and catalysis were quantified.
In a typical study of a poorly characterized protein...1. Physical & chemical methods to purify protein and to
analyze protein structure (some early examples): Dialysis and gel filtration, column chromatography of proteins Zone electrophoresis of proteins Estimation of protein and amino acid content Paper chromatography of amino acids and peptides High-V paper electrophoresis of amino acids and peptides Ion-exchange chromatography of amino acids and peptides Disulphide bond mapping Urea unfolding and stability tests Selective cleavage of peptide chains N-terminal sequence determination C-terminal sequence determination X-ray and later CD and NMR structures (with/without incipients)
2. Physical & chemical methods to analyze protein function Bioassays (enzyme kinetics, receptor-hormone, protein adsorption, cell
adhesion to protein layers) Comparative studies give insight to the S-F relationship!
An enzyme will not “reduce” the activation energy of a pathway! Like all catalysts, an enzyme will permit the reactants to follow an alternate, low–energy pathway.
CO → CO2
The alternative pathway reflects a new mechanism. Here, it proceeds via 2 or more intermediate steps.
2CO + O2
2CO2
Enzymes use similar tricks as non-enzymatic catalysts: E.g., bases, acids, metal surfaces, etc., PLUS some extra tricks, which are unique to its structure
overly simplified more correct
Enzymes in the Protein Family: Properties1. Monomeric or oligomeric or exist as part of a multienzyme complex2. Often require non-protein components (co-factors) for catalytic activity – activator eg. metal ion, co-enzyme, prosthetic group3. Efficient catalysts4. High Specificity5. High Stereospecificity6. Very sensitive to pH, temperature, dielectric (salts, solvent)
Industrial Uses of EnzymesTextile Industry – Cellulase for cottonDetergent Industry – Lipases and Carbohydrases for stainsFood Industry – Isomerase of glucose to fructose; lactase for lactose intolerant peopleOrganic Synthesis – Penicillin acylase; amino acid synthesis
1a. Oxidoreductases (all redox reactions) eg. Alcohol to aldehyde – catalysed by NAD oxidoreductase, aka alcohol dehydrogenase (plus NAD+ cofactor NADH)
1b. Transferases (transfer of methyl groups, glycosyl groups, phosphate groups, etc.) eg. creatine to phosphocreatine – catalysed by creatine phosphotransferase aka creatine kinase (plus ATP ADP)
1c. Hydrolases (hydrolytic cleavage of ester, amide and glycoside bonds by insertion of water) eg. glucose-6-phosphate to glucose plus phosphate – catalysed by glucose-6-phosphate phosphohydrolase, aka glucose-6-phosphatase
1d. Lyases (cleavage of bonds by mechanisms other than hydrolysis or oxidation; carbon-carbon lyases, carbon-oxygen lyases, carbon-sulfur lyases) eg. L-histidine to histamine plus carbon dioxide – catalysed by histidine decarboxylase
1e. Isomerases (racemizations, epimerizations, cis-trans isomerization) eg. D-ribulose-5-phosphate to D-xylulose-5-phosphate – catalysed by D-ribulose-5-phosphate 3-epimerase aka phosphoribuloepimerase
1f. Ligases (condensation of two different molecules at a new C-O or C-S bond, but coupled to the breaking of ATP) eg. L-tyrosine plus tRNA plus ATP to give L-tyrosyl-tRNA plus pyrophosphate – catalysed by L-tyrosyl-tRNA ligase aka lyrosyl-tRNA synthetase
Means of Quantification: Measure a change of S→P over time, many techniques
Rate measurements: Rate of formation of product or removal of reactant as amount/time e.g., M/s, mole/s, vol/s, g/ml/s, etc.
Try to measure these slopes!
Q: What do we call these measurements?A: Initial rates! Acquire data within a few minutes & within 1-5 mole% S conversion.
Q: Why measure initial rates?
Forward rate, S → P, has no interference:
1. No product inhibition is possible;
2. No reverse reaction is possible;
3. Enzyme instability is less of a concern; and
4. Be safe - Enzyme reaction models are more complex than ordinary kinetics: Invite errors when extrapolating non-initial rate data
Let us examine how the above theory has originated...
Historically Early studies (1895-1913) on the rates of the enzyme-catalyzed
reactions gave the following observations: 1. At constant substrate concentration, the rate of reaction was directly
proportional to the enzyme concentration. 2. At constant enzyme concentration:
a. The reaction rate was independent of substrate concentration. b. The reaction rate was directly proportional to the substrate concentration. c. The reaction rate was fractional with respect to substrate concentration,
with a value between zero and one.
In 1913, Michaelis & Menten proposed a scheme to account for the above observations: Enzyme only acts upon bound substrate, i.e., E & S must initially form a
complex, held together by physical forces. Assumptions:
E and S are equilibrated with ES, i.e., kcat << k-1
Breakdown of ES is 1st order so rate [ES] i.e. rate = kcat[ES]
Q: What are enzyme assays & how are they performed? SP
The Assay Any method that detects a change of physical property versus time:
Manometry, polarimetry, viscometry, NMR, MS, spectrophotometry, spectrofluoromethry and pH-stat. What is one pre-condition? The physical property should vary in proportion to S or P.
Direct assays Alcohol dehydrogenase can be monitored as a function of NADH
formation. NADH is strongly absorbent at 340nm. Is a buffer used? Hydrolases can be monitored as a function of proton formation (standard
ester cleavage). Is a buffer used here? Coupled Assays
If S & P are similar they cannot be directly used to assay. To get around this problem, a more distinguishable end product is made.
Target: With alanine aminotransferase; alanine + -ketoglutarate → pyruvate + glutamate. Using pyruvate dehydrogenase; pyruvate + NADH → lactate + NAD+ (NAD+ absorbs at 260nm). The coupled reaction should be faster than the principle reaction. WHY??
Sampling AssaysS or P is withdrawn at specific time intervals & quantified, e.g., by colorimetry or radioisotopy.
Experimental Target of a M-M assayTo measure 3 parameters: KM, kcat & kcat/KM. Do these carry a physical meaning?
Q: How do we carry out a typical M-M experiment? A: Measure the initial rates as follows:
With substrate concentration at least 200-500x greater than total enzyme concentration , measure KM& kcat directly. Carry out these measurements at 3-4 different pH values. Measure the specificity (kcat/KM) directly at many pH values, using 0.1pH unit intervals (construct a pH activity curve); In choosing your parameters, S must be at least 20x less than KM. Why? What is the significance of a pH activity curve?Repeat any of the above experiments in the presence of inhibitors, different S, activators, different environments, etc.
Q: How does your experimental scenario compare to the true situation in biological systems? Is there a biological relevance? Why do we conduct experiments in this way? rate = (kcat[E]o[S]o)/(KM+[S]o)
Hanes-Wolf plot
Michaelis-Menten kinetics
Conc of S (mM)
Initial rate (mM/s)
= KM/(kcat[E]o)
A closer look at kinetic scenarios: Probing ionizable groups, which are important for binding and/or catalysis?
(6)
Sample math treatment for 3 (apparent) ionizable groups that are important for binding and/or catalysis
= 0
0
20000
40000
60000
80000
100000
120000
140000
3 4 5 6 7 8 9
kcat/Km
pH
The pH activity profiles of cathepsin B. The substrates are acetyl-Arg-Arg-Arg-AMC (+), acetyl-Val-Arg-Arg-AMC (◊) and benzyloxycarbonyl-Arg-Arg-AMC (―).
Real example!
Thermokinetic background related to protein analysisThermodynamics: G, H, S, equilibrium constant Keq
Please delinate the relative importance of thermodynamic and kinetic contributions in the following scenarios
1. The right reaction releases energy faster than the left reaction. Q: Which videoclip shows the more exothermic reaction? A: Inconclusive! We cannot compare the molar enthalpy change from the videos.
2. True or false? All exothermic reactions are thermodynamically spontaneous and all endothermic reactions are thermodynamically non-spontaneous.A: False!
3. True or false? All thermodynamically spontaneous reactions yield a reaction & all thermodynamically non-spontaneous reactions fail. A: False!
Put away your weapons of mass destruction...
4. The thermite reaction is highly exothermic, H <<<0, the entropy change, S, is relatively unimportant, and the Gibbs energy change is highly negative, G <<<0. The reaction is thermodynamically spontaneous.Q: Why must you add a fuse to start the reaction?
5. The process H2O(s)→H2O(l) is highly endothermic (H>>0) Below is the evidence. Explain.
Time = 0min
Time = 60min
6. The reactant, nitrogen triiodide-NH3, sits at a high Gibbs energy level. Its products rest at a much lower energy state.
You must apply a physical shock before Nitrogen triiodide-NH3 explodes. Why?
NI3.NH3(crystal) → NH3(g) + ½ N2(g) + 3/2I2(g)
7. Liquid nitrogen evaporates. The process is thermodynamically spontaneous, endothermic & proceeds quickly. Q: How might you explain these comments?
N2(l) → N2(g)
8. The dissolution of ammonium sulfate in water is endothermic and readily proceeds under ambient conditions. Explain.
(NH4)2SO4 + bulk H2Os → 2NH4+
(aq) + SO42-
(aq) + a few less bulk H2Os
Why all the confusion?? Reason 1: Many terms and reactivity modelsReason 2: Misleading termsReason 3: Separate GS & TS concepts in chemical processes
At equilibrium, Ssys + Ssur is max.
Gsys Asys
UH
U PV
TSTS
kinetic Emic
potential Emic
rot vibrtransla
intermol. inter.
intramol. inter.
Let us progress until wearrive at the commonmodel to understandproteins...
Early measurements of U examined the link between enthalpy changes (H ≈ differences of bond energies) and reactivity
Why shouldn’t you predict reactivity using H?
A: H reports on the initial & final Ground States (GS) but not on the pathway (mechanism)
(There are other reasons too)
A
B
H
B
A
Reaction coordinate Reaction coordinate
H < 0 H > 0
Hfinal
HinitialExothermic Endothermic
What are some disadvantages of: the collision model? using Ea to predict reactivity?
Collision model: A kinetic view. Consider a potential barrier, Ea, between A & B. Rate const is kA→B = Ae-Ea/RT. (Later, A = Z)
A
B
U
Reactants collide with speed & good orientation.
In non-gases: PotE ≈ U, as (PotEf - PotEi) ≈ Uf - Ui
U ≈ H, as H = U + (PV)←very small
preexponential steric
Gibbs energy change: A way to explicitly incorporate entropy, S, to account for solvent effects, etc.
H-TSsys = Gsys
bomb calorimeter
most reactions
At equilibrium, Ssys + Ssur is max.
Gsys Asys
UH
U PV
TSTS
What is misleading by the term spontaneous?
G > 0G < 0
Spontaneity says nothing about energy barriers or chemical rates
Reaction CoordinateReactants Products
GibbsEnergy
Both processes arethermodynamicallyspontaneous
One is kinetically permitted,giving an observable rate,
and one is kinetically prohibitedby a high energy barrier
Reaction CoordinateReactants Products
Both processes arethermodynamicallynon-spontaneous
One is kinetically permitted,giving an observable rate,
and one is kinetically prohibitedby a high energy barrier, so we have 100% reactants
G≠
G
G≠
G
Transition State Theory: A kinetic element completes the Gibbs view.
State B
State A
GState A
State B
G‡
G‡
Reaction Coordinate
G
Changes of any state function are independent of path
State function
Not a state function
Problems with TS theory?
Reactant (A) proceeds through a high-energy transition state or activated complex to become product (B).
What is the weakness of predicting reactivity using only quantum tunneling?
Reaction Coordinate
Reactants Products
GibbsEnergy
Classic kineticbehavior
Tunneling (a 5A wavelength decays exponentially as it penetrates the barrier. If the barrier is not too long, R can reach the product sideof the hill without completely decaying away (i.e.,emerges on the product side with a non-zeroprobability density)
R,eg. +H P
O O-
HThe rate of proton transferoften has a significant tunneling component
5A
H
5A
Quantum tunneling kinetic view: The e- probability distribution of every particle is derived from a wave function
The above terms are related to large populationsCannot use TS theory to calculate the activity of “one” molecule or small groups of molecules, such as membrane proteins
Note: Reactions do tunnel, collision theory could apply
Let us examine a typical enzyme reaction...
G≠, H≠, S≠
G, H, SGA→B = HA→B – TSA→B
kA→B = (kBT/h) x e-G‡/RT
Keq = [B]b/[A]a = e-G/RT
To summarize: In protein systems, we assess thermodynamic & kinetic behavior in terms of G & TS theory (less use of Z or tunneling arguments)
G G
Uncatalyzed reaction Enzyme reaction
Reaction coordinate Reaction coordinate
S X+ P
E + S ES ES E + P=
ES≠
Enzymes lower G‡ (i.e., G‡ - GGS) in comparison to the uncatalyzed reaction
We shall simplify the notation even more...
Overall rxn is diffusion-controlled or rxn-controlled
Microscopic steps may be grouped intoPhysical binding (1st step; E + S → ES), and Chemical catalysis (2nd step; ES → ES‡ → E + P)
Dissect G‡ into enthalpic (H‡) and entropic (S‡) components:
k = (kBT/h) x e-G‡/RT can be written as k = (kBT/h) x eS‡/R x e-H‡/RT
where S‡ is the entropy of activation, Stransition state – Sground state and
H‡ is the enthalpy of activation, Htransition state – Hground state.
activationenergy offorwardprocess
GibbsEnergy
ProductsReactants
Reaction Coordinate
Ggs
GQ: How might you predict the free energy of activation, G‡?
Answer: Assess the enthalpic & entropic differences between:
1. reactant (ES at ground state) &
2: the activated complex (ES≠, at the transition state position).
A closer look at changing the position of G
E + P ESE + Sk1
k-1
k2
H
H
H
Reaction Coordinate
activationenthalpy oftwo forwardprocesses
HA
B
The enthalpy of activation , H‡, is always positive because bonds are being broken.
The entropy of activation, S‡, may or may not be favorable. Can you think of some examples?
Both parameters contribute to rate according to k = (kBT/h) x eS‡/R x e-H‡/RT
S
S
S
Reaction Coordinate
activationentropy oftwo forwardprocesses
S
A
Q: How might substrate-surrounding interactions affect the position of H?
(Hint: In solutions & solids, H ≈ U)
Reaction Coordinate
Polar solvent Nonpolar solvent
Enthalpy, H
Oil
Water
Q: If you ignore any entropic contribution, how might a change of H affect the Gibbs free energy position in solutions & solids?
Water
Oil
Gibbs Energy, G
Nonpolar solventPolar solvent
Reaction Coordinate
Reaction Coordinate
Reactants Products
GibbsEnergy
Equilibriumposition, before & after
Increasing theconcentration ofreactant
Q: What happens if you increase the chemical potential (i.e., the potential to do work) of a reactant?
Answer: Reaction is more spontaneous; equilibrium is even closer to the product side; transition state is reached earlier; activation energy is smaller; forward rate is greater
Q: Which principles do enzymes exploit to lower the position of the TS (& how)?
1. General acid catalysis, general base catalysis, electrostatic catalysis and electrophilic catalysis. All modes could stabilise charge accumulation in proceeding
2. Covalent or nucleophilic catalysis. A covalent activated intermediate is formed, e.g., a ping-pong mechanism. The high-energy mechanism is broken into energetically less-demanding steps.
The enzyme environment enhances the reactivity of nucleophiles such as serine hydroxyl groups, cysteine thiol groups, etc.
N
O
H
OH
O
HN
H
H2O
N
O
H
O-Enz
HN
H
O
O
Enz
H2O
Enz O-
OH
O
G
Cys
S-
HNNH
His
+
Cys
SH
(aq)
pKa = 9pKa = 3
4. Pre-reduction of ground-state entropies.The basis of this strategy is to minimize the ground state freedom of the ES complex during the chemical transformation phase of a reaction. In this way, the ascent to the TS will not require a major loss of freedom.
Strategy 1. Orbital steering.
Strategy 2. Decrease the number of reaction participants in the chemical transformation phase.
N
O
HAA
N
O
H
N
O
H
A
versus
(aq)
LSS≠ << RSS≠
+ versus +
EnzEnz
≠≠
CH3CH2O- ICH2CH3 versus+
O-
I
Enz
5. Formation of low-barrier H-bonds.A normal H-bond in the GS may become a low-barier H-bond in the TS if the pKa value of the enzyme group & the activated complex (as ES≠) are matched.
1 normal H-bond ≈1-5 kcal/mole1 low-barrier H-bond ≈ 25-40kcal/mole
ONH
H3C
H Enz-O
S
GSTS
S
pKa = 10
pKa = 16
O EnzH
H
H O
+N
H3C
pKa = 10
6. Binding energy considerations.Enzyme binding groups interact non-covalently with substrate at all points along the reaction coordinate. The energy term H is variable along the reaction coordinate!
E.g. 1, GS shape of S perfectly matches enzyme site
E.g. 2, TS shape of S is much more complementary to enzyme than GS shape of S (for enzymes that behave according to the TS stabilization model of catalysis).
10 goodcontacts
+
TS??
ES
E + SG
+
2 goodcontacts
10 goodcontacts
ESE + S
G
ES=
E + P
Conclusion: Ground-state binding shouldn’t be “extremely specific”, as is often assumed
Shape of S transforms along the reaction coordinate
GS TS
EnzS
SEnz
GS
E + P
=ES
G
E + SES
+
2 goodcontacts
3 goodcontacts
5 goodcontacts
8 goodcontacts
10 goodcontacts
6 goodcontacts
TS reached
In a well-evolved enzyme-substrate interaction, we see an increase of binding energy stabilization in proceeding to the TS
SEnz
Every “good” interaction lowers the position of H, etc.
10 good contacts
2
3
6
8
Q: Can you see evidence of binding energy participation in the TS of amide bond hydrolysis?Hint: Look at the changes of KM & kcat
ES binding energies can be grouped into three cases:
Case 1 (not shown here): ES has very strong GS binding (shape complementarity is exceptionally good in the ground state). Examples: HormonesCase 2: ES has poor GS binding and strong TS binding. Examples include carbonic anhydrase, acetylcholine esterase and catalase. Case 3: Modest GS binding and modest TS binding. Examples – Metabolic enzymes
Protein engineering has been used to investigate structure-activity & molecular recognition relationships to to make better protein productsQ: Why does Mankind wish to use proteins? Proteins accelerate chemical reactions Proteins form commercial products & improve other product properties Proteins enable novel processes
Some typical industrial applications: Bioreactors Textile treatment Medicinal and organic syntheses Protein drugs & drug delivery Biosensors Bioremediation Food preparation industries
Problems? Industrial constraints are often too demanding for native proteins. Consequences: Poor biological activity, short lifespan, limited reaction parameters, etc.
Q: What can we accomplish by using protein engineering? improve existing pharmaceutical proteins create superior high-value proteins with improved half-life create new proteins and pioneer new therapies improve desirable biological activities alter receptor specificity and binding activity reduce harmful side-effects and toxicities.
Barnase: Superimposed Xray/NMR, schematic and ribbon sketch
Interacting residues are observed
Q: Can mutations probe the stability of the folded state?
A: All residue interactions contribute to protein stability. By mutating 1 residue of an interacting pair, the Gibbs contribution of that pair to protein stability is assessed.
Eu, E'u
Ef
E'f
wildtype
mutant E'i
Ei
E'
E
G
Reaction Coordinate
=
=
How might you measure the thermodynamic unfolding/folding energy change of barnase?
In principle, all interactions contribute to protein stability. H2OGUnfolding is the free energy change (calculated), which accompanies barnaseFolded → barnaseUnfolded in water. Here are some examples:
Destroying parts of buried or solvent-accessible hydrophobic residues
Mutant # methyl(ene) GU
groups < 6Ǻ (kcal/mole)
IleVal55 5 0.30
IleAla55 16 1.15
ValAla10 37 3.39
IleAla88 55 4.01
LeuAla14 62 4.32
Destroying a solvent-exposed ionic interaction between Asp8, Asp12 & Arg110
Mutant GU
(kcal/mole)
AspAla8 0.89
AspAla12 0.31
AspAla8 & AspAla12 0.80
Mutation studies have validated the importance of some interactions. Can we use site-directed mutagenesis to engineer proteins with enhanced stability?
Yes! Bridged mutants show resistance to denaturants & thermal stability!
SDM may be aided by evolutionary clues left by Nature
With respect to Barnase, Binase has lost one amino acid (Gln2 → ) and has 17 different residues. The structure of Binase is slightly more stable than Barnase.
Hypothesis: Evolution may have selected some of the 17 amino acids because they promoted stability. If these amino acids are mutated into Barnase, the engineered Barnase may have higher stability...
Folding of Barnase - Overview Elucidating rules, which govern the folded conformation of
proteins, is of theoretical interest and practical importance particularly since advances in recombinant DNA methods have enabled the design and synthesis of novel proteins.
Although many physico-chemical approaches have been employed, the mechanism of protein folding remains unclear.
An approach, which combines the technique of site-directed mutagenesis with the more classical physico-chemical techniques, has been employed to address this problem.
By altering specific side chains in a folded protein, it is possible to correlate the contributions of their interactions towards the overall stability of the protein. Thermodynamic relationships, specifically Bronsted relations, are employed in this treatment.
Barnase, a relatively small 110 amino acid, monomeric extracellular ribonuclease of Bacillus amyloquefaciens serves as a model protein for this study.
Q: What was the experimental strategy? Choose a significant non-covalent interaction Make a subtle change to the interaction (e.g., Ser80 → Thr80; Ser85 → Thr85) Perform equilibrium/kinetic un/folding experiments and
compare the wt & mutant Rationale: All interactions contribute to protein stability - Some
form/break before the rate determining step of folding, whereas others form/break afterwards
Q: What is the general picture of the TS? Native-like, compact Some tertiary interactions lost Majority of 2˚ structure preserved Core1: weakened, esp. at Nterm 1
Core2: completely disrupted
Core3: fully intact
If we were to evaluate the unfolding pathway: 1st events: 3 of 5 loops unfold, Nterm of helices melt,
core1 weakens and core2 is destroyed; the remaining structure is disrupted later
EU → EI is faster than EI → EF;Thus, your measurements can examine EI → EF
Probing the rate determining TS of folding and an intermediate
For each mutation:Use the appropriate kinetic experiment (dilution of urea) &equilibrium foldingexperiment (urea) to probe the I state and TS of folding!
The state of interactions in the intermediate & transition state of Barnase with respect to the unfolded state; the last column shows the unfolding pathway for comparison
Q: What can be said about folding? Correlation between hydrophobic burial and early events All early regions inteact with sheet Early burial is hydrophobic, extensive and nucleated Late processes only interact slightly with sheet; no
core nucleation; some hydrophillic burial is noted
General statements: The refolding pathway is at least partially sequential 2˚ structure formation leads to local hydrophobic burial
and precedes most 3˚ interactions Consolidation of structure is gradual and earliest for 2˚
Concluding Remarks. When generalized to small globular proteins, folding proceeds by nucleation, i.e., local hydro-phobic collapse of core elements, followed by consolidation of hydrophilic interactions & 3º structural domains
A Closer Look at Chemical Modification of Protein Monomers in Aqueous (and
now also Organic or Dry-state) Environments and the Use of Paper-
Chemical modification and site-directed mutagenesis are complementary methods.
Rationale: Information obtained from chemical modification forms a base to design appropriate mutants. E.g. Chemical modification shows that 1 of 4 Met residues is
super reactive. To learn more abot the environment, use SDM to replace neigboring groups & observe the results…
Advantage of chemical modification in comparison to SDM? Fast, inexpensive, doesn’t require elaborate setup.
Disadvantage of chemical modification in comparison to SDM? Not very specific, and imposes a chemically reactive
environment. Can lead to drastic modifications. Modifications typically are limited to amino groups, carboxyl
Historical applications – chromatography and electrophoresis
Amino acid analysis
Peptide mapping
Disulfide bridge analysis
Analysis of other post-synthetic modifications, e.g., phosphorylation
Work-up of chemical modification experiments!
Protein/peptide/amino acid resolution and purification
Current applications
Same! Apparatus has been revised somewhat – e.g., HV tlc is a strong alternative to HV paper methods
Advantages of paper methods over HPLC and MS methods to identify and purify
1.Many samples can be run simultaneously
2.Multidimensional runs are conveniently setup; sample processing is possible in between runs!
3.Resolving power is great
4.Tolerance of potential interferents is high
5.Detection methods are potentially very sensitive and selective
6.Cost efficiency of equipment and experiments
7.Less need of skilled labor
8.MS and HPLC methods can be coupled if desired
The apparatus of paper chromatography
for biological samples
for simple analytes
You may discard the drying accessories; instead clip the paper horizontally at the bottom with zig-zag scissors - allows solvent to drop evenly to the bottom
suspended papers
trough with solvent
The apparatus of high-voltage paper electrophoresis
1. paper; 2. dielectric; 3. glass tank; 4. trough with buffer (top) and base with same buffer (bottom); 5. cathode (-); and 6. anode (+)
Types of volatile solvent systems
•BAWP
•Ammonia – organic
•Pyridine acetate buffer
•Formic acid – acetic acid buffer
Types of papers
•Whatmann 3MM for high loading, large peptides
•Whatmann 1MM for high resolution
Tlc alternatives as stationary phase
•Cellulose tlc plates
•Silica tlc plates
Protein sample preparation prior to spotting on paper
Amino acid analysisHydrolysis (acid typically; base hydrolysis if Trp is needed)Dry sampleReconstitute in a minimum amount of running bufferSpot the sample on paper
Identifying the number of arginine residues in BSAChemically block all lysine residues in 8M ureaDialyze away the ureaDigest all arginyl peptide bonds using trypsinDry sampleSpot the sample on paper
Separating the and chains of insulinIncubate protein in 95:5 formic acid/30%hydrogen peroxideDry sampleSpot the sample on paper
Origin X X X X XX X X X X
(-)
Submergedin buffer at base, pH 2.1
Submergedin bufferedtrough, pH 2.1
Direction of migration40V/cm
e.g. for HVPE
oo
o
o
oo
o
o
o
o
o
o
o
o
o
o
after migration
Identifying and quantifying bands after migration and drying of paper chromatograms
Strategies to identify•Intrinsic fluorescence•Radiolabeling and exposure of X-ray film•Fluorescent derivatization or colorimetric derivatization, e.g., ninhydrin
Strategies to quantify•Densometry of chromatographic images•Liberation from paper and subsequent analysis
How shall we see these?
n-Dimensional runs & the advantage of multi-dimensions
1-D
2-D
multi-D
X X X X X X
Xoo
o
o
rotate90degrees
o o o o
run 2nddimension
o
oo
oo
o o
oo
o
o o
oo
oo
run 1stdimension
2nd & subsequent dimensions may/may not be performed using the initial conditions
oooo
run 2nddimension
o
oo
after 1stdimension
o o o o
o o o o o
you are interestedin these seriessamples
cut out,stitch ontonew paper!
ooooo
o
o
o
o
o
oo
oo
o
now you are interestedin this vertical series cut out,
stitch ontonew paper,run anotherdimension
repeat asnecessary
You may chemically process the sample in between dimensions!
Q: Are there other factors, which affect apparent (macroscopic) reactivities? A: YES! Accessibility and steric considerations refer to the size &
amount of reagent used, whereabouts of reactive group, permeation time of reaction, and the conditions of reaction.
Nucleophilicity considers the base strength, solvation shell, and lone electron density, polarisability & conjugation of centers on protein groups.
Electrophilicity considers the electron density of protein groups and electron deficiency of reagents.
The “hard likes hard, soft likes soft” empirical relation does apply to some degree. Basically, the interaction of reacting orbitals and centers is in part determined by their “harness” or “softness.”
Structure-function studies generally follow this procedure: Choose to address a particular issue; Envisage a strategy to perturb the protein in order to investigate the
issue; Anticipate the best groups to modify in order to create the desired
perturbation; Choose the best reagent and conditions to specifically modify the
functional groups in question; Compare the change of properties of the modified protein to that of
the control protein; Form conclusions using the Okum’s Razor argument.
Local changes and Global changes: Chemical modifications of protein groups affect pI & pKa values, solubility, surface hydrophilicity & hydrophobicity, bioactivity, folding stability & global organization.
A: One application is to carry out structure-function studies Methods can be benign or destructive A typical protocol:
Label the native protein Determine a change of property, and Extract useful information on the basis of the results.
Data collected may be related to structure: E.g., a tyrosine specific reagent cannot react with a tyrosine residue; the
tyrosine may be buried. Data collected may be analytical.
E.g., Protein X does not react with a tyrosine-specific reagent in 10M urea; the primary sequence does not contain tyrosine.
Data collected may be related to function and/or structure and function. E.g. 1., 10 Tyr residues are solvent-accessible, but 1 predominantly reacts
with trace reagent; this Tyr is unusually reactive in the protein environment. E.g. 2., After Lys-93 is modified chemically, substrate X shows a reduced
Km value; Lys-93 plays a role in binding. If the role is direct, this Lys is likely near the binding site of the active center.
Q: What are the principle applications of protein chemical modification?
Adducts with different compounds such as PEG2000 Quantifying amino acids, functional groups, disulfide bridges, phosphate and other post-translational
modifications Various chemical reagents and chromatographic methods
Chemical modification is used to re-engineer proteins for improved performance in industry. The protein is characterized as much as possible The improved trait is defined A modification is envisaged to promote the property The protein is derivatized accordingly.
S-F problem?
InappropriateModification/ Conditions
Appropriate Modification/ Conditions
NOTE - Chemical treatment of biologicals need not necessarily be destructive and damaging. When conducting a structure-function study, your choice of reagent and protocol should not defeat the purpose of your study
Judging if the purpose of an investigation has been defeated• The meaning of defeating your experimental
purpose• Cases that are indifferent to over-reaction of
protein groups• Cases that require post-reaction validation
Distinguishing between apparent/macroscopic values and true/microscopic/theoretical values
• Kinetic measurements versus thermodynamic measurements
• Measuring a value via independent methods may give a true value
A time to chemically label and a time to chemically work-up: Distinguishing between the two steps
In a protein labeling experiment and its workup, we typically note:
1. Reactions conducted on native proteins are for labeling
2. Reactions conducted on protein derivatives are for labeling or work-up
3. Reactions conducted on unfolded native proteins are for labeling of controls
4. Reactions conducted on unfolded protein derivatives are for work-up
Acetylation using Acetic Anhydride or Acetyl Imidazole at pH 7.5-9 Acetylation blocks free amino termini, lysine side-chains and tyrosine side-
chains. Acetyl tyrosine is hydrolyzed at high pH values or transesterified above pH 6 by a strong nucleophile.
Acetylation of amino groups is usually quantitative only if the reaction is carried out in urea solution. Without urea, usually 60% of the lysine residues are modified.
This modification could have the following effects Decrease of protein solubility (Acetyl BSA is only soluble at pH < 5); Changes in biological activity (by either global structural changes or specific
changes in the active site); Dissociation of multimeric complexes (if the surface charges are required for
Maleiylation (left) using maleic anhydride & citraconylation (right) using citraconic anhydride at pH > 8Maleic anhydride and citraconic anhydride block amino groups but
can be removed by incubating the protein at low pH.Maleic anhydride is more difficult to remove & sometimes leads to
a minor side-reaction, in which Cys adds across the double bond.Citraconic anhydride is used to reversibly block amino groups and
to protect the amino groups from other chemical reactions. E.g., citraconylation will protect amino groups from subsequent oxidation by
hydrogen peroxide. Once Cys’s are oxidized, amino groups are regenerated. Citraconylation is the method of choice to temporarily solubilize
Trifluoroacetylation using Ethyl Thiotrifluoroacetate at pH 10 Ethyl thiotrifluoroacetate is used to reversibly block amino groups as in the case of
maleic anhydride and citraconic anhydride. But the derivative has a net zero charge. Furthermore, the group is removed under basic
conditions using sodium carbonate at pH 10.7 or 1M piperidine. E.g. 1., pancreatic ribonuclease was deactivated completely following
trifluoroacetylation yet there were no measurable structural changes. In carbonate buffer, activity was gradually restored.
E.g. 2., trifluoroacetylation caused structural changes in cytochrome c. Incubation in carbonate buffer restored full electron transfer ability.
Trifluoroacetylation can simplify protein sequencing if trypsin is used to cleave the protein
Trypsin normally cleaves after every free arginine and lysine residue, generating a complicated mixture. If the protein analyte is first trifluoroacetylated, trypsin can only cleave at the arginine bonds.
The fragments are isolated, deblocked and subjected to a 2nd tryptic digestion at the lysine sites
Amidination using methylacetimidate (left) or guanidination using o-methylisourea (right) at pH 7-10.Methylacetimidate forms stable derivatives with lysine residues
and amino termini. O-methylisourea reacts with lysine groups.Modification of amino groups increases steric bulk, while retaining the
positive charge. The pKa of derivatives shifts to values well above 11! Most modifications do not give significant structural changes.
Q: Suggest a test to postulate if the active-center lysine is the catalytic nucleophile or a binding group.
Q: Suggest another test to determine if a lysine and a neighboring aspartic acid maintain a crucial salt bridge. Hint: You will analyze the pH activity after reaction.
Ethoxyformylation using diethylpyrocarbonate (ethoxyformic anhydride) at pH 4 Ethoxyformic anhydride only reacts with imidazole groups at pH 4; it modifies
amino groups at basic pH values. The acyl-imidazole adduct is stable in water, particularly at pH 7, unlike other
acyl His derivatives. The group is removed by the action of H2N-OH at pH 7. EFA is used in molecular biology to battle against RNAse. EFA can rapidly inactivate many enzymes including trypsin and is used by
industry to cold-sterilize food. EFA can aid structure-function studies if histidine is important for bioactivity.
Q: You must decide if a Lys or a His is essential for catalysis. What experiment could you design using the reagents we have discussed thusfar?
Reductive dimethylation using H2C=O & NaBH4 (left) at pH 9; Variable methylation using ICH3 (right) at pH 2-10 Reductive methylation mono/dimethylates amino groups, retaining the positive
charge of the amine. Generally, structural changes are not observed following reductive methylation.
Reaction with iodomethane quaternizes amino groups with retention of positive charge. Met and Cys are converted to their sulfonium iodides. Tyr is methylated and His is converted to the dimethylimidazolium iodide.
Reaction selectivity is tuned by appropriate choice of pH & reaction medium. Unlike reductive methylation, iodomethane:
Puts a permanent positive charge on amino, imidazole and sulfide groups Removes the hydrogen bonding ability of tyrosine
Carboxyalkylation using iodoacetate (left) & carbamino-alkylation using iodoacetamide (right) at pH 2-10. Iodoacetate & iodoacetamide react similarly to iodomethane
except tyrosines are generally not modified.Derivatives are larger than iodomethaneDerivatives may be considered bulkier.
In the case of iodoacetic acid, a negative moiety is introduced. Iodoacetamide, iodoacetic acid and iodomethane are commercially
Reduction using mercaptoethanol or dithiothreitol (reduced form) at pH 8.Cystine bridges can be cleaved to afford two cysteines by the
action of mercaptoethanol or dithiothreitol. In the case of DTT, a stoichiometric amount of reagent is sufficient to bring
about the modification. The driving force for the reaction is formation of a stable 6-membered ring.
Reduction has been used to study bioactivity & structure. In many cases, reduced proteins exhibit changes of solubility & activity.Reoxidation of reduced cysteines sometimes reforms the correct disulfide
In competitive labeling, a modification ALWAYS reflects the state of the native protein. Why? Reagent is used in trace amounts
At most 1 group/protein is modified Thus, only the reactivity of the native protein is probed
Above: A typical experiment quantifies the statistical distribution of the kinetic reactivity of protein groups the reactivity of every reactable group is probed at different pH values. While the results describe apparent data, they are used to interpolate a group’s
local environment.
Molarradiolabelincorporation(apparentrelativereactivity) with respectto Phe-NH2
pH value of reaction
1-
0.5-
apparent pKaof reactive group
Apparent relative reactivity and pKa values of 3 proteingroups with respect to an external Phe-NH2 standardCompetitive Labeling
Many methods used to modify different functional groups
Generally the modifier is used in excess → proteins modified extensively.Onus is put on the investigator to prove that the results reflect the properties of the native protein.
Steps: 1.Tracer. Incubate a solution containing protein & standard (e.g., Phe-NH2) with a trace amount of 3H-reagent. Tritium incorporation is your probe. Carry out this operation at many pH values.
2.Normalization. Unfold the protein in urea and react the remaining groups completely with 14C labeled reagent. This protocol ensures that all protein and standard derivatives become chemically homogeneous.
3.Quantification. Extract the standard quantitatively into an organic phase and quantify the 3H/14C ratio for each pH value. Digest the protein using enzymes so that only one modified group is present per peptide. Migrate all peptides in an electric field. Identify peptide positions using autoradiography. Collect each band & quantify the 3H/14C ratio. Sequence the peptide in order to identify the derivative.
4. Interpretation. The 3H/14C ratio of protein & standard are compared. The reactivity of the standard reflects its aqueous environment & should parallel its titration curve. The reaction profile reflects the groups’ local environment so you should observe a “titration curve”, which illustrates the effect of the protein environment. This titration curve may deviate significantly from what would be expected in an aqueous environment. The data can thus be used to build a picture of the local environment.
Two data can be extracted for every group: The kinetic pKa of the group & the relative reactivity of the group can be assessed in comparison to Phe-NH2, which has a well-characterized reactivity and pKa that is devoid of any environmental influences. The two data help to assess if any potential steric considerations or stereoelectronic factors are out of the ordinary.
Below are some example results, which allow you to appreciate the power of this method:
e.g. Titration curve of Cys gives a pKa of 3. Perturbed pKa. e.g. Titration of Lys-29 gives poor reaction when reacted from pH 5 to
11. Buried. e.g. Titration of Lys-29 gives a continuous titration curve with an
interpolated pKa of 11 when reacted from pH 5 to 11 (A normal lysine is 10.5). Lysine is buried part of the time or otherwise perturbed.
e.g. Titration of Lys-29 gives a discontinuous curve when reacted from pH 5 to 11. First there is no reaction, then after pH 10.5 the reaction is very high. Conformational change and accessibility.
e.g. The N-terminal Histidine imidazole ring displays a pKa value that is equal to the pKa of its N-terminal amino group. Inductive effects and coupled reactivities.
The power of competitive labeling and paper methods
RECAP - Steps of competitive labeling: 1. Label each protein at most once, using a trace of tritiated reagent
His
NH2
LysTyr
SH3C
1000
1000 AcHisNH2
+100 *3-Me-I
95 *3-Me-OH
(aq)(aq)
SH3C
Me-3H*
Tyr Lys
NH2
His
(aq)
SH3C
Tyr Lys
NH2
His
Me-3H*
(aq)
SH3C
Tyr Lys
NH
Me-3H*
His
(aq)
SH3C
Tyr Lys
Me-3H*
NH2
His
AcHisNH2
Me-3H*999
997
111
in a pH "X" solution
AcHisNH21
Standard, with known pKa value & reactivity!
+
+ +
+
++
+
+excess *14-Me-I/12-MeI mixture
99% *14-MeOH/12-MeOH
in 8M urea pH 10 solution
+SH3C
Me
Tyr
Me
+Lys
Me
Me
Me
+N
Me
Me
Me
+His
MeMe
(aq)
+SH3C
Me
Tyr
Me
+Lys
Me
MeMe
+N
Me
Me
Me
+His
Me-3H*Me
(aq)
+SH3C
Me
+Lys
Me Me
Me
+N
Me-3H*
Me
Me
+His
MeMe
(aq)
+SH3C
Me
Tyr
Me
+LysMe
-3H*
Me
Me
+N
Me
Me
Me
+HisMe
Me
Ac+HisNH2
Me-3H*Me
999
997 1
11
Ac+HisNH2
Me Me1
+
++
+(aq)
MeTyr
(aq)
SH3C
Tyr Lys
NH2
His
(aq)
SH3C
Tyr Lys
NH2
His
Me-3H*
(aq)
SH3C
Tyr Lys
NH
Me-3H*
His
(aq)
SH3C
Tyr Lys
Me-3H*
NH2
His
AcHisNH2
Me-3H*
999
997 1
11
AcHisNH21
+
++
+
Step 2: Label all protein completely using excess low specific actitivity 14C-MeI to yield a chemically homoge-neous mixture
Step 3. Repeat steps 1 & 2 using more native protein; label many samples at different pH values.
Step 4. Prepare a positional marker protein; label native protein with high-specific activity 14C-MeI, then excess 12C-MeI in 8M urea
Step 4. Digest all methyl-proteins separately. Use 2-4 proteases.
Step 5. Separate the different peptides in an electric field along tlc or paper; use 1-4 dimensions; spot the marker peptides at the ends of the chromatogram.
Step 6. Expose X-ray film to the chromatogram (RT for 14C, -80˚C for 3H) to find the position of all substantially radioactive peptides.
Step 7. Find the 3H/14C ratio of each peptide band; sequence each to identify the reactive group (e.g., AlaTyr*Gln can only be Tyr18)
Step 8. Plot the reactivity (i.e., 3H/14C ratio) of each reactive group as a function of pH.
Subsequent Steps (for proteins of known 1˚ sequence):
cathode (-)
anode (+)
3kV,30min,pH 2.1
Peptide solutions spottedalong paper at origin
14C-marker peptides
x x x x x xx xx x x x x x x x x7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8
pH of labeling7.9 8.0 8.1 8.2 8.3 8.4 8.5
Paper chromatogram beneath;X-ray film superimposed on top
Recover & measure 3H cpm/14C cpm
e.g.1; peptide 8 @ pH 7.8 = 512/1890
e.g.2; peptide 7 @ pH 7.6 = 450/5680
e.g.3; peptide 6 @ pH 8.1 = 890/1543
. .
..
.
.
= radioactive ink spotted on Whatmann 3MM paper1
23
4
5
6
7
8
9
Peptide 6 was sequenced: AlaTyr*Gln indicates a Tyr reacted. There are 4 tyrosyl residues in this protein but the sequence data indicates Tyr18
Molar 3H/14Cradiolabelincorporation(apparentrelativereactivity) with respectto AcHis-NH2
pH value of reaction(referenced against known pKa of AcHis-NH2)
1-
0.5-
apparent pKaof reactive group
Apparent relative reactivity and pKa values of 5 proteingroups with respect to an external AcHis-NH2 standard
His102
Tyr18
Met1
Lys87
NH2
AcHis-NH2
In vitro manipulatin of protein monomers or their environments to
Goals: To formulate broad-scope protein preparations, which are: Cheaper More stable More catalytic Longer-lived More easily stored & transported More active at pH & temperature extremes
Locating/purifying thermophiles, etc.
Geneticmanipulation
Low-techchemical strategies
Native
search for robustness:
Approach 1: use proteins in water-free media
Approach 2: crosslink Approach 2: crosslink proteins togetherproteins together
Answer: NO! Recent evidence shows that protein structure and protein function may be very tolerant to strange environments!
Today, we will discuss the structure & function of proteins in reduced-water & water-free environments
Why does mankind wish to use biologically active proteins?Proteins accelerate chemical reactionsProteins improve product propertiesProteins permit novel syntheses
Here are some typical industrial applications:BioreactorsTextile treatmentMedicinal and organic synthesesProtein drugs & drug deliveryBiosensorsBioremediationFood preparation industries
Problem? Industrial conditions are often too harsh for proteins in the native state.Consequences: Poor biological activity, short lifespan, limited reaction parameters, etc.
Q: Good competitive inhibitors in water are poor competitive inhibitors in octane! Poor competitive inhibitors in water are excellent competitive inhibitors in octane! WHY?
In summary, when proteins are put into unusual environments they can survive!
Final Question: What are some advantages of using enzymes in water-free environments?
Novel reactions, which are not feasible in water, become posssible Use of “non-water” nucleophiles, e.g. transesterification and ester ammonolysis; Increased solubility of apolar substrates; andshifting thermodynamic equilibria to favor synthesis over hydrolysis, e.g., esterification and peptide formation.
Suppression of water‑mediated side-reactions. Alteration of substrate specificity. Enhanced thermostability of enzymes. Easy recovery of enzyme from low boiling solvents.
You may prepare protein drug formulations, which are not in water:
We saw some examples just before:Insulin Lysozymeintraperitoneal injections creams & gelstime-released matrix inhalable microcapsulesİnhalable microcapsules drop formulations
New Facts: Protein structure & function can be stable in unusual environments if you use the correct procedure! What Are The Results???? Cheaper More stable More catalytic Longer-lived More easily stored & transported More active at pH & temperature extremes
Established crosslinking media:A. In a solubilizing environment
Q: What are the characteristics of method A?Solution-phase crosslinking often leads to polydispersityEvents: Soluble monomers → Dimerization → Oligomerization →→→ Insolubilization (typically gelation)
Q: What are the characteristics of method B?Crosslinked enzyme crystals are biologically active catalysts with well-defined poresEvents: Soluble monomers → Crystallization → Chemical crosslinking →→→ Insolubilization
Q: What are the characteristics of method C?Crosslinking of lyophilisates leads to polydispersityEvents: soluble monomers → lyophilization at desired pH value → vacuum, heat and/or chemical crosslinker → oligomerization →→ insolubilization (powder resists swelling in solvents)
D: As a precipitate retaining the native structure
Q: What are the characteristics of method D?Events: Soluble monomers → Salt-out or insolubilize with miscible organics → Add chemical crosslinker → Oligomerization →→ Insolubilization (powder resists swelling in solvents)
Q: Let us please summarize approach 2: protocols, reagents & products
A. In aqueous solution
B: In the crystalline state
C: In the lyophilized state
D: As a precipitate retaining the native structure
Your choice of reagent? Prior history; Surface accessibility and steric constraints; & effective pH during the reaction conditions
A: glutaraldehyde, other simple aldehydes,
EDC.HCl, diimidates, difluorodinitrobenzene
B: glutaraldehyde, other simple aldehydes
C: volatile or organic soluble carbodiimides, acyl group activators, disulfide exchange, thermal induction of amide bonding, bifunctional acyl halides and similar reagents
Some ReferencesHarris, E.L.V. and Angal, S. eds., Protein Purification Methods: A Practical Approach, 1989, IRL PressHarris, E.L.V. and Angal, S. eds., Protein Purification Applications: A Practical Approach, 1990, IRL PressDeutsher, M.P., ed., Methods in Enzymology, Guide to Protein Purification, 1990, Academic Press
1. The following methods are used to purify protein:
Technique Exploited Protein PropertypH Precipitation Charge, pI valueAmmonium Sulphate Precipitation Intermolecular charge/
hydrophobic interactionsIon exchange chromatography ChargeHydrophobic Interaction chromatography HydrophobicityChromatofocusing Charge, pI valueDye Affinity chromatography Affinity for high MW dyeLigand Affinity chromatography Bioactivity/affinintyGel permeation chromatography Dynamic volume (size)
2. No single method is perfect, so you should use many together
Example manipulation:Starting purity = 10%, yield is 10mg of target proteinAfter AS precipitation, purity = 60%, yield is 8mg of target proteinAfter Chromatography, purity = 95%, yield is 4mg of target protein
3. Protein is characterized after purification: Final yield? MW? N- & C-terminal analysis, disulfide bridge analysis, etc.
With recombinant Human insulin:Lysis the cells, centrifuge, & collect your sample: A solution of insulin, other proteins, DNA, salts, metal ions, etc. You deterimine 1% insulin, 99% other proteinsand a total of 2g insulin in the batchHow to purify?
Protein purity and analysisQ: What is PURE ENOUGH?Q: What is the meaning of Purity? Free of other proteins? Free of ions? Free of DNA? Free of protease activity?
Q: In the case of insulin, what parameters should you analyze (and how)?Size (many)Primary sequence (many)N-terminus, C-terminus (many)Surface-accessibility of various groups (Chem. Mod.)Shelf-life at different humidities (Solubility)Amino group count (Kaiser test)Histidyl group count (Pauli test)pKa of ionizable groups (competative labeling)Secondary sequence (CD)Tertiary sequence (X-ray, NMR, receptor assay)
e.g. Purifying insulin as a drug productRoutes of application:Injection of suspended insulin under skinImplant materialsMicrocapsules containing insulin for lung absorption
What aspects of purity should you examine if you prepare insulin as a drug formulation?
SterilityStorage life and requirmentsBiological activityBioavailabilityMany of the previous criteria!
