Protein Chemistry & Engineering Lecture Series
Lecturer: Alpay Taralp, Materials Science & Engineering Program, Sabancı
University, Istanbul 34956; taralp@sabanciuniv.edu
Lecture Topics
Day 1, 3h
 Introduction to protein chemistry
 Strategies used by enzymes to accelerate reaction rates
Day 2, 3h
 Protein stability elucidated and enhanced via protein engineering
 Protein folding & unfolding probed via protein engineering
Day 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 function
Day 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 performance
Day 6, 2.5h
 In vitro manipulation of protein monomers or their environment to enhance performance (contin.)
 A closer look at optimizing protein function in non-aqueous environments
 Protein purification and related analytical methods
 Short examination
Lecture Breakdown
Day 1
 Introduction to protein chemistry
o A brief review of protein structure
 Physico-chemical traits of the amino acids
 Properties of the peptide bond
 Definition of an oligopeptide, polypeptide and protein
 Factors determining protein structure & stability
 Levels of structure in proteins
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 3-D structural representations
o Different protein families
 Protein classes with different functional roles
 Structural similarities and differences
 Noteworthy examples showing the diversity of physico-chemical attributes
o What is protein chemistry?
 Classic definition, previous contributions to science and current role
 The merit of structure-function elucidation
 Physical methods to probe structure and function
 Chemical methods to probe structure and function
 Influence upon other biochemical fields
 Genomics, i.e., molecular biology
 Proteomics, i.e., protein crystallography
 Strategies used by enzymes to accelerate reaction rates
o Enzyme properties
o Quantifying enzyme rates
 Types of assays
 Michaelis-Menten rates and analyses
o Enzyme classification according to biological function
o A brief review of thermokinetic parameters and their merit in protein investigation
 Significance of thermokinetic parameters at the level of protein structure &
function
 Thermokinetic changes: G, H, S, G≠, H≠ and S≠
 Absolute values of G, H and S depicted diagrammatically
 Interrelating the thermokinetic parameters
 A light-hearted look at established kinetic theories
 Collision theory
 Transition state theory
 Quantum tunneling
o Rate acceleration
 The principle of ground state elevation
 The principle of transition state stabilization
o Similarities & differences between enzymatic and non-biological catalysts in their modes
of action
o A detailed look at enzymatic strategies to accelerate rates
 The evolution of Gibbs energy, enthalpy and entropy along the reaction coordinate
 A discussion of strategies to decrease activation energy, G≠
 Structure-induced minimization of S≠
o Orbital steering of catalytic groups
o Union of bimolecular participants prior to chemical transformation
 Electrophilic, metal-ion and general acid & base catalysis
 Low-barrier H-bonds
 Nucleophilic catalysis and promotion of multi-step pathways
 Structure-promoted activation of active-site functional groups
 Binding energy stabilization
o Misconceptions linked to enzyme ground-state specificity
o The meaning of shape-complementarity
o The energetics of favorable and unfavorable interactions
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o The onset of tight binding in proceeding to the transition state
Day 2
 Protein stability elucidated and enhanced via protein engineering
o Efficacy of interactions in the folded state quantified according to location and type
 X-ray mapping of interacting residues
 The rationale to subtly perturb interacting groups via mutation
 Equilibrium un/folding measurements of wild-type and mutant Barnase
 H-bonding groups & their contribution to stability as a function of location
 Hydrophobic interactions & their contribution to stability as a function of
location
 Ionic interactions & their contribution to stability as a function of location
o Enhanced stability following intelligent mutagenesis & biomimicry of a related structure
 Disulfide bridges mutated into Barnase
 Mimicry of Binase, a structural homologue, to yield a Barnase mutant with superstabilizing interactions
 Accelerated mutagenesis and screening
 Protein folding & unfolding probed via protein engineering
o Protein folding & unfolding probed via mutagenesis of interacting residues
 A discussion of interactions, which define the final protein conformation
 Dispelling the myth that folded structures are rigid in solution
o Some native structures are more flexible than others
o The correlation between protein flexibility and crystallizability
 The energy considerations that determine a protein to fold and its energy profile
 Protein folding is clearly directed
 Timeframe of folding versus the conformational possibilities
 Misfolded proteins and the prion diseases
o Globular to fibrous transitions & changes of physico-chemical traits
 Elucidating the protein folding problem, i.e., the 2nd half of the genetic code
 Key questions
 Techniques to assess the mechanism and pathway
 Motivation to understand the rules, which govern folding
Day 3
 Protein folding & unfolding probed via protein engineering (continued)
o Protein folding & unfolding probed via mutagenesis of interacting residues (continued)
 A light-hearted look at Barnase
 Properties & merit of Barnase as a model protein to conduct folding studies
o Primary structure
o Secondary and tertiary structure
o Convenience of expression
 Strategy and protocol used to probe the folding and unfolding pathways
o X-ray structures to map out the interacting residues
o Subtle perturbations of interactions via mutagenesis
o Validation of Barnase mutants as a good test sample
 X-ray structural analysis
 NMR validation of 3-D structure
 Bioassays
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o Schematic comparison of wild-type and mutant Barnase along a
reaction coordinate
o Differentiating between GF-GU and GF-U
 Schematic representation of GF-GU
 Schematic representation of GF-U
 Advantage of adopting GF-U
o Equilibrium & kinetic un/folding of wild-type and mutant
 Use of tryptophan fluorescence to characterize structure
 Techniques employed to determine energy changes
o The use of Bronsted relationships to assess structure
 Bronsted catalysis equation and the meaning of 
 Bronsted catalysis equation re-defined for protein systems
using 
 Afold GA-U/GF-U; Aunfold GA-F/GU-F
o GF-U described using energy terms
o Limits imposed on equating the meaning of 
&
 The shape of the rate-determining transition state of
unfolding
 Experimental design and rationale
 Results after numerous mutations of interacting pairs
 The shape of a transition state & intermediate during folding
 Experimental design and rationale
 Results after numerous mutations of interacting pairs
 Summarizing some rules that mediate the folding process
Day 4
 The combined power of in vitro chemical modification and paper-supported
chromatography as a probe of structure and function
o History of chemical modification of proteins
 Practical uses of chemical modification: Historically and currently
 Chemical modification as a structure-function probe
 The notion that structure perturbs function and function defines structure
 Chemical modification as a compliment of enzyme kinetics
 Chemical modification as a lead-in to effective mutagenesis studies
o Paper chromatography and paper electrophoresis
 The history of paper methods applied to protein investigation
 Advantages of paper methods over HPLC and MS methods to identify and purify
 Materials and methods for any paper technique
 Solvent systems, different grades of paper & tlc alternatives
 Protein samples and their preparation prior to spotting on paper
o Acid & base hydrolysis
o Enzymatic processing
o Performic acid, cyanogens bromide & other methods of cleavage
 Developing tanks
 Identifying bands and spots following migration
o Intrinsically marked samples
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 Radioactive labeling and autoradiography
 Fluorescent residues
o Post-migration staining
 Separations
o One-dimensional runs
o Two-dimensional runs (diagonal separations)
 Use of same or different solvent systems
 Secondary chemical or enzymatic derivatization of protein
samples before migration in the 2nd dimension
 Spray methods
 Dip methods
o Multi-dimensional runs
 Combining science & art: Where is the seamster?
 Advantages of multi-dimensional runs
o The chemical modification of protein groups
 Reactability of protein residues is a function of
 pH value
 solvent-accessibility
 intrinsic reactivity modulated by stereo-electronic contributions of the
folded state
 Potential changes anticipated/observed in the protein following reaction of specific
residues
 Local changes
 Global changes
 Questioning the significance of your measurements
 Distinguishing between apparent/macroscopic values versus
true/microscopic/theoretical values
o Kinetic measurements versus thermodynamic measurements
o Measuring an apparent value using unrelated techniques can yield
the true value
 Judging if the purpose of an investigation has been defeated
o The meaning of defeating your experimental purpose
o Cases that are indifferent to over-reaction of protein groups
o Cases that require post-reaction validation
 A time to label and a time to work-up: Distinguishing between the two steps
 A typical protein labeling experiment and its workup
 Reactions conducted on native proteins are typically for labeling
 Reactions conducted on protein derivatives are typically for labeling or
work-up
 Reactions conducted on unfolded native proteins are typically for labeling
of controls
 Reactions conducted on unfolded protein derivatives are typically for
work-up
 A detailed look at typical protein modifiers (crosslinkers excluded) and some
potential stereo-electronic changes of the protein derivative
 Acetic anhydride, succinic anhydride, maleic anhydride and citraconic
anhydride
 Carboxyanhydrides
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Ethylthiotrifluoroacetate
Methylacetimidate and O-methylisourea
Ethoxyformic anhydride
Formaldehyde and borohydrides
Alkyl halides, haloketones and haloamides
Diazoglycinamide
Carbodiimide and amine
Mercaptoethanol and dithiothreitol
Day 5
 The combined power of in vitro chemical modification and paper-supported
chromatography as a probe of structure and function (continued)
o A closer look at competitive labeling
 Advantages over classic chemical modification techniques
 Rationale and method
 Diagrammatic extraction of apparent reactivity and pKa values
o Competitive labeling and paper methods can solve those “impossible” problems
 Investigating the solvent-accessibility, reactivity & pKa value of every ionizable
group of the L12 protein subunit, while in the native state, i.e., residing as a 50protein subunit in E. coli.
 Discussion of the rationale, method and results
 Could “modern methods” have produced the same quality of results?
 In vitro manipulation of protein monomers or their environment to enhance performance
o Proteins applied to industry
 Antibodies
 Therapeutics
 Enzyme processes
 Examples of enzyme-catalyzed reactions in industry
 Advantages over non-enzymatic processes
 Drawbacks over non-enzymatic processes
o Discussion of current goals to better integrate enzymes in industry
Day 6
 In vitro manipulation of protein monomers or their environment to enhance performance (contin.)
o Employment of diverse strategies to realize these goals
 Extracting and purifying extremophiles from Nature to use under harsh industrial
conditions
 Laboratory-scale mutation of available strains, screening, and bulk expression of
improved strains for industrial use
 Post-translational manipulation of off-the-shelf proteins for industrial use
 Off-the-shelf proteins deployed under non-aqueous conditions
o Advantages of utilizing enzymes in non-aqeous conditions
o Dispelling biases related to the use of protein in non-aqueous media
 Chemical modification of off-the-shelf proteins for industrial use
o Crosslinking methods
 Advantages of crosslinked proteins over free proteins
 Crosslinking of protein solids
 Crosslinking of protein monomers
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
Mechanistic discrepancies between the crosslinking
methods
o Modification of protein monomers
 Derivatizations under aqueous & non-aqueous conditions
 Chemical basis for altered reactivity, pH optima, thermal
stability and shelf-life
 A closer look at optimizing protein function in non-aqueous environments
o Protein structure variations upon dispersion in non-aqueous media
o Parameters that affect catalysis
o Tricks to recover aqueous-like bioactivity under non-aqueous conditions
 Protein purification and related analytical methods
o The three types of proteins and some classic methods to purify them
 Properties exploited
 Series use of different methods
o The need to establish the meaning of purity
o The relation between increasing purity and lowering yields
 Short examination (schedule - open)
Reference Materials:
1. Lundblad, R.L., Techniques in protein modification, CRC Press, 1995.
2. Wong, S.S., Chemistry of protein conjugation & crosslinking, CRC Press, 1991.
3. Darbre, A., Practical protein chemistry: A handbook, Wiley, 1986.
4. Methods in enzymology, Academic Press, Vols. 11, 25-27, 47-49, 61, 91, 117, 130, 131, 135-137.
5. Bailey, J. L., Techniques in protein chemistry, Elsevier, 1962.
6. Lundblad, R. L., Chemical reagents for protein modification, 2 nd ed., CRC Press, 1991.
7. Walsh, G., Headon, D.R., Protein biotechnology, Wiley, 1994.
8. Mean, G., Feeney, R.E., Chemical modification of proteins, Holden Day, Inc., 1971.
9. McGrath, K., Kaplan, D., Protein-based materials, Birkhauser, 1996.
10. Koskinen, A.M.P. and Klibanov, A.M., Enzymatic reactions in organic media, Blackie, 1996.
11. Suckling, C.J., Gibson, C.L., Pitt, A.R., Enzyme chemistry: Impact & applications, Blackie, 1998.
12. Magdassi, S., Surface activity of proteins: Chemical & physicochemical modifications, Marcel Dekker, 1996.
13. Fersht, A.R., Structure & mechanism in protein science, Freeman, 1999.
14. Crieghton, T.E., Protein structure: A practical approach, 2 nd edition, Oxford University Press, 1997.
15. Wüthrich, K., NMR of proteins & nucleic acids, Wiley, 1986.
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