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Lecture Notes - 2 7.24/7.88J/5.48J The Protein Folding Problem
Handouts: An Anfinsen paper Reading List
Anfinsen Experiments
The Problem of the title refers to how the amino acid sequence of a polypeptide chain determines the folded three-dimensional organization of the chain:
- Does the sequence determine the structure??
- Protein Denaturation
- Emergence of the Problem
- Ribonuclease A
- Refolding of ribonuclease in vitro.
A. Protein Denaturation/Inactivation
One of the features that identified proteins as distinctive polymers was
1) Unusual phase transition in semi purified proteins
When exposed to relatively gentle conditions outside the range of physiological
- heat
- pH
- salt
- organic solvents, e.g. alcohols
Lets consider the familiar transition I mentioned last week;
- Heat denaturation; cooking the white of an egg
- Acid denaturation; Milk turning sour
Macroscopic changes in bulk solution; scatters visible light, increase in viscosity - white of egg: >>heat; opaque, hard; cool down; no change: Coagulation, Aggregation, precipitation, denaturation:
One of the components is egg white lysozyme: activity assay; hydrolysis of bacterial cell walls:
Activity remaining vs. temperature on same axes This transition: is Denaturation. These transitions were in general found to be irreversible: activity was not recovered upon cooling:
Historically the study of the folding and unfolding of proteins emerged from trying to understand this unusual change of state or phase transitions.
B. Emergence of the Protein Folding Problem
In the period 1958-1960, the first structure of a protein molecule - myoglobin, the oxygen binding protein of muscle - was solved by John Kendrew in 1958, followed soon after by the related tetrameric red blood cell oxygen binding protein, hemoglobin, solved by Max Perutz, both of the British Medical Research Council Labs in Cambridge, U.K.
“When the folding of the hemoglobin chain emerged from the model building process, it was shocking. The crystallographers expected each molecule to have high internal symmetry. In fact as Kendrew said, myoglobin is almost nothing but a complicated set of helical rods sometimes going straight for a distance then turning a corner and going off in a new direction;.much more complicated and irregular than most of the early theories of the structure of proteins had suggested.”
What’s so surprising; Well like the epicycles every educated person knew that heavenly movements had to be circles; When Kepler showed that orbits were elliptical - a radical departure.
Same with proteins; assumed without even being aware, highly regular, formed crystals, diffracted;
When it turned out the fold was irregular!!??
Question immediately arises as to how that fold was accurately specified? Since proteins crystallized, many if not all in that fold to high precision.
As it became clear that what genes specified was sequence of amino acids,
1) How was fold determined?
Emerged as central question in modern biology.
Initially, very unclear:
- Since both proteins and DNA helices, perhaps DNA as direct template? Popular, No experimental support emerged.
- mRNA as template? No physical interaction found.
- Ribosome as template? Yes, but proteins different from each other, ribosomes the same.
- Soluble catalysts? Maybe, but most catalysts, proteins, how would their structure be determined?
- No template!? Mature fold determined by the amino acid sequence directly:
The view that 3-D structure is determined by 1-D amino acid sequence, rested for three decades on a series of enormously influential experiments carried out by Christian Anfinsen, Frederick White, Michael Sela, and Edgar Haber at the National Institutes of Health. Behooves us to examine carefully those initial experiments.
So not by chance that RNAse was so amenable to purification and characterization evolved property of protein.
RNAse was protein used to develop very basic methods of amino acid composition analysis,
- Gel filtration - sephadex and
- DEAE chromotography,
- Edman reagent
- Tryptic mapping
- many other techniques
b) Disulfide Bonds
Stanford Moore and William H. Stein “Chemical Structures of Pancreatic Ribonuclease and Deoxyribonuclease” Science , 180 , 4 May 1973, 458-464.
“The sharing of knowledge among academic scientists and industrial designers of instruments and manufacturers of ion exchangers has played an important role in the progress of biomedical research in this field”
Bovine Ribonuclease A: From pancreas and intestines of cows. Major species is
Description of Bovine Pancreatic Ribonuclease A:
- 124 amino acids.
- 13,700 daltons;
- 37% beta strand, 23% alpha helix, rest coil
8 cysteines as four disulfides;
N--Cys26 - Cys40 – Cys 58 - Cys65 - Cys 72 – Cys84 - Cys 95 - Cys 110 – C00H.
26-84 (between beta strand and helix) 40-95 (between loops connecting beta strands.) 58-110 (helix to strand at the end of sheet) 65-72. (loop to strand) Reactivity of S-S bonds quite heterogenous
Quick Review of SH and S-S chemistry:
The thiol group of Cys is most reactive of any amino acid side chain. HN – C – S-H C= It ionizes at slightly alkaline pH; the thiolate ion is reactive form;
For free amino acid pka of 9.0-9.5. In Proteins pk 8.5;
but pk very sensitive to local electronic environment, so cannot assume this within proteins.
Reacts with very rapidly with alkyl halides such as iodoacetate or iodoacetamide to give stable alkyl thio ether derivatives;
Thiols form a variety of complexes with metal ions, Organic mercurials such as para chloro mercuri benzoate.
Reaction with latter can be detected by spectral changes; former by incorporation of radioactive amino acids.
Cl- with Hg+. make heavy atom derivatives for crystallography. Change in absorption max allows easy quantitation.
Sulfur atom is also susceptible to oxidation by oxygen in a reaction that is probably catalyzed by trace heavy metals.
End products is CH2 - S - S - Ch air or other oxidants, to the sulfoxide:
Note that this reaction is multiple step: P-C-S-S--P + S-C-C-OH >>C-S-S-C-C-OH + -S-C -P
Mechanism of Catalysis: Are S-S bonds key to catalysis??
In trying to understand the catalytic activity, first question was, does it depend on oxidized S-S bonds?
So attempted to prepare fully reduced Rnase: Standard methodlology for S-S Reduction: Purified 4-S-S Rnase + Excess Mercaptoethanol >>
Rnase 4 SH reduced, but 2 S-S remained, refractory tp reduction:
Then
M. Sela, F. H. White Jr, and C. B. Anfinsen (1957) Reductive Cleavage of Disulfide Bridges in Ribonuclease Science, 125, 691-692.
Highly Purified RNase:
- 8M urea, H2N – C (=O) – NH
- 0.2 M Beta-mercaptoethanol
- pH 8.5,
- 23-24 oC,
- Purge with Nitrogen
Purge with nitrogen for 15 minutes [Why??], then
incubate in stoppered tube for 4 hrs at room temp.
Reduced, inactive, and [presumably, fully unfolded]:
Precipitate protein with acetone, on ice: removes urea, mercaptoethanol : For reoxidation , dissolve to 2mgs/ml, add 0.02 M Na2HPO adjust to pH8.0 with a few drops of NaOH
Oxidize in g250 ml graduated cylinder, with air coming in through tube, 1 bubble/2-5 seconds!
Regained active Ribonuclease!!
Critical Question: Is refolded material equivalent to starting Rnase??
- Reoxidized material shows same ultraviolet 2) absorption spectra – tyrosines and tryps in same environment;
- chromatographically material similar to starting. Major A peak and minor B peak. Same elution.
- Same specific activity enzymatically.
Are disulfides the same? Carried out digest with proteases Subtilisin and Nagarse>> paper electrophoresis/chromatography.
Same S-S peptides spots.
UV spectra of reduced has peak slightly shifted from native, presumably because of altered environment disrupts tyrosine OH hydrogen bonds.
This reoxidized material was crystallized into two of the well known crystal forms of the proteins. Those crystals diffracting to good resolution and the same lattice constant, indicating the same space group indicating very similar structure}.
Characterizing and optimizing reaction conditions and examining dependence on protein concentration:
A third series of improvements in the procedures were described by C. B. Anfinsen and Edgar Haber: Studies on the Reduction and Re-formation of Protein Disulfide Bonds J. Biol. Chem, 236 , 1361-1363.
Careful examination of effects of reaction variables on yield:
In order to oxidize, reduced chains have to be separated from reducing agent. Key improvements:
- Stop reduction reaction by lower pH to 3.5 with glacial acetic acid. After reduction,
- Isolate reduced chains by column chromotography, rather than acetone precipitation: o Cross-linked dextran sephadex G-25 column equilibrated in 0.1M acetic acid. o To separate reduced protein from residual reducing agent dump thioglycolic acid.
- Dilute, rather than bubble- denaturation at air/buffer interface?
Fraction Number In effort to increase yield of activity, and to decrease insoluble material,
Identified three additional experimental variables:
- protein concentration; optimal yields at low protein concentrations
- thiolation of amino groups by impurities in sulfhydryl agent
- inactivation of protein by cyanates generated from urea solution
Protocol. Take fractions from column, adjust to PH 8.5 and allow to stand for 20 hours.
Table 1. Oxidation of reduced RNase at various protein concentrations
Conc. of Red. Prot. % Yield Sol. Act of Sol. % Recovery 7.0 27 31 8 4.8 42 70 29 2.3 87 75 65 0.9 100 77 77 0.35 100 94 94
Activity of soluble is expressed with respect to an equivalent concentration of native RNase.
Note that inactive chains must fall into two categories: insoluble and soluble: What do these states represent? What does it mean to have a soluble folded RNAse that is enzymatically inactive? Mispaired disulfides!!?
Evidence: Reoxidize in presence of urea!! Reform 4 disulfide bonds, about regain less than 1% of activity. Presumably scrambled ribonuclease.
