Lecture Notes Biophysics 204 Parts 1-3
Robert Fletterick
February 9, 2016
Protein - Protein Interactions
Part 1 Background biology and a look at some examples of protein
interfaces
Among the molecular interactions of proteins, their associations with other proteins are critically
important. On average a protein domain of about 200 amino acids makes interactions with 3 to
5 other protein domains. This implies that the 30,000 or so proteins in eukaryotic cells engage in
about 100,000 interactions. Sometimes stable assemblies of tens of proteins form transiently to
provide functional machines, such as transcription complexes.
Protein-protein interfaces are often dynamic, changing with small molecule binding to one
partner, or changing by covalent modifications such as phosphorylation or sumoylation
(covalent linkage of a SUMO protein (Small Ubiquitin-related MOdifier) to a target protein.
The interfaces are usually specific much like the specificity found in enzyme active sites and
large. Our reason to study the structure and chemical basis of these interactions is that they are
important in almost all aspects of molecular and cell biology.
Introduction
A few diverse examples of protein associations will demonstrate variations and are
instructive and teach us certain qualitative principles. The proteins that will we mention
in the following are not among those examples that will be treated in depth in the later
lectures.
Classification
There is no convenient to group the types of interfaces aside from the obvious one of
permanent, such as found in obligate homodimers, and transient such as found in
regulators of protein kinases by cyclins or inhibitors- focus paper 4. These differences
will be noted later.
One way of classification is by the function enabled by the association.
When proteins come together, the phenomenon can be considered to be primarilycatalytic, for assembly, capture to localize, or for purposes of stabilization or folding.
1. Catalytic- e.g. two proteins associate to form an active enzyme. This is common, for
example the dimerization of Tyr kinase signaling complexes (EGF receptor) at the
plasma membrane or intracellular kinases ( eg, protein kinase A) that interact with
regulatory proteins to form a complex.
Only the complex will catalyze phosphorylation of specific Ser, Thr, or Tyr side chains to
control the activity of the targeted protein substrate.
There are about 500 protein kinases that use phosphorylation to change the active site
structure or associations of the substrate (targeted) protein. These changes are usually
by allosteric rearrangements, manifest by the appearance of the phosphate somewhere
special in the tertiary structure in the substrate protein.
Example The most studied example is protein kinase A, PKA, which phosphorylates
substrate proteins at specific serines: PKA is found as an assembly of four subinits of
the form R2C2.
Engineering activation is interesting: the catalytic subunit C is inhibited by having an R
chain peptide segment, a mimic of the substrate, with Ala, not Ser presented to C’s
active site.
2. Assembly- identical subunits often form oligomers that are usually symmetric, the
most common association being dimers. [Whenever a dimer is found that does not have
twofold symmetry it is noteworthy.
For some oligomers, such as the dehydrogenase tetramer, the assembly is for integrity,
for other assemblies the active sites are spit between subunit interfaces; thymidylate
synthase. Example- G proteins, such as the oncoprotein Ras, are unusually poor at
hydrolyzing GTP and require GAP, G activating proteins.
How does GAP activate the G protein RAS? Ans. Arg from GAP added to active site of
Ras.
An amazing special case- virus coat proteins assemble with interfaces that are
functional even though they are deformable by pH or changes in Ca ion concentrations.
Virus coat proteins employ a remarkable trick. They are also deformable depending on
their potential binding partner. They are adaptable in their formation of interfaces. These
coat proteins form both pentamers and hexamers- using the same binding set of amino
acid residue interactions. The same residues can form interfaces around a five fold or
six-fold axis by adjusting atomic positions accordingly. Loosely, this is called
quasiequivalence.
Allosteric interfaces form another sub class, e.g., glycogen phosphorylase only
exists as a dimer and hemoglobin forms tetramers. For these two, and many others, the
interfaces bind effector molecules and rearrange not only the interface, but also the
tertiary structures and active sites and alter activity. In glycogen phosphorylase
dimers, both AMP and phosphoserine of the N terminus bind between the subunits and
activate glycogen breakdown by the enzyme.
To put this in perspective we will compare three homologs; those from bacteria,
yeast and mammals, in order of increasing complexity of allosteric regulation.
Jenny L. Buchbinder, Virginia L. Rath, and Robert J. Fletterick. Annu. Rev. Biophys.
Biomol. Struct. 2001. 30:191-209. Structural Relationships Among Regulated And
Unregulated Phosphorylases
Figure 1. Ribbon diagram of MalP dimer a glycogen phosphorylase homolog from E coli.
This is the precursor non allosteric version of the allosteric eukaryotic enzymes. The
binding site for maltodextrose, two glucoses linked by the same chemistry that links
glucoses in glycogen, is shown in magenta. The PLP cofactor- a vitamin B6, (yellow) is
covalently linked to the side chain of Lys-680 within the nucleotide binding fold
subdomain of the C-terminal domain. The active site of the enzyme is located in a
crevice between the N-terminal and C-terminal domains and is shown with bound
maltose (purple), glycerol (purple), and sulfate (pink).
E. coli phosphorylase is not regulated at the interface or elsewhere. The eukaryotic
homologs are regulated by Glc-1P, Glc-6P, purines, phosphorylation, glycogen, ADP,
AMP, ATP, metal ions and most importantly glucose.
Figure 2. Comparison of (A) phosphorylated (RCSB Protein Data Bank entry 1YGP) and
(B) unphosphorylated yeast phosphorylase. The phosphorylase dimer is depicted as a A
so-called “Connolly surface” (a modified solvent accessible) with one monomer colored
blue and the other purple. N-terminal residues 1-22, corresponding to the N-terminus of
muscle phosphorylase, are shown as a ribbon in white. The unique N-terminal extension
of yeast phosphorylase (residues -1 through -39) is drawn as a ribbon in pink. The
structure of yGPa is of a truncated form of the enzyme, which contained a deletion of the
first 22 N-terminal residues; therefore, only residues -1 through -14 of the N-terminal
extension are shown. The structure of yGPb is of the full-length enzyme; however,
residues -12 through -22 of yGPb were disordered and are not shown. In the
unphosphorylated enzyme, the N-terminal extension blocks the entrance to the active
site. Phosphorylation results in the movement of the N-terminal extension to an allosteric
site at the subunit interface where it displaces the inhibitor, glucose 6-phosphate
(orange). Thr-10, the site of regulatory phosphorylation is colored pink. The position of
the active site is indicated by the PLP cofactor (yellow) and in yGPa, by the additional
presence of a bound phosphate (pink). Hydrophobic residues (Phe-252, Leu-254, Phe257, Tyr-262, Tyr-163', Val-278', Tyr-280', Pro-281', Phe-285' and Leu-291'), shown in
coral, condense to form a hydrophobic cluster near the active site in yGPa.
Consider two features of the effector changing the shape and function of the
protein. One is the trigger following binding; the second is the response in
conformational stability in response to the trigger. You can imagine the features
which permit the enzyme to be stable when activated might be conserved whereas
the triggers and linkage mechanisms might change in evolution.
Consider triggers- a ligand binding or a phosphorylation. The test of understanding
would be to engineer a new binding site for ligand which still sends the enzyme into the
active conformation. For phosphorylase, protein interactions can be engineered to make
a novel allosteric switch. X-ray analysis showed that for mammalian enzymes, the two
phosphorylase subunits come closer together, and that change at the interface
rearranges the subunits and the configuration of the dimer. The tertiary structure
changes to become an active catalyst. The details are intricate, about 200 H bonds
rearrange. The energy driving stabilization turns out to be simple. The driving force for
the changes is hydrophobic, though the trigger is by AMP binding or Ser Pi binding at
the subunit interface.
The experimental test was to stop AMP activation by removing two AMP binding side
chains, calculated to be in the right position, so that if they were mutated to His, they
would bind Ni metal ion. Thus Ni was found to fit, and the enzyme was no longer
activated by AMP, but it could be activated by Ni ion- to about 10 % of AMP level of
activation. (Identification of the molecular trigger for allosteric activation in glycogen
phosphorylase. Michelle F. Browner, David Hackos, Robert Fletterick: Activation of
protein function through phosphorylation can be mimicked by the engineering of specific
metal binding sites. Nature Structural Biology 1, 327-333 (01 May 1994) )
The figure shows the structures of the three activated enzymes, by Ni, AMP or Ser Pi at
the subunit interface:
3. Capture- for regulation of activity or signaling. The example here is for protein
inhibitors for protein degrading enzymes.
Proteases are often kept from anarchy by binding to proteins that are protease inhibitors.
Ecotin is the most interesting example of a protease inhibitor as it functions a dimer
within a tetramer complex of the form E2P2. Ecotin is a member of the class of proteins
that David Eisenberg has termed domain swapped. This means that a segment of one
subunit extends a domain that packs into the second subunit. For ecotin a critical
element of its function is that the C terminal 15 amino acids are packed with the
symmetry related subunit.
Figure 3. Ecotin in yellow and magenta shows domain swapped C terminal strands. It
forms a tetramer with two proteases shown in green and blue. Ecotin binds the active
site, primary, and the terminal helix, secondary site, of the serine protease accounting
for the serine protease fold specificity.
Ecotin is one of the most amazing proteins known! It looks like any typical  sheet
protein, but ecotin is impervious to every serine protease, including the voracious
digestive enzymes. Even though ecotin will inhibit nearly all serine proteases of the
chymotrypsin fold, regardless of specificity and regardless of sequence divergence.
Two that are inhibited: fiddler crab collagenase and human trypsin are 30 % identical
in amino acid sequence. Collagenase degrades collagen, an especially stable structural
protein, and trypsin can work at low or neutral pH’s to break most any polypeptide chain
with an Arg or Lys. Yet ecotin strongly inhibits both! Surprisingly a few serine proteases
are immune: ecotin will not inhibit thrombin, but it is not digested, [Wang SX, Esmon CT,
Fletterick RJ. Crystal structure of thrombin-ecotin reveals conformational changes and
extended interactions. Biochemistry. 2001 Aug 28;40(34):10038-46.]
Figure 4A. The structure of cow thrombin assembled into a tetramer with two
monomers of ecotin. Ecotin does not bind without the mutation M84R. in shown as
cartoons of secondary structures. Light chain (L) and heavy chain (H) of thrombin are
shown in green, with catalytic triad in red. Ecotin is shown in purple; disulfide bonds and
calcium ion are highlighted in yellow and orange. The symmetry mate that helps to
construct a full tetrameric complex is shown in gray.
Figure 4B. A close-up of the interface between thrombin and ecotin M84R at the active
site of the protease denotes all surrounding surface loops in thrombin (the 37's, 60's,
99's, and 148's loops) and the two primary site loops in ecotin (the e50's and e80's
loops).
Ecotin was made to inhibit thrombin by changing a singe amino acid, the one presented
to the primary specificity pocket. A point mutation (M84R) in ecotin results in a 1.5 nM
affinity for thrombin. The crystal structure of thrombin with ecotin M84R mutant shows
that surface loops surrounding the active site cleft of thrombin moved to permit inhibitor
binding. Thrombin and ecotin M84R interact in two distinct surfaces. The loop at
residue 99 and the C-terminus of thrombin contact ecotin through mixed polar and
nonpolar interactions. The active site of thrombin is filled with eight consecutive amino
acids of ecotin and demonstrates thrombin's preference for the thrombin cleavage site.
What is the advantage to the ecotin tetramer? A dimer is a pair; a tetramer can be a
network.
Pay attention to networks in biology!
A chain, such as the typical dimer of protease protease-inhibitor, is only as strong as its
weakest link, while a network, as found in the tetramer is stronger than its strongest link!
Figure 5. Comparisons of conformations of (a) the 60's, (b) the 148's, and (c) the 99's
(d) the 37's loops bound with different inhibitors. Bovine thrombin structures, 1ETR.pdb
(green, bound to small inhibitor 2MQPA), 1BBR.pdb (blue, bound to fibrinopeptide
2 7-
16), 1TBR.pdb (yellow, bound to rhodinin), 1TOC.pdb (purple, bound to ornithodorin),
1UVT.pdb (gold, bound to small inhibitor MB14.1248) and the ecotin bound thrombin
molecule (red), are superimposed based on core residues. Trp60D in all structures in (a)
are shown in full stick model.
Engineering ecotin is easy. A single chain scEcotin can be made by a tandem repeat
of ecotin. A different type of monomer, one half the size of mEcotin or normal ecotin
can also be made. Ecotin’s domain swapped interface is required for it to function
physiologically but ecotin can be made into a single chain monomer or a single chain
dimer by extending the C terminus with two amino acids, allowing a tight turn and
allowing the C terminus to fold back onto the molecule.
Figure 6. Design of mEcotin and scEcotin. The dimeric structure of uncomplexed WT
ecotin is shown with one subunit colored by secondary structure and the other colored
gray. The dotted red lines represent the modeled mutations. mEcotin consists of an AlaAsp-Gly insertion after residue 130 to form a  turn. scEcotin involves a Gly-Gly-Gly
linker between the two subunits. Residues on one subunit are labeled prime and on the
other labeled non-prime. The side-chains are shown for the two Trp residues of each
subunit, 67 and 130.
Figure 7 Structure of mEcotin. (a) Ribbon plot of a 2.0 Å structure of mEcotin, showing
the  strands in yellow, helices in magenta, and flexible loops and turns in blue. The
introduced new turn is labeled red. The structure demonstrates the conservation of the
flattened  barrel fold and the folding back of the C-terminal arm onto the barrel.
We will see that antibodies have specialized genetically variable and mechanically
dynamic assemblies of loops just like those found in ecotin that capture their target
proteins called antigens.
Inter cell signaling is often by protein hormones that bind to cell surface protein
receptors as another form of assembly. We will study one case, growth hormone and
its receptor in detail.
Scaffold proteins or domains. Some proteins consist of soley one or many domains
used to nucleate assembly. Large and important classes of protein domains are used to
create functional multiprotein complexes. These domains come in many shapes.
Several families of proteins are used in protein network signaling or for anchoring the
protein to the appropriate location, e.g. the inside of the plasma membrane. Examples
are SH2, (SRC homology) SH3, PDZ, Zn finger (protein type), Fork head homology
and PHD (pleckstrin homology domain).
4.
Reforming- templates, e.g., chaperone proteins.
Figure 8 Each molecule of SptP binds as an unfolded polypeptide to three SicP chaperones. Two
views (a and b) of the SptP–SicP complex as cartoons related by a 90° rotation about a horizontal
axis.
SicP is shown with  helices as solid cylinders, and  -strands as thick arrows. The helices of
SptP are shown as ribbons, with the two different polypeptide chains shown in red and blue.
The non-interacting middle two molecules of SicP at the center of the complex are shown in
magenta, and the outer two SicP molecules of the tetrameric chaperone arrangement are shown
in green.
The SicP chaperone homodimer pairs are therefore mixed with one green and one magenta
molecule.
The secondary structural elements of SptP are labeled, and the four important regions of contact
between SptP and SicP are labeled A–D in orange (for one SptP molecule).
Regions C and D at the carboxyl terminus of the chaperone-binding domain are separated from
regions A and B by a long helix (H3) that makes few contacts with SicP, and runs across the
homodimer pair from the outer SicP chaperone to the middle chaperone of one homodimer pair.
From C. EREC STEBBINS AND JORGE E. GALÁN
Nature 414, 77 - 81 (2001) Maintenance of an unfolded polypeptide by a cognate
chaperone in bacterial type III secretion.
Two points about relevance of our studies:
1. MOST proteins are oligomers. In copying DNA to RNA, transcription, several complexes of
20 to 30 proteins form on DNA. The machine is a cluster of proteins, dynamic and often loosely
held and the interfaces consist of changing protein-protein interactions.
2. A select set of protein interfaces may be appropriate for pharmaceutical development
provided that formation of the interface is critical for function and is formation can be blocked by
binding a small organic molecule, a drug.
3. Most proteins form from domains of a hundred or more amino acids. Most domains form
interactions with 5 or more proteins. Some proteins, like p53, Androgen receptor and actin
interact with 25 to 100 proteins.
Specificity Versus Affinity
In Biology some processes require great specificity, does this demand tight affinity? Antibodies
which can be very specific for their epitopes on target proteins typically bind tightly. Other
proteins like actin or p53, bind to 10 or more proteins using the same surface so affinity can is
usually weak (1- 10 micromolar).
Part 2
Discovering protein to protein interactions- Qualitative and
quantitative
1. Protein affinity chromatography- pioneered by B. Alberts.
Formosa, T et al, 1991, Meth. Enzymol. 208:24-45.
Strategy - attach a specific bait protein to a gel matrix and flow over a clean (after high speed
spin) cell extract. Non-interacting proteins flow through and interacting ones stick to be later
eluted by altered conditions.
How to make this work:
Build an attachment handle onto the protein. Attach the protein to a chemically active gel matrix
covalently or attach a high affinity antibody for the protein to the gel matrix, or add a six tandem
His extension, called a His tag, or add a protein flag in the sequence at either the N or C
terminus, eg a GST (glutathione S transferase- very soluble, very stable) fusion. Care must be
taken to have protein and cell extract relatively pure. Controls are not possible. Extracted
proteins must be tested in assay of function. This technique is limited to protein assemblies that
are more tightly bound than about micromolar Kd’s.
2. Immunoprecipitation:
An antibody is created by monoclonal technology. The antibody, Ab, is added to a cell extract,
with the Ab recognizing the protein target itself, or a tag that was added to the protein to more
easily purify it, such as six repeated His residues at the N terminus. Ab-protein complex is
fished out by beads that themselves bind to Fc portion of all antibodies- these are Protein A
coated beads. Protein A binds specifically to Fcs.
Finally, gel electrophoresis or mass spectrometry of the purified proteins can then identify
binding partners.
Can you think of a problem? In spite of years of effort this technology could not be made to
work to find binding partners for RAS.
Question- what is wrong with using a single MAb to your favorite protein?
Ans. The MAb may cover the binding interactions! Abs can be made to any surface of a protein,
so you would use three different Abs.
3. Two hybrid system
Fields, S and Sternglanz, R. (1994) The two hybrid system: an assay for protein protein
interactions. TIG 10(8):286-292.
Modular proteins often control DNA transcription, a DNA binding part, a DBD, and a protein or
ligand binding part- the transcriptional activator. Activation of transcription recruits two or more
protein partners to the DNA at the right place.
The DNA response element- defined as say six consecutive nucleotides of a designated
sequence, binds a protein X linked to a DBD. One of the two hybrids is therefore- DBD1-X.
The reporter gene being controlled, (produces a blue color if transcribed, for example) also
needs the RNA polymerase to be assembled through a transcription activator, TA, that must be
recruited to the DBD site.
The second hybrid is of the form Protein-Y and is covalently linked to TA. Y-TA. Transcription
requires formation of TA-X:Y-DBD1 onto DNA and so reports the X:Y interaction. Sequence
the DNA in the blue yeast to find the identity of Y.
Problems with this technology are well known and limit the applicability, yet the method is
almost always used. The principle difficulties are that:
1.
Extracellular proteins may not be stable in the yeast nucleus.
2.
Fusion proteins may auto activate giving false positives.
As you expect, the recovered potential partners are unreliable as the technology is nonlinear
and non quantitative: it may also pick up extraneous proteins so that other reasons for
considering the protein partnership must be evaluated.
4. Phage Display
M13 is a bacteria virus or phage. It tolerates changes and is still infective. M13 has inserted into
its genome, a sequence coding for a foreign protein that on the mature phage is expressed as a
coat protein. The grown phage are bound to a bead, containing the protein of interest and the
phage that stick are isolated and grown and the DNA sequence is determined to identify the
protein that sticks.
Libraries can be used.
Technical problems can be overwhelming. Panning and washing are art and careful attention is
essential to finding a signal.
A successful example:
Lowman et al. 1991. Selecting high affinity binding proteins by monovalent phage display.
Biochemistry. 30: 10832-10838.
5. Spin down assay commonly called pulldowns or SPA.
Pulldown assays are not quantitative but have the advantage of showing a physical interaction
even when the proteins can be difficult to prepare or purify. Few systems can synthesize any
protein on a preparative scale, a limitation to an important goal in biotechnology. Three
strategies are used: chemical synthesis (limitations are folding, chemistry issues, size and
others), in vivo expression (limitations are induction and stabile product in the heterologous
cell), and cell-free protein synthesis. Cell-free translation systems can synthesize proteins with
near in vivo rates and can express proteins incompatible with cell physiology. Cell-free
translation systems are however inefficient and unstable. Wheat embryos can synthesize
proteins in a dialysis bag used to continuously supply cellular substrates and provide removal of
byproducts. Such systems can show active translation for three days, yielding 1–4 mg of active
proteins.
Even the simple reticulocyte assay is effective as shown for GST labeled androgen receptor
binding to 160 kilodalton protein SRC2: The expression vectors for the GST-AR (amino acids
646-919) and SRC2 are simple to make. SRC2 labeled with 35S methionine is easily produced
using the TNT-Coupled Reticulocyte Lysate System (Promega, Madison, WI). The GST fusion
of the receptor is expressed in E. coli and purified through its GST tag protein (the protein fusion
is anchored to a solid support -agarose-glutathione beads. Only small amounts of material are
needed because the signal is radioactivity.
For binding assays, bead suspensions containing 10µg of GST fusion protein are incubated with
a few l of 35S-labeled SRC2 in buffer containing 2µg/ml bovine serum albumin to protect the
proteins (BSA binds thousands of small molecules better than most proteins!). After incubation
for 2hrs at 4°C, beads were washed (three times) with buffer and heated to 100°C for 3 min.
Bound proteins are separated by SDS-PAGE and visualized by autoradiography.
Even weak interactions can be measured. There is a relatively weak interaction between Ras
and the GTPase-activating protein, GAP, neurofibromin (NF1). The complex between the
catalytic domain of NF1 and the GTP-form of Ras dissociates rapidly. Skinner RH; Picardo M;
Gane NM; Cook ND; Morgan L; Rowedder J; Lowe PN. Direct measurement of the binding of
RAS to neurofibromin using a scintillation proximity assay. Analytical Biochemistry, 1994 Dec,
223(2):259-65.
SPA technology requires no separation step as implied in the first example above. Ras was
complexed with labeled GTP and was mixed with NF1 fused with glutathione S-transferase
(GST), anti-GST, and protein A-coated SPA beads. The complex is centrifuged via the beads
and the proteins can be counted, or visualized on PAGE.
GST-Ras fusion protein was bound to protein A-coated SPA beads. This technology is readily
extended to the measurement of other protein-protein interactions.
Measuring Affinity- Experimental:
The analytical ultracentrifuge of years ago was archived to the back room. But it was reborn as
a small sleek unit with appropriate software. The AUC and chromatography are important tools
in showing and measuring affinity. A vintage paper using traditional methods,
Becerra SP; Kumar A; Lewis MS; Widen SG; Abbotts J; Karawya EM; Hughes SH; Shiloach J;
Wilson SH; Lewis MS.
Protein-protein interactions of HIV-1 reverse transcriptase: implication of central and Cterminal regions in subunit binding. Biochemistry, 1991 Dec 17, 30(50):11707-19.
HIV reverse transcriptase (RT) purified from virions is composed of a 51,000 Mr
polypeptide and a 66,000 Mr polypeptide thought to be in heterodimer structure and are
identical except for a 15,000 Mr C-terminal truncation.
Recombinant RT’s as the approximately 66,000 Mr polypeptide (p66) or as the
approximately 51,000 Mr polypeptide (p51) in analytical ultracentrifugation studies in 0.25
M NaCl at pH 6.5 revealed that p66 was in monomer-dimer equilibrium with KA of 5.1
x 104 M-1.
p51 failed to form dimers and behaved as a monomer under these conditions.
Mixing p66 and p51 polypeptides resulted in a 1:1 heterodimer with KA of 4.9 x 105 M-1.
This KA indicates a tighter complex.
These results on formation of the P66/P66 homodimer and P66/P51 heterodimer were confirmed
by gel filtration analysis using FPLC Superose-12 columns.
Binding between p66 and individual p66 segment polypeptides also was observed using an
immunoprecipitation assay.
Binding between p51 and p66 in this assay was resistant to the presence of approximately 1 M
NaCl, suggesting that the binding free energy has a large hydrophobic component.
Note that it is easy to adjust salt and pH in the AUC to probe type of interactions
Individual RT peptides p51 and p66 form a 1:1 heterodimer and suggest that the central
region of p66 is required for this subunit binding; the C-terminal region (15,000 Mr) of
p66 appears to be required also, as p51 alone did not form dimers.
The analysis of UC data can be difficult- See Creighton, page 267 or Cantor and Schimmel
p591 for a discussion.
Chromatography is more readily interpretable:
Quantifying protein to protein interactions kinetics and equilibrium constants remains difficult.
Part 3 Methods -Surface Plasmon Resonance
The focus paper uses SPR to measure protein associations.
SPR is used for measuring kinetics, that is on rate and off rate of the ligand and from these
binding constants may calculated by ratio of the two rates.
The predominant experimental methods use the BIA core machine from Pharmacia. The
machine is expensive and employs an unusual signal and detector system – surface plasmon
resonance.
The interaction of the evanescent wave with free electrons of metal (plasmons) decreases
reflected intensity. The resonance phenomenon, constructive amplification, occurs only at a
particular angle of incidence, which is an unusually sensitive function of the refractive index.
Ultimately, the refractive index is dependent on the concentration and molecular weight of the
solute near the metal. The resonance angle is monitored and measured to several significant
figures. Note that light does not pass through the detection volume.
In the BIA core, the detector is on an integrated cartridge with continuous buffer flow and
injection of defined aliquots of sample over the active surface. The gold film is under a 100nm
dextran phase to produce a hydrophilic surface. Sensitivity is about 1 x10-4 degrees in a range
of 3°. In the experimental measurement, the smaller macromolecule is immobilized on the gold
dextran surface and the other molecule is flowed over the surface while the resonance angle is
measured (hence the change in refractive index reporting the new molecular weight of the
complex that is formed.) It is simpler to measure changes in response than to measure
absolute values.
SPR can be exquisitely sensitive. There are two choices for the stationary and moving
phases, called the ligand and analyte. Since the moving phase contains the mass that is
to be caught, sensitivity will increase if the larger molecule is flowing over the attached
smaller molecule. So for measuring a peptide –protein complex, one might put the
peptide on the immobilized plane and flow the protein over it. However, newer
instrumentation is very sensitive. Glycogen phosphorylase dimers of molecular weight
200,000 were in the immobilized phase and flowing over it were compounds of about
200 daltons. The experiment produced fine signals as shown in the following reference:
Ekstrom JL, Pauly TA, Carty MD, Soeller WC, Culp J, Danley DE, Hoover DJ, Treadway
JL, Gibbs EM, Fletterick RJ, Day YS, Myszka DG, Rath VL. Structure-activity analysis of
the purine binding site of human liver glycogen phosphorylase. Chem Biol. 2002
Aug;9(8):915-24.
Figure 1. SPR Analysis of the Caffeine/HLGPa Interactions(A) Binding responses were
measured for caffeine (500 μM–2 μM by 2-fold dilutions) injected over a surface to which
21,000 RU HLGPa had been coupled.(B) Equilibrium analysis of the caffeine/HLGPa
interaction. Equilibrium binding responses for caffeine in the absence (filled squares)
and presence (filled circles) of 50 mM glucose were independently fit to a 1:1 interaction
model (solid lines) to determine equilibrium dissociation constants.
Figure 2. Purine Site Screen
For each of the 18 compounds tested, the RU values at equilibrium versus ligand
concentration were fitted to a 1:1 interaction model. The resulting fitted curves are
superimposed and listed in order on the right starting from the curve closest to the upper
left (riboflavin) to the curve closest to the lower right (allantoin), with the labels color
coded to match their respective binding isotherms. For clarity, only the curves for
riboflavin, caffeine, and uric acid are labeled directly.
Advantages and UsesLabel Free
Small Volume
Kinetic on and off rates
Thermodynamic analysis
Biacore technology has been used for:
Interaction analysis for measuring associations of Protein, DNA, RNA, Peptide, Small
Mol, Cell, Membranes
Functional Proteomics
Epitope mapping
Screening for protein interactions or ligand binding
Ligand Screening
The experiment is a real time analysis with individual on and off rates determined - ka
= kon & kd = koff
–
And the equilibrium constant is KD= kd/ka
The instrument has three important components as shown above.
The gold dextran surface on the chip has special properties:
ligand
Specific layer
Dextran layer
Linker layer
Biocompatible
Low non-specific
binding
Gold
Glass
Robust~ 100 runs on the
same surface
Most importantly, Immobilization must create a functionally active surface.
The ligand can be linked to the chip in many ways; most commonly via a linker system:
Streptavidin : Biotin
Anti-Biotin : Biotin
RAMFcg : MAb
Anti-GST : GST
Ni2+•NTA : 6xHis
The ligand can also be linked chemically:
Amine
Ligand Thiol
Surface Thiol
Maleimide
Aldehyde
The SPR detection system relies on the resonance of surface plasmons at the conductorinsulator interface.
The response units in the sensogram are 0.0001 degrees, corresponding to about 1 picogram
of mass per square millimeter. Note that the intensity shifts between I and II; it is II where the
analyte binds to the ligand. The second inset graph show the typical sensogram as a function
of time.
The sensogram shows many features of the binding event, summarized here:
Controls are important components of protein interaction analyses. To show that the
interaction is specific we should have:
Control surface
Positive and negative control compounds
Positive inhibition
A critical ontrol surface is necessary to correct for bulk response and show binding is not
nonspecific.
A positive inhibition test will verify specificity.
Recognizing non specific binding can be difficult. The causes also include a
dirty chip, attachment to dextran or problems with the reference cell. One way
to check is to repeat the runs at different temperatures, pH, or salt
concentrations. The nonspecific binding will be affected by detergent or salt
concentration depending on the nature of the interaction. Good Biacore data
requires careful development steps.
Data analysis is done with the Biacore software. A range of concentraions
should be used and as ususal, study the residuals to the fitting to look for
systematic errors.
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