Marisa Fernández

mmfernan@ffyb.uba.ar
fernandezmarisa398@gmail.com
IMBS 2024
Overview Immune Response

Th
17
 Reagents

In solution Solid Phase

AUC Model Fitting

SPR

ITC Experimental data

Surface Plasmon Resonance
 What surface plasmon resonance (SPR) is good for?

 specificity of molecules interactions

 on and off rates (kinetics)

 binding strength (affinity)

 Thermodynamic characterization

Principle

SPR-based instruments use an optical method to

measure the refractive index near (within ~300
nm) a sensor surface.
 angle I = reference cell
angle II = reactive cell

1- Change in the refelected light angle. The signal is shown in Resonance

Units (RU)
2- Changes are related with the analyte concentrataion and the streght of
the interaction
Biacore T100
 D
RU
A
E
R

RU
2400

2200 Time (s)

2000

1800

1600
Response at different concentrations
Response

1400

1200

1000

800
0 50 100 150 200 250 300 350 400 450 500
Time s
pH Scouting. Optimal pH to obteim a proper copupled
o the chip
BIAevaluation
 What surface plasmon resonance (SPR) is good for?

Principle

SPR-based instruments use an optical method to

measure the refractive index near (within ~300
nm) a sensor surface.
 Steady State Affinity
(SSA)

Specific binding Response in the equilibrium

Raective cell

RU
RU RU

2400 1200
1180
2200 1000

2000 800
1130
1800
600
Response

1600 1080 400

1400
200
1200 1030
0
1000
-200
800 980 0 50 100 150 200 250 300 350 400 450 500
0 50 100 150 200 250 300 350 400 455000 0 50 100 150 200 250 300 350 400 450 500 Time s
Time s Time s

K *C*Rmax
Re q = A
(KA*C*n+1)
Non linear regression
SE or standard
desviation

25
 Koff
Kon Regeneration

Global fit

Association Dissociation

KD = koff/kon
 RU Plot
500

450

400

Relative Response - binding

350

300

250 Sample

200

150

100

50

0
C onc m g/ m l

RU Plot
800

700

600
Relative Response - binding

500

400
Sample
300

200

100

-100
C onc m g/ m l
 What surface plasmon resonance (SPR) is good for?

Principle

SPR-based instruments use an optical method to

measure the refractive index near (within ~300
nm) a sensor surface.
The equilibrium constant for the reaction is
determined the standard free energy
change:
ΔG° = RT ln KKeq
Structural Immunology
 A single molecule

•Relationship between structure and function

•Evolutionary pattern

A complex

•Determine the interface of interaction

•Hot spots involved in the interaction

•Speculate about putative binding site

 X ray diffraction
Limitations
•Crystal genesis is a random process

•Amount of protein

•Crystal structure is an snap shot of the molecule

•Crystal contacts and biological contacts

•Determine the unit cell

•You can not determine the position of the H

•A crystal is a solid with a regular repeating
internal three-dimensional arrangement of atoms.
•This periodic arrangement can be exploited to
determine molecular identity and structure when
the crystal is exposed to x-rays.
•X-rays are used because their wavelengths
correspond to inter-atomic distances.
 X-ray sources

Diffractometer and Synchrotron

•The mounted crystal is then irradiated with a beam of monochromatic X-

rays.

•The brightest and most useful X-ray sources are synchrotrons; their
much higher luminosity allows for better resolution. They also make it
convenient to tune the wavelength of the radiation, which is useful for
multi-wavelength anomalous dispersion (MAD) phasing.

•Synchrotrons are generally national facilities, each with several

dedicated beamlines where data is collected around the clock, seven days
a week.

•Difractometers at house: rotating anode type source

 Recording the reflections

•When a crystal is mounted and exposed to an intense

beam of X-rays, it scatters the X-rays into a pattern of spots
or reflections that can be observed on a screen behind the
crystal.

•The relative intensities of these spots provide the

information to determine the arrangement of molecules
within the crystal in atomic detail. The intensities of these
reflections may be recorded with photographic film, an area
detector or with a charge-coupled device (CCD)
•Pattern location and intensity are used to
determine size and composition of the molecule
respectively. The phase relations of the diffracted
beams are resolved mathematically before a model
structure is deduced (referred to as the "Phase
Problem"). Molecular Replacement, Heavy Atoms

• Using computer software (ccp4i), structure

parameters are systematically adjusted to give the
best fit between observed intensities and
calculations from the model structure (CNS, Xtal
View)

•The final determination yields atom identities and

positions in the unit cell and bond lengths and
angles derived from the atom positions.
•Probably the most famous X-
ray diffraction image is the
photograph of the B form of
DNA taken by Rosalind Franklin
in May 1952
 PyMOL

•It is an open-source, user-sponsored, molecular visualization system

created by Warren Lyford DeLano and commercialized by DeLano
Scientific LLC.

•It is suited to producing high quality 3D images of small molecules

and biological macromolecules such as proteins.

•According to the author, almost a quarter of all published images of

3D protein structures in the scientific literature were made using
PyMOL.

•PyMOL is one of few open source visualization tools available for use
in structural biology.

•The Py portion of the software's name refers to the fact that it

extends, and is extensible by the Python programming language