Biophysics and Bioengineering for Pharmaceutical Sciences
Introduction to Biophysics and Bioengineering
Biophysics = branch of science concerned with the application of physical principles and methods to biological problems
1892 – Karl Pearson introduced the term (“biophysics not improbably has an important future”)
Physics = a science that deals with matter and energy and their interactions - generic principles that control nature
Drug = a substance used to treat dysfunctional human body (and other biological organisms)
Proteins = primary functional elements in human body drug has to target protein to correct for a dysfunction
Protein = matter drug = matter drug interacts with proteins (matter with matter - = physics)
Birth of biophysics: finding protein structure (Kendrew & Perutz – first to defined structure of protein)
You need to know the structure of a protein in order to be able to design a drug for it
Biophysics fields: cell biophysics, biophotonics, structural biology, biomechanics, molecular biophysics, theoretical biophysics,
systems biophysics, nanobiophysics
Bioengineering = the application of engineering principles, practices and technologies to the fields of medicine and biology
Engineering = the application of science and mathematics by which the properties of matter an the sources of energy in nature
are made useful to people | = the branches of science and technology concerned with the design, building and use of
engines, machines and structures
Engineering vs. Science:
- Creating new things or processes using existing knowledge - Creating knowledge
- Need/application driven - Hypothesis driven
- Results are expected to be implemented - Results are expected to be published
Bioengineering field: biomaterials, bioinformatics, medical imaging & bio-optics, rehabilitation engineering, cell & tissue
engineering, bioinstrumentation, biomechanics, biotransport & drug delivery, genetic engineering, biosensors & electronics,
neural engineering, bionanotechnology
Biophysics and bioengineering are growing fields (bioengineering rose after biophysics had become a thing)
Donald Ingber: organ-on-chip | David Weitz: microfluidics | Bob Langer: nanoparticle technology | Alexander van
Oudenaarden: application of biophysics to medical problems | Uri: theoretical biophysics, systems biology
Initiatives: Wyss institute – for organ-on-chip | Max Planck – for physics and medicine
Physics for pharmacy ≠ medical physics
Medical physics: CT-scan, MRI – instruments for diagnostics
Use physics to understand a system and then use understanding to develop drug
Consortium ATOM was created to address challenges in pharmaceutical sciences
Eroom’s law = cost of developing a drug is high and increasing – time to develop is long, much money needed, start with many
drugs and most of them fail new innovative approaches needed use physics and engineering
Molecular Biophysics for Pharmaceutical Sciences
Molecular biophysics: I. Structural biology (bulk analysis) II. Single-molecule biophysics (single molecule)
Structural biology = a branch of biophysics concerned with the molecular structure of biological macromolecules, especially
proteins and nucleic acids
Importance: function of biological macromolecules is intricately related to their 3D shape & structure
Molecule structure - protein:
Primary – sequence (which amino acids in what order?)
Secondary – local folding (α-helix, β-sheet)
Tertiary – long-range folding (how chain folds like a rope)
Quaternary – multimeric organization (when multiple proteins come together to form a complex, e.g. ribosome)
Supramolecular – large size assemblies
Watson & Crick – structure of DNA | Perutz & Kendrew – first structure of protein | Pauling – α-helix | Rosalind Franklin
– X-ray diffraction of DNA (α-helix experimentally) | Dorothy Hodgkin – structure of B12/insulin
Tertiary protein structure: protein folding 3 approaches to measure protein folding
1. Experimental determination (X-ray crystallography, NMR)
2. Comparative modeling based on homology - compare unknown with known structure if sequences are similar
3. Ab initio (de novo) prediction - make model of polymer that moves & with interactions find minimum energy / in
time find structure that is energetically & entropically best (Molecular Dynamics simulation)
X-ray crystallography: used to determine 80% of structures
- Requires: high protein concentration, crystals (pure protein needed to get crystals) - impurity = no crystallization
- Solubilization of the over-expressed protein
- Structure determination by diffraction of protein crystals
- Size of molecule: no theoretical limit
Crystals act as a 3D grating & produce diffraction diffraction pattern contains complete info on placement of
scatterers (= electrons in atoms)
By Fourier transforming the diffraction pattern, we obtain info on the structure of the molecule in the crystals
, Nuclear Magnetic Resonance (NMR):
- Magnetic field applied to proteins in solution
- Does not require crystallization
- Solubilization of the over-expressed protein (must be soluble)
- Structure determination of a molecule as it exists in solution
- Size-limit is a major factor
PROs: high resolution info | does not require a protein crystal & not affected by crystal contacts | can be used to
study flexible /dynamic proteins | reflects conformational averaging
CONs: requires high conc of soluble protein (to generate signal, because not super sensitive) | cannot be applied to
large proteins (800kDa max) | cannot be used with amyloid fibrils (not soluble)
Measures nuclear magnetism or changes in nuclear magnetism in a molecule
NMR spectroscopy measures the absorption of light (radio waves) due to changes in nuclear spin orientation
Light = electromagnetic wave (when you apply light to sth, you apply a magnet to it)
NMR only occurs when a sample is in a strong magnetic field
Different nuclei absorb at different energies (frequencies)
Atom has spin = nucleus of atom acts like little magnet interacts with external magnet (H1, C13)
Not all atoms have this magnetic property (spin) - C 12 does not, but its isotope C13 does
Spin interacts with externally applied magnetic field spin orients towards certain direction at a specific
frequency an additional oscillating magnet can interact with the spin H 1 or C13 atoms absorption of energy by
system that depends on environment (neighborhood) of atom of interest (e.g. everything around specific H atom
affects interaction) because of this you can learn about the neighborhood of atom & by putting neighborhoods
together, about the structure of the protein
Spin interacts with neighborhood through J-coupling or chemical shift
Molecules in solution rotate very fast many signals: average out simplifies equation of interactions (equation
becomes linear if proteins rotate fast) interpretable extract structure of protein from equation
The larger the protein gets, the slower it rotates complex equation (not linear) data not interpretable
Disadvantages:
o X-ray crystallography: producing enough protein for trials | crystallization time & effort (& difficult)
o NMR: producing enough labeled (H 1, C13) protein for collection | size of protein | assignment process is slow & error
prone
Bulk measurements: average of measured quantity is obtained by averaging you may lose information!
Single molecule measurement: distribution of measured quantity is obtained (e.g. different points in cell cycle)
Signal of 1 molecule is very weak (close to noise) difficult to measure signal
Put single-molecule measurements together to get same result as with bulk measurement (no info lost)
An individual enzyme may exist in ≥ 2 states of activity that are not revealed by bulk studies (e.g. speed of RNA polymerase
transcription)
Static heterogeneity = different enzyme molecules function at different rates (e.g. a lame popu & a fast popu)
Dynamic heterogeneity = a given single enzyme can switch between different rates
If we have heterogeneity in a dynamic system and heterogeneity is important use single-molecule analysis
If we want to get an average idea of dynamic system use bulk analysis
Single-molecule techniques:
1. Atomic Force Microscopy (AFM)
2. Laser traps (optical tweezers)
3. Single molecule fluorescence
Atomic Force Microscopy (AFM) ------for single-molecule
imaging------------------------->
- ‘feels’ surface of sample with tip of the cantilever by touching & moving over
sample with cantilever tip to understand what object is
- When cantilever tip touches surface, cantilever bends (laser) light that shines on back of cantilever and goes to mirror,
will move, because angle will change detected by photodetector
Tilted light (object touched) can be seen with computer
Angle of light (shining on back of cantilever) only changes when cantilever tip touches sample light location
changes surface is reached
When surface is reached, don’t move to much, because this can break tip or sample go backwards & start
scanning surface
When object on surface is reached, cantilever bumps into it cantilever will go above object (if cantilever is soft
enough) & touch surface of molecule
- Sample in liquid (substrate) for biology
- Can be done on: single molecule & cell
- Resolution depends on size of cantilever tip (50 µm, 10µm, 10 nm, etc.)
