Biophysik

=> Dynamic viscosity describes friction in a fluid

=> friction force = force holding back the particle

- increases with increasing speed v of the particle

- friction coefficent for a neutral sphere in aqueous solution is proportional to the size of the particle (radius r) and to a material property of the solvent that is called viscosity

=> Reynold‘s number R = ratio between inertial force and frictional force

- R is small -> friction is very important, as is the case for a small molecule in aqueous solution

- R is larger -> inertia becomes more important sometimes causing turbulence in the medium. As long as no turbulence appears we have laminar movement of the fluid

=> Microorganisms swim at low Reynold‘s numbers of 10^-2 to 10^-5

-> molecules are in continuous,random motion ➔ „Random walk“

-> Random walk is caused by kicks from solvent molecules

-> thermal energy drives a random walk of molecules, particles (molecules collide with each other)

-> thermal energy is determined by the Boltzmann constant kB times the temperature T —> E = kB*T

equation for the diffusion constant = Boltzmann distribution / Stokes friction coefficient

Unit: m^2/s

=> With the Stokes‐Einstein equation and the definition of particle flux (Fick‘s law) -> compute time scales for purely diffusive transport processes for various sized particles

Polymers properties:

• elasticity

• diffusion

• osmotic pressure

Hyaluronic acid: very flexible chain

Aktin, IMF: semi‐flexible filament

Collagen, mikrotubules: semi‐stiff rods

Scaling law = describes how the property changes with polymer size n

=> e.g.: scaling law for the size of single‐stranded DNA —> Scaling law: Rh ~ N^v

-> exponent v describes how much polymer segments interact with each other

-> The experiments are done with fluorescence correlation spectroscopy (FCS) in order to measure polymer size from observed diffusion processes

-> v=0.588 : 0‐solvent(ideal polymer withexcluded volume)

v>0.588 : good solven t(repulsive forces)

v<0.588 : bad solvent (attractive forces)

Flory’s characteristic ratio = alternative way of describing deviations between real polymers and the FJC model

-> describes the stiffness of polymer chains: the larger number the more stiff

= segments are assumed to be completely free – no self avoidance

-> Problem: Some features of a real polymer are not taken into account. E.g. chemical bonds typically only allow free rotation and no change of bonding angles in any direction. Furthermore, atoms cannot penetrate each other. Therefore the space occupied by one segment of the polymer can be seen as excluded volumen for other polymer segments.

• Corrections to the FJC model: Freely Rotating Chain

-> In the freely rotating chain there are fixed bond angles and variable torsional rotation angles

—> Attractive, repulsive and entropic forces can balance, such that the polymer behaves like a FJC => Cn = 1

—> All forces are influence by temperature, solvent, or other environmental conditions => A polmer is behaving ideal at the theta‐temperature or in a theta‐solvent θ

-> polymer is treated as a continuous rubber band with a certain stiffness

-> the stretched conformation (gestreckte Konformation) has the lowest energy. Any bending requires some external force (which could be due to thermal energy) —> Jede Biegung erfordert eine gewisse äußere Kraft

-> good description for semiflexible polymers where structural changes are due to competing influences from stiffness and thermal motion

• Freely Jointed Chain (FJC): n monomers of fixed length b, each having an independent orientation -> flexible polymer

• Worm‐like Chain (WLC): continuous model with persistence length, in which the orientation of the chain tangent is changing over contour lengths greater than LP -> semiflexible polymer

• Gaussian Chain: n monomers connected by harmonic springs -> very similar to FJC

single‐molecule measurements of end‐to‐end distances:

• Electronenmicroscopy

• AFM

• Force spectroscopy

-> measuring its mechanical behavior —> measure the forces needed for stretching the polymer or pulling the polymer ends apart

• Streching a polymer decreases entropie: work has to be done against Brownian motion

• The polymers response to external forces is described by molecular elasticity

-> Force needed to unfold a protein: 150-300 Piconewton

• RNA folding: complex RNA system has a 2‐state system -> process of folding/unfolding

-> Computing the average transition times and taking into account that the equilibrium constant for a 2‐level system is equal to the ratio between transition rate constants for folding and unfolding -> Free Gibbs Energy (delta_G(0)) can be computed

• DNA supercoiling occurs when the molecule relieves some helical stress by twisting around itself

• Over‐twisting (right turning): negative supercoiling

• Under‐twisting (left turning): positive supercoiling

-> Polymerases introduce DNA supercoiling

-> Topoisomerases reduce DNA supercoiling (TopoI: break 1 strand of DNA, ATP independent; TopoII: break 2 strands of DNA, ATP dependent)

= a device to manipulate dielectric particles with focused laser light

-> important in force spectroscopy

-> allow simultaneous measurement of force and extension (i.e. distance between bead and surface)

-> beads position can be visually observed in a microscope and measured at high accuracy

-> The force is determined by how much the bead is relocated from the center of the laser focus

-> Since the laser focus can be treated like a spring holding the bead in its center position, force can be determined with a good knowledge of the spring constant k (Da der Laserfokus wie eine Feder behandelt werden kann, die die Perle in ihrer Mittelposition hält, kann die Kraft mit einer guten Kenntnis der Federkonstante k bestimmt werden)

-> For a sufficently good calibration round, dielectric beads made from silicon or polystyrol are used

• applying controlled magnetic forces to magnetic beads attached to biomolecules -> a magnet is used to to generate a magnetic field & manipulate a magnetic bead with applied force and turn it in any direction

• allow to add rotation to a single, connected molecule

• control both tension (force applied along the length of the molecule) and torque Drehmoment (rotational force applied to twist or unwind the molecule). Einheit: Newtonmeter

-> used to apply force and torque while at the same time measuring extension and rotation of the bead‐molecule system

-> e.g. Pull and Twist DNA

-> action of single topoisomerase molecules (& state of supercoiling in dsDNA) has been studied with magnetic tweezers -> As soon as a single topoisomerase binds the DNA molecule and enzymatically changes the DNA’s state of supercoiling, this is observed as a change in extension

-> uses electric fields to manipulate charged particles or molecules

-> By observing the position of a fluorescent particle with a fluorescent microscope and a camera, a fast feedback system can be set up to drag the particle back to the center position, using electric fields, whenever it drifts away

-> position the trapped object with nanoscale resolution

= a system which tends to maintain a prescribed relationship of one system variable to another by comparing functions of these variables and using the difference as a means of control

Open loop control: the control action from the controller is independent on the process variable ("process output“)

Closed loop control: the control action from the controller is dependent on feed back from the process in the form of the value of the process variable

• 3 components: proportional (P), integrative (I), differential (D)

-> Each of them computes a part of the total system input u(t) depending on the measured error e(t) and fixed parameters (K_i, K_d, K_p)

= spontane Emission von Licht kurz nach der Anregung eines Materials durch Licht. Dabei sind die emittierten Photonen in der Regel energieärmer als die vorher absorbierten

= property of certain substances to absorb light energy at one wavelength (usually shorter, higher energy light) and then emit light at a longer wavelength (lower energy light)

= process by which a fluorescent molecule loses its ability to emit light after being exposed to intense light = irreversible loss of fluorescence from a fluorophore

Fluorophores = molecules that can absorb light at a specific wavelength and then re-emit it at a longer wavelength, producing fluorescence

1. Absorption of Light: When a fluorescent molecule absorbs light (laser in fluorescence microscopy) at its excitation wavelength, it enters an excited state

2. Emission of Light: In the excited state, the fluorescent molecule can emit light at a longer wavelength (lower energy), which is detected and used for imaging in fluorescence microscopy

3. Photobleaching: However, when a fluorescent molecule is exposed to intense light for an extended period, it can undergo irreversible chemical changes that prevent it from returning to its ground state and emitting light. As a result, the fluorescence signal from the molecule reduces over time, leading to a decrease in image quality and potentially affecting the accuracy of quantitative measurements

=> E.g. Contribution of Reactive Oxygen Species to the Photobleaching of Organic Fluorophores

=> Strategies to mitigate photobleaching: reducing the intensity and duration of illumination, using fluorescence dyes with higher photostability, and implementing techniques such as fluorescence recovery after photobleaching (FRAP) to study dynamic processes while minimizing photobleaching effects

• Light Intensity and Exposure Time

—> High-intensity light: exposure to high-energy light, especially in the ultraviolet (UV) or blue spectrum —> increases the likelihood of photobleaching

—> Longer exposure times can increase the accumulation of reactive species (e.g., free radicals) that can destroy fluorophores

• Fluorophore Properties/Concentration: Photostability, Fluorophore chemical structure

• Oxygen Concentration

• Chemical Environment: Extreme pH levels accelerate photobleaching

• Temperature: high T —> photobleaching

= describes the intensity of transitions between electronic states, focusing on the nuclear motion within a molecule during these transitions

—> It plays a key role in understanding the intensity and likelihood of electronic transitions, especially in the context of absorption and fluorescence spectroscopy

= experimental technique to study the movement and dynamics of molecules within living cells

Principle of FRAP

1. Fluorescence Labeling: Molecules of interest within a cell or a biological sample are tagged with a fluorescent marker. This can be achieved using fluorescent proteins (like GFP) or fluorescent dyes

2. Photobleaching: A specific region of the sample is subjected to a high-intensity laser beam, which causes the fluorescent molecules in that area to lose their ability to fluoresce

3. Recovery Monitoring: After photobleaching, the sample is continuously monitored to observe the movement of non-bleached fluorescent molecules into the bleached area. The recovery of fluorescence in the bleached region is recorded over time —> Monitor the return of fluorescence in the bleached area using time-lapse microscopy

4. Data Analysis: The rate and extent of fluorescence recovery are analyzed to derive quantitative information about molecular mobility, including diffusion coefficients and binding interactions

Applications of FRAP

1. Membrane Dynamics: Studying the lateral mobility of proteins and lipids within cellular membranes.

2. Protein-Protein Interactions: Investigating the binding and dissociation rates of protein complexes.

3. Intracellular Transport: Analyzing the movement of organelles and vesicles within the cytoplasm.

4. Gene Expression Studies: Understanding the dynamics of transcription factors and other nuclear proteins.

5. Drug Discovery: Evaluating the effect of drugs on molecular mobility and interactions within cells

Advantages of FRAP

• Non-Invasive: Allows the study of live cells without disrupting their normal functions.

• Quantitative: Provides precise measurements of molecular dynamics.

• Versatile (Vielseitig): Can be applied to various biological systems and different types of molecules.

Limitations of FRAP

• Photodamage: High-intensity laser used for bleaching can cause damage to the cells.

• Complex Analysis: Data interpretation can be challenging and often requires sophisticated mathematical modeling.

• Limited to Fluorescent Molecules: Only applicable to molecules that can be fluorescently tagged.

= a class of proteins that can absorb light at specific wavelengths and then emit it at longer wavelengths

—> Direct genetic labeling of target proteins in cells

- Green Fluorescent Protein (GFP): Originally derived from the jellyfish Aequorea victoria

- Enhanced GFP (eGFP): a brighter and more photostable variant of GFP, often used in various applications

- Red Fluorescent Proteins (RFPs): Derived from various marine organisms, these proteins emit red light —> mCherry (Discosoma species) and DsRed are popular examples

Applications of Fluorescent Proteins

1. Live-Cell Imaging: FPs allow real-time visualization of cellular processes in living cells, tissues, and organisms. They can be used to tag proteins, organelles, and other cellular structures.

2. Protein Localization: FPs are fused to proteins of interest to study their localization and movement within cells. This helps in understanding protein function and interactions.

3. Gene Expression Studies: FPs can be used as reporters to monitor gene expression levels in real time. The intensity of fluorescence correlates with the activity of the promoter driving FP expression.

4. Fluorescence Resonance Energy Transfer (FRET): FRET uses pairs of FPs to study protein-protein interactions, conformational changes, and other molecular events at close proximity.

5. Fluorescence Recovery After Photobleaching (FRAP): As mentioned earlier, FPs are critical for FRAP experiments to study the dynamics of molecular diffusion and interactions.

6. Super-Resolution Microscopy: Advanced imaging techniques like STORM and PALM utilize FPs to achieve resolutions beyond the diffraction limit of light microscopy.

= advanced optical microscopy technique that allows for high-contrast imaging of events occurring at or near the cell membrane

—> By exploiting the phenomenon of total internal reflection, TIRF selectively illuminates and excites fluorophores in a thin region of the sample, typically within 100-200 nm from the glass-water interface, making it particularly useful for studying membrane dynamics, cell-substrate interactions, and molecular events at the cell surface.

Principles of TIRF Microscopy

1. Total Internal Reflection: When light traveling through a medium of higher refractive index (e.g., glass) hits an interface with a medium of lower refractive index (e.g., water or cytoplasm) at an angle greater than the critical angle, it undergoes total internal reflection. This generates an evanescent wave that penetrates a short distance into the lower refractive index medium.

2. Evanescent Wave: decays (zerfällt) exponentially with distance from the interface, typically exciting fluorophores within a few hundred nanometers of the interface. This selective excitation results in high-contrast images with minimal background fluorescence from deeper within the sample.

= powerful spectroscopic method for measuring distances in the 2-10 nm range

—> It relies on the non-radiative transfer of energy from a donor fluorophore to an acceptor fluorophore, which occurs when the two are in close proximity (2-10 nm)

—> provide valuable information about molecular interactions, conformational changes, distances within and between proteins

Principles

1. Energy Transfer: FRET occurs when the excited-state energy of a donor fluorophore is transferred to a nearby acceptor fluorophore through dipole-dipole coupling. This transfer is highly distance-dependent, making FRET a sensitive molecular ruler for nanoscale distances.

2. Spectral Overlap: The emission spectrum of the donor fluorophore must overlap with the excitation spectrum of the acceptor fluorophore for efficient energy transfer.

3. Efficiency: The efficiency of energy transfer (𝐸) is described by the Förster equation:

-> R0​ = Förster distance (the distance at which energy transfer efficiency is 50%)

-> 𝑟 = actual distance between the donor and acceptor

Where does r^6 distance from FRET come from

= comes from the physics of dipole-dipole interactions between the donor and acceptor molecules involved in the energy transfer process. The energy transfer rate is proportional to 1/r^6, which reflects the probability of the donor transferring energy to the acceptor through dipole-dipole coupling

Example

=> Recovery after Acceptor Bleaching (FRAP) => monitoring the recovery of fluorescence in the acceptor molecule after it has been photobleached, which can provide insights into molecular interactions and dynamics

=> Avoid Synchronization of Ensembles to study conformational changes => Single-molecule FRET (smFRET)

-> offers detailed insights into the heterogeneity and dynamics of molecular processes

-> Single-Molecule Sensitivity: smFRET measures FRET efficiency at the level of single molecule pairs, providing information about individual molecular events and conformational states

Alternating Laser Excitation (ALEX) spectroscopy = used in smFRET to improve the accuracy and reliability of FRET measurements

-> By alternately exciting the donor and acceptor fluorophores, ALEX allows for the direct measurement of both donor and acceptor fluorescence intensities, as well as the identification of fluorescent molecules that contain both fluorophores

-> This approach helps to distinguish (unterscheiden) between true FRET events and other fluorescent signals, such as those from donor-only or acceptor-only species

Principles of ALEX

1. Alternating Excitation: the excitation light source alternates between two wavelengths — one that specifically excites the donor fluorophore and another that specifically excites the acceptor fluorophore. This alternation occurs rapidly, typically on the millisecond timescale

2. FRET Efficiency Measurement: By measuring the fluorescence intensities of the donor and acceptor during their respective excitation periods, ALEX allows for precise calculation of FRET efficiency (𝐸)

3. Stoichiometry Measurement: ALEX also provides information about the presence of donor-only, acceptor-only, and donor-acceptor (FRET-capable) species, allowing researchers to determine the labeling stoichiometry

Correlative EM & dSTORM —> 10 nm

dSTORM —> 100 nm

STED microscopy —> 10 µm

Lattice Light-Sheet microscopy —> 100 µm

—> primarily limited by the diffraction of light. This limit restricts the ability to distinguish two closely spaced objects or points

Range: resolution range of around 200–300 nm laterally and 500–700 nm axially

-> Diffraction limit at 1,5

Rayleigh Criterion defines the resolution limit of an optical system

—> describes the smallest distance between two objects where they can still be seen as distinct points, based on the diffraction of light

1. Deconvolution Microscopy

-> Recalculate the true structure

-> Knowing the PSF, deconvolution algorithms allow to compute are constructed image with improved resolution

Problems

• PSF is not known in all details

• Noise is a stochastic process

• Large computation capacity is required

2. Confocal fluorescence microscopy (improvement about 30%)

• smaller pinhole -> better resolution

• but limitations in the signal-noise-ratio (SNR)

3. Two-photon microscopy

• Further decrease the noise level

4. Image scanning microscopy (Airy scan microscopy)

• improves confocal resolution by ~ 40%

• The distribution of the emitted light is imaged simultaneously for every position of the excitation focus in the sample. The image is then reconstructed by pixel re-assignment

Core idea: to bypass the diffraction limit by employing various optical, computational, or physical strategies to more precisely determine the position of fluorescent molecules within a sample

• Resolution extension by the moiré effect —> transform the low into high frequency informations

• superposition of high-frequency periodic structures produce a down-modulation of the frequencies

• Relies on multiple acquisitions and computation-intensive postprocessing

• Image Nuclear Pore Complex

• Lateral resolution:~80 nm

• allows: multicolor, 3D, live cell

• requires a doughnut-shaped intensity profile —> Focal spots in the middle

• BUT: the illumination to excite fluorescence can kill cells —> If the radiation intensity is too high, it will kill the cells

Photoactivated Localization Microscopy (PALM)

-> PA-GFP (photoactivatable green fluorescent protein) can be activated by UV light from a non-fluorescent state to a fluorescent state —> selective for labeling of proteins & cells

-> 1. photoactivate molecules -> 2. Image activated molecules -> 3. localize molecules -> 4. photobleach & record positions

Stochastic Optical Reconstruction Microscopy (STORM)

-> Photoswitching of organic dyes by thiols

-> On or off stage depends on oxygen levels

-> On-rate: •Oxygen concentration (250 µM) •Thermal stability

-> Off-rate: • Excitation intensity • concentration of reducing agents

Point Accumulation for Imaging in Nanoscale Topography (PAINT)

-> Able to detect more photons

-> Resolution about 7-12 nm

-> Illuminate only a small area -> fluorescently labelled probes diffuse in a solution and start to fluoresce upon binding to the object of interest

• hydrogels are used for swelling

• expand the whole sample and then labeling

• Measure the whole cell, single cell analysis -> 25-nm-resolution imaging

1. Fixation

2. Labeling: labeled biomolecules are covalently attached to a swellable polymer network

3. Gelation/digestion: sample is embedded in a dense, highly cross-linked polymer gel

4. Expansion

Fab fragment (~50 kDa)

• linkage error ~6 nm /each

• modulators of functionality

pAB/sAB (~150 kDa)

• linkage error ~15 nm /each

• modulators of functionality

Nanobodies

• commercially available

• linkage error ~4 nm

• Similar affinity

• Higher penetration

• High stability

• Easy to modify

mEOS family

• similar size as FPs (-25 kDa)

• compatible with SMLM(photoconvertible)

• linkage error ~ 5 nm

Fluorescent proteins

• easy accessible (genetic encoding)

• moderate size (-27 kDa)

• linkage error ~5 nm

• terminal labeling

• Fusion proteins

• Contain chromophore (formed by multiple aa)

• Cover broad spectral range (UV to NIR)

• Can be modulated (e.g. photoactivation or photoswitching)

• Can be used as sensors (environment,proximity, …)

SNAP/HALO/protein tags

• similar size as FPs (-25 kDa)

• compatible with org. fluorophores(photoconvertible)

• linkage error ~ 5 nm

HaloTag -> derived from the haloalkane dehalogenase enzyme DhaA from Rhodococcus rhodochrous

-> Chloroalkane reactive linker (HTL)

SNAP tag -> O6-alkylguanine-DNA-alkyltransferase (hATG)

-> O6-benzylguanine (BG)

CLIP tag -> O6-alkylguanine-DNA-alkyltransferase (hATG)

-> O2-benzylcytosine (BC)

Affimers

• based an natural proteins (e.g. Stefin A)

• Two loops form binding surface

• Variablized binding loops

• No intrinsic cysteines (disulfidbonds)

Aptamer

• single-stranded nucleic acid molecules, typically RNA or DNA

• bind to specific target molecules with high affinity and specificity

• involves conformational recognition

Microtubules = part of the Cytoskeleton

• Function: transport system (vesicles, organelles, macromolecules), cell divison, dynamic instability

1. How to introduce an unique amino acid?

• Genetic Code Expansion

-> Einführung neuer, nicht-kanonischer Aminosäuren (ncAAs) in Proteine —> post-translationale Transkription

-> Ein orthogonales tRNA/Synthetase-Paar aus einer tRNA und einer Aminoacyl-tRNA-Synthetase, das spezifisch für die ncAA ist —> nicht mit den natürlichen tRNAs und Synthetasen des Wirtsorganismus interagiert

—> Diese tRNA wird von der orthogonalen Synthetase spezifisch mit der ncAA beladen

—> Stoppcodon (oft UAG = "Amber"-Codon) wird in die DNA-Sequenz des Zielproteins eingeführt, an der Stelle, wo die ncAA eingebaut werden soll -> orthogonale tRNA erkennt dieses Stoppcodon und bindet es, wodurch die ncAA ins Protein eingebaut wird

—> Zielprotein mit dem eingefügten Stoppcodon wird exprimiert und detektiert

2. How to specifically attach a fluorophore?

• Click Chemistry: Photoclick Cycloaddition, CuAAC, SPACC, SPIEDAC

-> Bioconjugation: couple 2 molecules together

—> Reactions:

• Oxime Ligation

• Staudinger Ligation

• Native Chemical Ligation

• Unnatural Motifs

• Cycloadditions: 1,3-dipolar Cycloadditions, CuAAC, SPAAC, Diels-Alder Cycloaddition, Photoinduced Tetrazole-Alkene Cycloaddition

- Time averaging analysis of spontaneous fluctuations of fluorescence signals dF

• Autocorrelation function: compares fluorescence signals of single species with themselves for different lag times

• Crosscorrelation function: compares fluorescence signals of different species in the same manner

• fluorescent binding site of molecules

—> molecular identity, concentration, molecular interactions, environmental changes, conformational dynamics, and excitation properties

Key Principles:

FCS is based on the temporal fluctuations in fluorescence intensity that occur when fluorescently labeled molecules move in and out of a small, defined observation volume (typically created by a focused laser beam). These fluctuations are analyzed mathematically to extract information about the behavior of the molecules

FCS Measures:

• Diffusion Dynamics: measure how fast molecules diffuse in different environments, providing insights into molecular mobility in membranes, cytoplasm

• Molecular Concentration: By analyzing the frequency and magnitude of fluorescence fluctuations —> estimate the concentration of fluorescent molecules in the observation volume

• Binding Interactions: detect changes in molecular diffusion rates that occur when molecules bind to one another -> studying molecular interactions: protein-protein or ligand-receptor binding

• Molecular size or conformation changes

Steps:

1. Fluorescent Labeling the molecules of interest (proteins, lipids, nucleic acids, etc.)

2. Observation Volume: A laser is focused to create a tiny observation volume, typically in the nanometer range. Molecules entering or leaving this volume cause fluctuations in fluorescence intensity

3. Data Collection: Fluctuations depend on the diffusion rate, molecule size, and interactions

4. Correlation Function: The recorded fluctuations are analyzed using a mathematical function called the autocorrelation function, which quantifies how the intensity changes over time are correlated with themselves

Miss Out: —> Forces Acting within the Denatured State!

Paradox:

-> protein folding cannot occur by random sampling of all possible conformations

-> proteins must fold via a more directed or guided process, follows specific pathways, driven by the energetics of the system

-> Statistical description of protein conformation

-> Only native interactions count

-> simulation of folding with amino acids represented as beads on a lattice --> „Lattice simulation“

Gibbs free energy: calculate the energy between 2 states (Denatured & Native)

-> Enthalpy: Sum of all interactions (Coulomb, Polar, van der Waals, H-bonds)

-> Entropy: Sum of all degrees of freedom (Chain mobility, Ordering of water: hydrophobic effect)

—> driven primarily by a weakening of the hydrophobic effect and changes in the balance of thermodynamic forces

Hydrophobic effect: forces which stabilize the native structure of proteins

-> nonpolar (hydrophobic) amino acid residues tend to avoid water and cluster together in the protein's core

-> low temperatures —> strength of the hydrophobic effect become weak -> H2Omolecules become more ordered and form more stable hydrogen bonds with each other -> prevent hydrophobic groups to aggregate -> protein's core destabilizes -> unfolding

Hot:

-> Entropy of solvent H2O high

-> Hydrophobic effect high

-> Ordered water molecules are released by hydrophobic association

Cold:

-> Entropy of solvent H2O low

-> Hydrophobic effect low

-> No increase of entropy upon release of ordered water molecules by hydrophobic association

• Preventing Aggregation

-> bind to exposed hydrophobic regions of newly synthesized or unfolded polypeptides, preventing them from interacting inappropriately with other hydrophobic regions of other proteins

• Assisted Folding within Isolated Compartments

-> Chaperonins GroEL/GroES system provide an isolated environment where proteins can fold without risk of aggregation

• Unfolding and Refolding

-> Hsp90: if proteins are misfolded, some chaperones can help by partially unfolding them and giving them a second chance to fold correctly

• Heat Shock Response (high temperature or oxidative stress)

-> chaperones (e.g. Hsp70, Hsp90) refold damaged proteins or target them for degradation if they cannot be rescued

-> structure prediction through sequence comparison

-> DeepLearning e.g. Alphafold

Diffusion-limited reactions:

-> A reaction reaches its maximum speed, if every collision of reacting particles yields (ergibt) a product

-> But reactions of biomolecules are rarely diffusion-limited

Reasons:

➢ Correct orientation of molecules is required (dS)

➢ Forces act between molecules (dH)

➢ Activation barriers need to be overcome (dH*)

➢ accelerates a reaction by 10^6 to 10^12 fold!

➢ works under mild, physiological condition

➢ is highly specific and stereo-specific to substrates

➢ can be regulated (e.g. by allostery)

—> acceleration but do not change the thermodynamic equilibrium

Solution 1: cell penetrating peptides (CPPs)

-> usually cationic & hydrophobic peptide carriers that promote passive and active cellular uptake

CPPs limitations:

• low efficacy

• endosomal escape

• toxicity

Solution 2: Lipid Nanoparticles (LNPs)

-> Lipid-based materials that encapsulate and deliver therapeutic molecules, such as RNA-based drugs, to target cells in the body. LNPs have gained significant attention with their success in the COVID-19 vaccines

LNPs limitations:

• stability

• immunogenicity

• tissue-specific targeting

• economic production

• clearance

Solution 3: Passive Diffusion: Peptides ➔ Mimetics

1. Isothermal Titration Calorimetry (ITC)

2. Surface Plasmon Resonance (SPR)

3. Biolayer Interferometry (BLI & SCORE)

4. Microscale Thermophoresis & Temperature Relatied Intensity Change (MST & TRIC)

5. SwitchSense & Fluorescence Proximity Sensing (FPS)

6. Fluorescence Polarisation (FP)

-> measure the heat change that occurs during a molecular binding event in solution

Parameters can be extracted:

1. Binding affinity (Ka​): The strength of the interaction between the molecules

2. Enthalpy change (ΔH): The heat released or absorbed during binding, which reflects changes in enthalpy

3. Stoichiometry (n): The number of binding sites

4. Entropy change (ΔS): The change in disorder or randomness in the system, indirectly calculated using the equation: ΔG=ΔH−TΔS (ΔG = Gibbs free energy)

How it works:

1. Experimental Setup: a sample cell and a reference cell—kept at constant temperature. One of the interacting molecules is placed in the sample cell, while the reference cell contains a buffer solution.

2. Titration: A syringe injects small, precise amounts of a second molecule into the sample cell containing the first molecule. If the two molecules interact, heat is either released (exothermic reaction) or absorbed (endothermic reaction)

3. Measurement: The calorimeter measures the heat change associated with each injection, which is proportional to the extent of the molecular interaction

-> measure molecular interactions in real time

1. Biosensor Tip: BLI employs a biosensor tip (a fiber optic-based sensor) coated with a biolayer (usually with an immobilized molecule such as a protein or antibody). This biosensor is then dipped into a sample solution containing the molecule of interest.

2. Interference Pattern: When light is shone onto the biosensor tip, some of the light reflects off the surface of the biolayer, while the rest travels through the layer and reflects off an internal reference layer. The interaction between these two reflected light beams creates an interference pattern.

3. Binding Event Detection: As molecules from the solution bind to the immobilized molecules on the biosensor surface, the thickness of the biolayer changes. This change causes a shift in the interference pattern, which is detected by the instrument and converted into real-time binding data.

4. Association and Dissociation: The system monitors both the association phase (as the molecules bind) and the dissociation phase (as they unbind when the biosensor is moved into a blank solution).

5. Kinetics and Affinity: The resulting data is analyzed to calculate kinetic parameters such as the association rate constant, dissociation rate constant, and the equilibrium dissociation constant, which reflects the binding affinity of the interaction.