Lecture 16 Adsorbed Proteins

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Proteins hierarchical structure

Primary structure

Secondary structure

Tertiary structure

Quaternary structure

Protein dynamics

Protein bonds are vibrating, twisting, reorienting

molecules

amino acid side chains

Amino acid side chain

Charged (acidic/basic)

Non-charged, polar

Non-charged, non-polar

Define relevant biological components

Cell cytoskeleton

Cellular membrane

Integrin

Extracellular matrix

Tissue

List in chronological order the biological component interaction with biomaterials

water

proteins

cells

Identify properties that influence protein adsorption

Implanting artificial material vs donor tissues (allografts)

Protein adsorption may alter the material interface

Study protein adhesion on specific materials and the resulting cell attachment to determine biocompatibility

Rapid Adsorption Kinetics and Irreversibility

Study protein adhesion on specific materials and the resulting cell attachment to determine biocompatibility

Protein adsorption may alter the material interface

Polystyrene cell culture dish

Expose to protein solution, water no longer beads up

Rapid Adsorption Kinetics and Irreversibility

Proteins initially adsorb quickly

More difficult over time for proteins to find and fit onto

surface

Monolayer of proteins

Competition in protein adsorption

Competition in protein adsorption

Specific proteins can have varying affinities for different

materials

Affinity for specific surface chemistry and mass

concentration in bulk phase determine competition of

protein adsorption

Vroman effect

Protein adsorption is transient

Molecular spreading events: conformational and biological changes in adsorbed proteins

Molecular spreading events

: conformational and biological changes in adsorbed proteins

"Soft" proteins may change shape and are more adsorptive

"Hard" proteins are more stable in bioactivity

"Soft" proteins may

change shape and are more adsorptive

"Hard" proteins are

more stable in bioactivity

Study protein adhesion on specific materials and the resulting cell attachment to determine biocompatibility

Typically study with in vitro experiments

Typically study with in vitro experiments

Single protein

Multi-protein

Single protein in a

buffer solution

Multi-protein solution like

blood plasma

tissue integration means

control:

-water -(small high concentrations) (1st arrives) (surface characterization)

-protein-> major research area in vitro testing

-cells

-tissue-> in vivo

how they all integrate within the tissue

Protein 

chain of amino acids

Protein primary structure

- peptide bonds, covalent bonds

-strong sequence of amino acids

-sets stage for secondary structure

protein secondary structure

- common interactions between carboxyl group + amine group of different amino acids (create the alpha helix or beta pleated sheet) are weak bonds can break apart spotted in tertiary stcture

so like 20% beta sheet and 8--% alpha or any other ratio

p

protein tertiary structure

- 3D shape of max stability and lowest energy

protein dynamic change shape in heat and interact with diff things

weak interactions between amino acids so like van der waals forces and hydrogen bonding

protein quaternary structure

- subunits many multichains, peptide

interaction between carboxyl and amine grupsthat cause interactions withing the beta sheet leads to formations that are alpha or beta get interactionsbetween amino acids between carboxyl and amine

Describe protein structure and dynamics

bonds twist and reoriant covalent dont break

but common for all structures to break and be lost

3rd and 4th postive chanrge and negative charge polar

amino acids only thing different is the

R group

can be nonpolar-methyl groups or hyrogens no charge

can be postive charge- NH2+ or carboxyls

polar groups-OH and amine groups Nh-

3d shape of protein is dependent on

amino acid interactions /behavior

nonpolar (hydrophobic) amino acids would be in the

center

of the protein

since interact less with environment

gives shape of the protein

-so where these are located in peptide chain would create shape since they all like to hang out in the center to avoid water

polar postive charge and negative charge all

outside or near outside

Cell cytoskeleton

formed by protein builds shape of cell structural support rebar of cells long structures proteins

long,strong proteins

dont need to know the specific

Cellular membrane

envelope of the cell, provides protection

have proteins in the cell membrane only certain things cana pass like water but cant pass can pass through channels made by proteins

Integrins

protein in the cell membrane and passes thorugh the cell membrane where it attaches the cell cytoskeleton to the tissue cytoskeleton which is called the extracellula matrix

(ECM)(- portein and tossu that provide support to the tissue))

is key to good cell integration

Extracellular matrix

integrims attach to this

Tissue

want integration

are cells an ECM (tissue)

-not just cells since strength comes from ECM and cells are not structural or strong the strength of bonesand stretchiness is because of ECM

cells and ECM found in each tissue is different

-implant medical device

-injury

-> initiates a wound healing response (can lead to tissue integration if go well if goes badly can lead to foreign body response)

-

as we cause injury we are

crushing capillaries

cutting vasculature

blood would come to the

injured area

blood-plasma (water ions and proteins)

can cause a clot made with fibrinogen (protein causes blood clotting in plamsma)

-

-serum=plasma-fibrinogen (serum is easy to work with to avoid clots from forming)

plasma and serum are both water+ios and proteins

separated by

density

bottom red blood cells (oxygen)

buffy area with white blood cells (platelets)

plasma(water+ions+proteins(salts an enzymes) all dissolved

is high concentration comes first and proteins are dependent on

water since hydriphobic vs hydrophilic

Water

(low molecular weight) diffuse fastest, reach biomaterial

first

Proteins

(from blood serum)

come to the materials and start interacting with the material then they spead

often proteins change shape with adsoption (adsorption-weak interactions between the protein and material surface)(more likely to happen with hydrophibic since distrupting the order )

-proteins spread out and increase attachment points between protein and material

shape of binding sites is key

cells

 attach to certain proteins ex immune and white blood cells issue not tissue creates immune repsonses

like to attach are host tissue cells cells belonging to that tissue know to create ECM for that tissue

from blood or tissue area and attach to our adsorbed proteins via integrins then when attach spreads out and forms ECM then tissue differentiation (DNA->RNA->protein which happens to be ECM)

order of biological components

plasma (top)

WBC

RBC (bottom)

cells

cell would love to bind to

diff proteins and all around are integrins and integrins have specific binding site shape and in that binding site are specific amino acids so specific amino acids might have some positive or negative charges and provide selectivity on which protein to bind to

diff cells diff integrin types like skin or bone diff since binding to proteins in that are

once the protein adsorbs into the surface can change

shape (the protein may loose its shape of the binding site) so the triangle changes into a square

if the protein loses its shape it can change its

bioacitvity therefore where it binds to and can open new binding sites or remove binding sites

on a surface if hydrophobic particles are likely to interact with one another and adsorb and can become a binding site for integrins and once get absorbed can change shape so binding

protein configuration is

dynamic

binding site shape may change

when get adsorption can lose bioactivity but can also gain bioactivity what lost it can gain it and reverse so

bioactivity changes

when implant material is coded with proteins some might

change shape some might not get a monolayer of proteins adbsorbed onto a surface

if cell comes up on material, it recognizes the protein on it not the

type of material since it is coated with proteins

cells interact with the

protein layer'

so its which porteins track and attach diff cells onit

would like to have host tissue cells

- bone cells or skin cells or muscle cells (whtever cell is supposed to be in region it is being implanted into) its bad if attract and attach white blood cells, red blood cells, platlets

important protein layer

can we preadadsorb proteins?

integrins

- specific binding site that would bind to a protein and attach well

integrins bind to

3 specic amino acids (RGD)

in a ton of proteins and bind to different intgrins

problem is that RGD is

super common in many proteins and

binds to many different integrins also

binds to platelets and cause blood

cloths as well

does not combine specifity needed

(RGD is promiscus amino acid sequence)

Protein adsorption may alter the material interface

Polystyrene cell culture dish

Expose to protein solution, water no longer beads up

Polystyrene cell culture dish

- water beads up (high contact

angle)

if have protein layer can change

hydrophobic or philic property

Expose to protein solution, water no longer beads up

protein adsorption leads it to be hydrophilic so protein layers can change behavior of surface

protein adsorption leads it to be hydrophilic so protein layers can change behavior of surface

polystyrene cell culture dish (if put water beads up but if expose to blood serum in cell culture water spreads out

Rapid Adsorption Kinetics and Irreversibility

Proteins initially adsorb quickly

More difficult over time for proteins to find and fit onto

surface

Monolayer of proteins

Competition in protein adsorption

Competition in protein adsorption

Specific proteins can have varying affinities for different

materials

Affinity for specific surface chemistry and mass

concentration in bulk phase determine competition of

protein adsorption

Vroman effect

Protein adsorption is transient

have material first interact is

water then small MW then high concentration proteins

one of the most common small me and high conc proteins is

albumin and there are blood serum proteins (proteins dissolved in blood

is most common out of all main job is control osmolarity in and outside the cell keep blood cell isotomic and is filler doesnt bind or do anything hangs around onmaterial surface but does nothing does not help in cell intergration) only has one or two points of connecting

3rd is getting larger MW and lower concentration proteins- bigger proteind

less in blood serum like fibernectin or fibernogen big proteins that have alot of points of contact with material (higher MW more liely to have more interaction points ad more likely to stay addsorbed)

these are weak interactions like vander waals forces and often break so chances of albumin readsorbing is high because there is only one or two points while te larger has more interactions that allow it to break and reform thus staying over for the long term

because of diffusion (smaller and high cocnentration meets material first vs other combo that we get this order but over thwe we have Varman effect (protein absorption dynamics larger would replace smaller and stay longer because more inreraction points)

protein adsopbrtion is transient so can

get adsorbed but also if "soft" too since it has weaker teritary,secondary and quernaty structure since it can unfold onto surgace and stay adsprbed while a rigid has more covalent bonds and more likely to stay in place

so protein more likely

bind to cells

soft spread out more interaction

but binding site integrations change

wont bind to a cell

this is also looked for in enzymes

"hard"-

proteins more likely to leave surface but also more likey to maintain bioactivity

example protein and binding site

available one adsorbed on biomaterial surface and might open a cryptic binding site that was not open before but now is

study by looking at single proteins in vitro

-adsorption rates

-conformational changes in proteins

-multi protein solutions so coat material with serum or plasma and then see protein competition

ex on surface would like to have fibranectin (protein known for good cell/bone attachment) we do not want fibrinogen which leads to platelet attachment causing blood cloths (bad!)''']

both have RGD on them but these control cell integrim bindings

protein adsorption

controls good and bad outcomes and biocompatibility

protein can either be

stable (hard) or unstable (soft) effect electrical charge and adsorption

add functional groups to surface as well

Cell cytoskeleton

is a fundamental, structurally highly organized, and functionally integrated assembly of proteins within the cell.

It forms the major contractile structure in most cells and plays a critical role in cellular mechanical functions, adhesion, and signaling

Cell membrane

critical boundary that defines the cell, mediates interactions with the environment, and is central to several biological processes, including mechanotransduction, molecular transport, and immunity.

Integrins

are a major class of transmembrane adhesion receptors that play a pivotal and indispensable role in mediating cell-Extracellular Matrix (ECM) interactions and regulating critical cellular functions

link the ECM to the cytoskeleton (specifically actin filaments) through focal adhesion complexes. This linkage transmits mechanical forces from the ECM to the nucleus, which can alter chromatin structure and regulate gene expression

form a mechanical linkage between the ECM (via adhesion sites called focal adhesions) and the internal cytoskeleton (actin filaments). They regulate vital cellular functions, including cytoskeletal organization, proliferation, differentiation, apoptosis, and migration

Extracellular Membrane (ECM)

vital, complex, and dynamic biological material that serves as the microenvironmental niche in which cells reside, adhere, and function within tissues and organs. It is far more than just a passive scaffold; it actively regulates cell behavior and tissue integrity through a continuous exchange of physical and biochemical cues

proteins mediate cell adhesion, including Fibronectin (presenting the crucial RGD sequence) and Laminins (a major constituent of the basement membrane

Tissue

scaffolds are designed to serve as an artificial Extracellular Matrix (ECM) to restore, replace, or regenerate tissues lost due to disease or injures

Allografts

biological tissues or fragments obtained from a human donor or cadaver and transplanted into a recipient patient. They are widely used in medicine, particularly for replacement therapies where autologous (patient-derived) tissue is unavailable or unsuitable, or where synthetic materials fall short

Bulk proteins

the vast array of macromolecules present in biological fluids (like blood plasma and serum) that are critical reactants in the host response to implanted biomaterials.

Even if a protein has a high intrinsic affinity for a surface, it will not be present in high amounts on the surface if it is

present in low bulk concentration compared to other competing proteins.

For example, albumin, despite being a lower affinity protein than fibrinogen, is adsorbed to the surface in similar amounts because its bulk concentration is much higher, driving it onto the surface according to the law of mass action.

Adsorbed proteins

are a layer of biomolecules that rapidly and tenaciously form on the surface of any material exposed to a biological fluid (such as blood plasma or serum), and they are the primary determinant of the subsequent biological response to an implanted synthetic material

converts an inert, synthetic surface into a biologically recognizable material

are irreversibly bound to the surface; washing the surface with buffer does not remove them. This tight binding is due to the large size of the protein molecules, resulting in multipoint attachment where many weak, noncovalent bonds (like van der Waals, electrostatic, and hydrophobic interactions) form simultaneously between the surface and the protein, making the probability of all bonds breaking at once very low

The Vroman Effect

: This is an unusual competitive phenomenon where a protein (most notably fibrinogen) is initially adsorbed at high levels but is subsequently displaced by later arriving, more surface-active plasma proteins (such as high-molecular-weight kininogen).

is thermodynamically driven to lower the overall free-energy state of the system

Blood serum

(often referred to interchangeably with plasma in general physicochemical property discussions)

is the fluid component of blood defined by the absence of fibrinogen, a key coagulation protein

Blood plasma

is the aqueous medium component of blood that contains a complex mixture of proteins, ions, and organic compounds.

It is crucial in blood-material interactions (BMI) because it is the fluid phase from which proteins adsorb onto foreign surfaces, initiating coagulation and other host responses

is defined as the fluid component remaining after the blood has clotted, meaning it is plasma without fibrinogen

Due to the absence of fibrinogen (a large protein), the viscosity of serum is slightly less than that of plasma

Soft proteins

refers to proteins characterized by low thermodynamic stability.

These proteins exhibit specific behavior when interacting with surfaces:

• Adsorption: Soft proteins tend to adsorb more readily and more tenaciously than "hard" proteins, which are more stable.

• Conformation: Due to their low stability, soft proteins are prone to unfolding or conformational changes upon adsorption. These changes can be so profound that some soft proteins (e.g., lactalbumin) may exhibit complete unfolding when preadsorbed onto certain surfaces, such as polystyrene or hematite.

Hard proteins

are defined as proteins with high thermodynamic stability. In contrast to "soft" proteins, a hard protein is more stable to unfolding in solution in response to denaturing conditions, such as elevated temperature.

Key characteristics include:

• Adsorption Resistance: Hard proteins tend to be less adsorptive than soft proteins.

• Conformational Integrity: They are resistant to adsorption-induced unfolding or denaturation

Why do scientist study protein adsorption?

it is the fundamental event that determines the host's subsequent response to any synthetic material placed in a biological environmen

How can we control protein adsorption?

preventing nonspecific adsorption (creating nonfouling surfaces) or directing specific interactions (creating bioactive surfaces).