Grad School: Biochem (Tertiary Structure - Serebryany Lecture)

Tertiary Structure

three dimensional arrangement of atoms, secondary structural elements arrange to form tertiary structures

Do hydrophobic or hydrophilic residues prefer to be exposed in membrane proteins?

hydrophobic, trapped in oil

Protein Domains

independently folding and evolving proteins structures that are all part of 1 polypeptide

How can protein domains lead to diversity in protein function?

protein domains are reshuffled during evolution, create vast protein diversity and novel cellular functions

Protein Folding

proteins cycle through many folding intermediates and molten globule state, frequently an assisted process

Molten Globule

general folded state found first then residues lock into place

Do larger or smaller proteins have more ways to misfold?

Larger

Chaperones

proteins that control/assist folding of other proteins, can be associated with ribosome

What was Anfinsen’s hypothesis about how a peptide chain accessed a well determined 3D structure?

a protein’s structure is encoded in its sequence

What angles at Ca define orientation of planar peptide bonds?

Phi and Psi angles

Phi Angle

The rotation angle around the bond between the nitrogen atom and the a-carbon

Psi Angle

the rotation angle around the bond between the a-carbon and the carbonyl carbon

Are all angle combinations possible?

No, some angle conformations clash and lead to steric hinderance

Ramachandran Plot

these allowed phi and psi combinations, revealing energetic favorability based on physical space constraints.

In terms of phi and psi angles how do secondary structures form?

secondary structures are formed when consecutive residues adopt similar phi and psi angles

What secondary structure does the green region correspond to?

B-sheets

What secondary structure does the red region correspond to?

a-helix

What secondary structure does the grey region correspond to?

left handed a-helix

What are a-helices stabilized by and which direction so the side chains point?

stabilized by hydrogen bonds and side chains point out

Dipole on a-helix

delta positive by N-terminus, delta negative by C-terminus

What are B-sheets stabilized by and which direction so the side chains point?

stabilized by hydrogen bonds and their side chains point up and down from the H-bonding plane

Foladases

bind misfolded or unfolded proteins to allow refolding, Hsp70

Holdases

hold onto unfolded/misfolded proteins without ATP energy source to prevent aggregation, a-crystallins

Chaperonins

barrel-like complexes providing isolated environments for folding, GroEL

Disaggregases

Use ATP to pull aggregated proteins apart through a pore to re-attempt folding

When does folding typically begin?

often begins during synthesis, N-terminus of protein may fold before C-terminus is synthesized

How fast is protein translation

5-50 seconds

How many residues can fit in a ribosome exit tunnel?

40 plus residues or even an entire small folded domain

How can translation rate be regulated?

rare codon distributions can regulate translation rate because there are less corresponding tRNA causing brief ribosomal stalling

What are three known types of structured aggregates?

Native-state, domain swapped, amyloid

Native State Aggregates

sickle cell anemia, clusters or assemblies formed by proteins that largely retain their functional, native conformation rather than unfolding into random structures

Domain Swapped Aggregates

form when identical protein molecules partially unfold and exchange structural segments with each other, Eye lens turbidity leading to cataracts

Amyloid Aggregates

fibrous protein structures, cross-beta-sheet architectures, form when soluble proteins misfold and stick together, almost all folded proteins have the capability to form these, neurodegeneration

Prion-like Propogation

templated protein misfolding where misfolded monomers act as seeds to induce conformational change in native proteins (ex. SOD1)

X-Ray Crystallography

requires crystal formation, uses electron density maps from X-ray diffraction patterns to determine atomic model

Nuclear Magnetic Resonance (NMR)

uses a magnetic field to orient atomic spins either with (low energy) or against (high energy) the magnet. it is also sensitive to local chemistry.

Nuclear Overhauser Effect Experiments (NOE)

a specialized NMR spectroscopy technique used to determine 3D molecular structure, stereochemistry, and conformation

NOE experimental process

take assigned protein sequence to determine structural constraints. the structure is then determined by calculating models that satisfy the constraints

Cryo-EM

rapidly freezes dilute samples and images individual macromolecular particles without needing crystals

Sequence Based Structure Prediction

AlphaFold, RoseTTAFold

Do proteins need to adopt a stable conformation?

No some proteins are intrinsically disordered such as a-syn whose aggregation is linked to dementia

why does protein folding matter for medicine?

a protein's three-dimensional shape determines its biological function, and structural errors or misfolding cause severe diseases and guide modern drug design

How to force a protein to switch conformations

alter its internal energy landscape or apply an external stimulus that stabilizes an alternative structural state, ligand binding, post translational modifications, changing local pH or temperature