Biochemistry I :protein Structure, Synthesis, Targeting And Degradation

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Protein Structure Determination Methods

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- X-ray Crystallography

- Liquid-State Nuclear Magnetic Resonance (NMR)

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Protein Structure Determination Methods: less direct methods

Back 2

- Fiber Diffraction
- Cryo-Electron Microscopy
- Solid-State NMR

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Protein Structure Prediction is usually done by

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- X-ray Crystallography

- Liquid-State Nuclear Magnetic Resonance (NMR)

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X-ray Crystallography is similar in principle to

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- sonar

-You locate something (moth/atoms) using waves (sound/X-rays).

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To boost the signal for X-ray Crystallography so that we can measure it,
and to make it easier to calculate all those atomic positions,
we would?

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scatter off of a regular array of proteins, a crystal.

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In X-ray Crystallography, for each orientation of the crystal (typically, every 1º), we collect

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- a picture with hundreds of dots (also called “peaks” or “reflections”).

- Taken together, one typically measures 20-40 thousand reflections; their intensities are our data

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In X-ray Crystallography, The data collected is used to

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- get the electron density

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In Liquid-State Nuclear Magnetic Resonance, Many nuclei are little

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- magnetic dipoles (“bar magnets”), including 1H, 15N and 13C

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In Liquid-State Nuclear Magnetic Resonance, When nuclei are placed in an external magnetic field, they precess like ...

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- little gyroscopes

- We can measure that precession frequency very, very accurately

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In Liquid-State Nuclear Magnetic Resonance, The precession frequency of nuclei depends on

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- the nucleus’ environment

Different environments = discernible frequencies

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In Liquid-State Nuclear Magnetic Resonance, What is the basic strategy of figuring which peak corresponds to which nucleus?

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- by measuring frequencies, get spatial constraints, especially on distances between hydrogens and orientation in a global reference frame.

- Identify structures that fulfil those constraints, plus other structural data. Take mean.

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What are the 5 Structural Constraints Determined by NMR

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1. Short Distances (<5Å) between hydrogens (methyl groups)

2. Global Orientation of a Bond Vector (esp. N→H)

3. Hydrogen Bonds

4. Dihedral Angles

5. Angle between Two Bond Vectors → Dihedral Angles

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Significance of Short Distances (<5Å) between hydrogens (methyl groups)in Structural Constraints Determined by NMR

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- Nuclear Overhauser Effect (NOE’s) (1980’s)
- Main Data that Define 3º Structure

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Significance of Global Orientation of a Bond Vector (esp. N→H)in Structural Constraints Determined by NMR

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- Residual Dipolar Couplings (mid-1990’s)
- Good for Refinement & Relative Orientations of Domains

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Significance of Hydrogen Bonds in Structural Constraints Determined by NMR

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- Through-Space J-couplings(late 1990’s)

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Significance of Dihedral Angles in Structural Constraints Determined by NMR

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Through-Bond J-couplings (1970’s)

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Significance of Angle between Two Bond Vectors → Dihedral Angles in Structural Constraints Determined by NMR

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- Cross-Correlated Relaxations (late 1990’s)

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X-ray Crystallography vs. Liquid-State NMR: Which one is better?

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- X-ray structures are more precise (“higher resolution”)
30000 data vs. 400 data

But…
1. X-ray crystallography requires crystals (duh!)

2. NMR captures dynamics, fluctuations

3. NMR works well on proteins with floppy loops

4. Guaranteed to work if protein is sufficiently soluble

5. Good preliminary check whether the protein is folded

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Pros of Liquid-state NMR vs. X-ray crystallography?

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1. X-ray crystallography requires crystals (duh!)

2. NMR captures dynamics, fluctuations

3. NMR works well on proteins with floppy loops

4. Guaranteed to work if protein is sufficiently soluble

5. Good preliminary check whether the protein is folded

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What is Fiber Diffraction?

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Crystallography w/out the crystal

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3 Facts of Fiber Diffraction

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- Many fewer data than crystallography

- Need to exploit other known data:
*Chemical bond lengths, angles
*Known molecular interactions

- Success stories:
* DNA, filamentous phage

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Basic Principle of Cryo-Electron Microscopy

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Use software to combine thousands of 2D images into 3D structure

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Facts about Cryo-electron Microscopy

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- Present resolution: 5-30 Å

- At best, you can make out where the helices are, but side chains are invisible
and b-sheets are big blobs

-Requires very little material

- Good for large, regular assemblies of proteins

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What process gives the best results of Protein Structure Prediction?

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- Homology modeling gives best results (<2 Å agreement for >70% identical sequences)

- Glorified plagiarism
*copy coordinates between matching residues
* some sophistication in matching residues

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In Protein Structure Prediction, Anfinsen’s experiment gives hope that we can:

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predict protein structure computationally

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In Protein Structure Prediction, what’s the energy function?

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- Efficiency vs. Accuracy
* Physics-Based Potentials –
* Too high-resolution
* don’t know when you’re close
- Knowledge-Based Potentials work OK

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The Big Picture of How Proteins Came to Be?

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- Before the stars, there were H and He atoms

- Nuclear reactions require heat; universe cooled off too fast to make heavies

- Nuclear reactions in the first stars led to the formation of heavier elements of the periodic table (mainly 2nd period, such as C, N, O)

- Dying, these stars exploded in supernovae, making even higher elements (mostly 3rd and 4th period and higher, such as Ca, K, Fe, Zn, Ni and Co)

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The Earth was assembled from the big bang

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- scattered remains about 4.5 billion years ago (no wasted time!)

- “We are all stardust” (Joni Mitchell or Carl Sagan)

- Meteor bombardment kept boiling oceans until ~3.8 Gya

- Biochemistry seems to begin ~3.5 Gya

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The universe is ______ billion years old; Big Bang

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13.7 ± 0.2 billion

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RNA world hypothesis of how we got proteins?

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- RNA can fold, and catalyze reactions (ribozymes)

- Simple rules for copying, stable structures

- Major cellular systems for editing and manipulating RNA

- Many fundamental enzyme co-factors and important small molecules in biochemistry seem to be based on RNA

- Origin uncertain; may be complicated, involve other molecules

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But why proteins from RNA? RNA doesn’t fold well, still floppy

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-Proteins stabilize RNA folding, makes it better enzyme

- Surface proteins can protect the RNA from chemical attack

- DNA likely came after, since only proteins do transcription

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Proteins are made by ______ in the _________

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- RNA

- ribosome

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RNA is the intermediary between

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- DNA and proteins

- DNA → mRNA → Protein (central dogma)

- Genetic code (differs slightly among organisms!)

- Codons (3 DNA nucleotides = 1 amino acid); start and stop codons

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What is a Ribozyme?

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A Ribosome

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Many pharmaceuticals target the ______ of bacteria

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ribosomes

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What carries amino acid to the ribosome

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tRNA

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Ribosome matches mRNA codon to what and this does?

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- to anticodon,

- lengthens polypeptide chain; few mistakes

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Protein Targeting - How are proteins shipped to their proper location within the cell

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Classic destinations:

- Endoplasmic reticulum (N-terminal “signal peptide”)

- Chloroplast, mitochondrion, peroxisome (N-terminal “transit peptide”)

- Nucleus (NLS=nuclear localization signal, + but not necessarily N-terminal)