Chapter 6 The Three-Dimensional Structure of Proteins

Homework: 1, 9,12,

Proteins: Higher Orders of Structure

• The structural variety of human proteins reflects the sophistication and diversity of their biologic role • The maturity of a newly synthesized polypeptide into a biologically active protein requires that it be folded into a specific 3-D arrangement or conformation

Conformation vs. Configuration

• Configuration deals with the arrangement of specific bonds about individual atoms • Conformation refers to the spatial relationship of every atom of a molecule • Proteins were initially characterized by their gross characteristics

Initial Characterizations

• • • • • • Soluble proteins Globular proteins Fibrous proteins Lipoproteins Glycoproteins Metalloproteins

Currently classification and Folding

• Now, proteins are classified based on similarity or Homology, of residue sequence and structure • • Typical proteins could have >10 50 conformations possible But since they fold as they form, it is easier to achieve biologically active conformation

Orders of protein structure

• Primary Structure, 1 o • Secondary Structure, 2 o • Tertiary Structure, 3 o • Quaternary Structure, 4 o

Secondary Structure

• • • • The number of possible secondary structures is restricted The is only free rotation about 2 of the 3 bonds in the backbone Phi angle Psi angle

Alpha Helix

• • • • • Figure 6.3a, page 164 Both Phi and Psi angles are defined as well as distance it rises per turn Complete turn averages 3.6 residues R groups face outwards Mostly only righthanded a -Helix seen in nature due to mostly L-amino acids used!

Stabilizing the helix

• • • • • H-bonds van der Waals in core Proline is only found in 1 st turn. Why?

When present elsewhere, disrupts helix and forms a bend Most hydrophobic R groups are on one side and hydrophilic on the other. Effect?

The Beta Sheet

• • • • As opposed to the helix, the amino acids are extended to form a zigzag or pleated pattern The R groups alternate up/down for a joining residues Stabilized by H-bonds, but without proximity Figure 6.3b, page 165

Types of Beta Sheets

• • Parallel- adjacent segments proceed in same directions as far as amino to carboxyl Antiparallel- Adjacent chains proceed in opposite directions, as in 6.3b.

• Most have a right hand twist, they are not flat • Depictions, see figure 6.16, page 179

Loops and Bends

• • • Approximately half the residues in a protein are in either a helix or beta sheet.

The other half are in less ordered conformations such as Loops, Bends, etc.

Turns and Bends are short segments that connect secondary structures

Beta Turn

• Beta turns involve four Amino Acid residues with the 1 st residue H-bonded to the 4 th residue resulting in a tight, 180 degree turn • Fig 6.18 page 181 • Proline and Glycine are often present in beta turns. Why?

Loops

• • • Loops are regions that contain more AA residues than minimally required to connect adjacent secondary structures Although they have no set conformation, they do serve key biological roles There are some conformations that are secondary structures interacting, but not enough to be termed a tertiary

Super Secondary Structures

• Helix-Loop-Helix – Provide the oligonucleotide-binding portion of DNA-binding proteins such as repressors and transcription factors

Back to Loops

• • • Loops are mainly found on the exterior portion of a protein.

– Exposed to solvent – Readily accessible sites for recognition and binding While the structure may be irregular, they still exist in specific conformations Conformations are held together by H-bonds, salt bridges, and hydrophobic interactions

Tertiary and Quaternary Structure

• • • • 3 o refers to the entire 3D conformation of the protein Domain-a section of protein structure sufficient to perform a particular chemical or physical task such as binding of a substrate or ligand Proteins may have one or more domains.

Fig 6.27 page 192

Tertiary and Quaternary Structure

• • In some cases, proteins are assembled from more than one polypeptide or protomer Quaternary structure defines both the composition and spatial relations between these multiple chains.

Designations

• • • Monomeric proteins consists of one chain Dimeric proteins consists of two chains For Dimeric and above, there are different kinds: – Homodimers-contain two copies of same chain – Heterodimers- contain two different chains

Designations

• Greek letters are used to identify the different chains and subscripts indicate the number of each unit.

Stability of 3

o

and 4

o

structures

• • • • • Primarily stabilized by non-covalent forces Hydrophobic interactions H-Bonds Salt-bridges Etc • Think Velcro!!!

Determining 3D Structure

• • Two main techniques: X-Ray crystallography • NMR Spectroscopy

X-Ray Chrystallography

• Key is to get a good crystal!!!

• Then bombard the crystal with x-rays and acquire a diffraction pattern • The diffraction pattern, along with the primary structure can be used to deduce the 3D structure.

X-Ray Chrystallography

• • • Computers are getting better and better at interpreting X-ray diffraction patterns Major stumbling block remains the requirement of inducing highly purified samples of the protein to crystallize Several lines of evidence, including the ability of some crystallized enzymes to catalyze chemical reactions, indicate that the majority of the structures determined by X-ray crystallography represent the structures of proteins in free solution.

NMR Spectroscopy

• Many common nuclei can be “seen” by NMR • Chemical shifts depend on the nuclei, what functional group it is in, and neighboring NMR nuclei

2-D NMR

• Shows which nuclei are near each other.

Protein Folding

• • • We have said the number of possible conformations of even small peptides is very large Thermodynamics dictate the formation of the conformation The desired conformation is usually energetically favored

Protein Folding

• • • Even with this energetically favored situation, it would take billions of years for a protein to explore all possible conformations to find the favored state So folding has to take place in some sort of guided environment First, as protein leaves the ribosome, short segments fold into secondary structures which are directed by the primary sequence

Protein Folding

• • • Second, the aqueous environment of the cell drives all hydrophobic side groups to the middle of the protein creating a molten globule Regions of secondary structure rearrange to form the mature conformation.

While this process is ordered, it is not rigid

Definitions

• • • In vivo- in the cell In vitro- in test tube Denatured- unfolding of a protein • Aggregates- disordered complexes of unfolded or partially unfolded polypeptides held together by hydrophobic interactions

Chaperones

• • • Chaperone proteins help fold over half the proteins in mammals They work by covering up hydrophobic groups, shielding them from the aqueous environment, thus preventing aggregation Chaperons can also rescue misfolded proteins.

Higher Orders

• • Disulfide bonds help stabilize tertiary and quaternary structures even though they are non-specific.

The protein disulfide isomerase is present to continually break and reform disulfide bonds.

X-Proline Peptide Bonds

• • • • All X-proline peptide bonds-where X represents any residue-are synthesized in the trans configuration.

However, of the X-Proline bonds of mature proteins, approximately 6% are cis.

The cis configuration is particularly common in Beta-turns.

Isomerization from trans to cis is catalyzed by the enzyme proline-cis,trans-isomerase.

Alzheimer’s Disease

• • Refolding or misfolding of protein endogenous to human brain tissue, b -amyloid, is a prominent feature of Alzheimer’s Disease.

Levels of b -amyloid become elevated, and this protein undergoes a conformational transformation from a soluble helix-rich state to a state rich in beta sheets and prone to self aggregaton.

Mass Spectrometry

• • • • • Separates molecules based on MW Peptides are vaporized Applied charge propels the molecules down a bent tube Molecules interact with magnetic field in tube Where the molecules hit the side of the tube, which is the detector, gives their mass

Mass Spectrometry

• Since all A.A. except Leu and Isoleu have different masses, all AA present can be determined • Post translational modifications can also be determined with this method

Posttranslational Modifications

Modification

Phosphorylation Hydroxylation Methylation Acetylation Myristylation Palmitoylation Glycosylation

Mass Increase (Da)

80 16 14 42 210 238 162

Advances

• The methods of vaporization vary and recent advances have allowed for larger peptides to be used.

• Book discusses “Time of Flight” MS.

Make sure you read!!

• • • Make sure you read Ch. 6. Remember, you are responsible for material not covered in lecture.

Example: Fibrous Proteins: Structural materials of Cells and tissue. Be able to compare and contrast Karatins, Fibron, collagen, and elastin.