Modeling of protein turns and derivation of NMR parameters related to turn structure Megan Chawner BRITE REU Program Advisor: Dr. Dimitrios Morikis Department of Bioengineering University of California, Riverside

Outline Background My Project Results Conclusions Acknowledgements

Protein Structure: All proteins are made up of twenty amino acid building blocks into a sequence = primary structure

Protein structure: sequence folds into  -sheet,  -helix, random coil loops and various types of turns stabilized by atomic interactions (e.g., H-bonds) = secondary structure Anti-parallel  -sheet  -helix Primary structure: GPLLNKFLTT Primary structure: EKQKPDGVFQE Strand 1 Strand 2 Inter-strand H-bonds C=O(i)…H-N(i+4) H-bonds 1 helix turn = 3.6 a.a.

Protein Structure: three-dimensional protein folds are stabilized by long range interactions = tertiary structure Turns introduce reversibility in the direction of other elements of secondary structure, such as  -helices or  -sheets 3 amino acids =  -turn 4 amino acids =  -turn  -turn  -turn

i-1 i i+1 ii ii ii  -sheet Ramachandran plot (  ) plot defines secondary structure Backbone torsion angles:  Turns  -helix

Protein Structure Determination: uses Nuclear magnetic resonance (NMR) spectroscopy to get NMR observables, which are converted to NMR-derived structural parameters Nuclear Overhauser effects (NOEs)  inter-proton distances 3 J(H N -H  )-coupling constants   -torsion angles Karplus Equation (Karplus, 1959, J Chem Phys) NOE equation (Wuthrich, 1986) r i,j  inter-proton distance  c  rotational correlation time A=6.98, B=-1.38, C=1.72 (Wang and Bax, 1996, JACS) NOE < 5 Å  through-space interactions  inter-proton distances 3 J(H N -H  ) = 3-chemical bond coupling  through-bond interactions   -torsions

Amino Acid iAmino Acid i+1 NCC C OR H NCC C O H R ii ii 3 J(H N -H  )  i+1  i+1 HH ii H N (i)-H  (i) H N (i)-H N (i+1) H N (i)-H  (i+1) 3 J(H N -H  ) = 3-bond   -torsion NOE < 5 Å  distance in space H  (i)-H  (i+1) H  (i)-H N (i+1) Relations of experimental observables and structural parameters d  N (i,i+1) d NN (i,i+1) d N  (i,i) d  (i,i+1)

3 J(H N -H  ) (Hz)  ( o ) Cis  =0 o  =60 o  =90 o  =150 o Newman Projections N C=O C=O H H C  N C=O C=O H H C  N C=O C=O H H C  N C=O C=O H H C   =-90 o  =-30 o Trans  =180 o  =-120 o Solution of Karplus equation using MatLab  -helix  -sheet NC  H  H Cis NC  H  H Trans NC HH Cis NC  H  H Trans C NC  C  Cis C NC  C  Trans C NC  C  Cis C NC  C  C NC  C  C NC  C  Trans C NC  C  C NC  C  Chawner & Morikis, in preparation

My Project Goals: To use NMR-derived parameters (inter-proton distances and  -torsion angles) to create databases of expected NMR observables (NOEs and 3 J(H N -H  )- coupling constants) for ideal  - and  - turns with statistical deviations. Bottom line: we are back-calculating NMR observables. Remember, during structure determination, NMR-derived parameters are obtained from NMR spectroscopic observables, NOEs and 3 J(H N -H  )-coupling constants. Use: Rapid protein turn structure identification by examination of raw NMR observables, without a complete structure calculation.

Color code: Blue: N Light blue: H Gray: C Red: O Color code: Blue: N Light blue: H Gray: C Red: O VIII I I’ II’ II         H-bond C  -C  I I’ II’ II         H-bond C  -C   -turns Computational modeling of ideal  -and  -turns according to torsion angles using DeepView Classic  -turn criteria Distance: C  (1)-C  (4) < 7 Å C=O(1)…H-N(4) H-bonded Distance: O(1)-N(4) < 3.3 Å Distance: O(1)-HN(4) < 2.4 Å Angle: O(1)-H(4)-N(4) almost linear ± 35 o Torsion angles ( o ) Type 22 22 33 33 I II I' II' VIII Chawner & Morikis, in preparation

Torsion angles ( o ) Type 22 22 Direct Inverse directinverse  -turns Computational modeling of ideal  -and  -turns according to torsion angles Classic  -turn criteria Chawner & Morikis, in preparation

Nuclear Overhauser effects (NOEs)  inter-proton distances Characteristic  -turn distances H  (2)-H N (4): (i, i+2) H  (2)-H N (3): (i, i+1) H  (3)-H N (4): (i, i+1) H N (2)-H N (3): (i, i+1) H N (3)-H N (4): (i, i+1) Characteristic  -turn distances H  (1)-H N (3): (i, i+2) H  (1)-H N (2): (i, i+1) H  (2)-H N (3): (i, i+1) H N (1)-H N (2): (i, i+1) H N (2)-H N (3): (i, i+1)  -turn  -turn

Torsion angles ( o )D < 7 ÅH-bond distance (Å)H-bond angle (°) Type 22 22 33 33 C  (1)-C  (4)O(1)-N(4)O(1)-H N (4)O(1)-H(4)-N(4) I II I' II' VIII Torsion angles ( o )D < 7 ÅH-bond distance (Å)H-bond angle (°) Type 22 22 C  (1)-C  (3)O(1)-N(3)O(1)-H N (3)O(1)-H(3)-N(3) Direct Inverse Marginal H-bonds present because of larger deviations from linearity Test of compliance of molecular models with ideal turn criteria Not present H-bond present Chawner & Morikis, in preparation

Inter-proton distance (Å) Type H N (2)- H N (3) H N (3)- H N (4) H  (2)- H N (3) H  (3)- H N (4) H  (2)- H N (4) H  (2)- H  (3) H  (3)- H  (4) H N (2)- H  (3) H N (3)- H  (4) I II I' II' VIII Torsion angles (°) Inter-proton distance (Å) Type 22 22 H N (1)- H N (2) H N (2)- H N (3) H  (1)- H N (2) H  (2)- H N (3) H  (1)- H N (3) H  (1)- H  (2) H  (2)- H  (3) H N (1)- H  (2) H N (2)- H  (3) Direct Inverse Ideal  -turns Ideal  -turns Molecular models: measured distances corresponding to characteristic NOEs

We classified the inter-proton distances as corresponding to strong, medium, weak and very weak NOE intensities: Å = strong Å = medium Å = weak Å = very weak Relative NOE intensities Type H N (2)- H N (3) H N (3)- H N (4) H  (2)- H N (3) H  (3)- H N (4) H  (2)- H N (4) H  (2)- H  (3) H  (3)- H  (4) H N (2)- H  (3) H N (3)- H  (4) ISSMMWVW N/OVW IIVWSSMMW N/O I'SSMMWVW II'VWSMMW N/OVW VIIISWMSN/OVWWN/OVW  -turns Relative classification of NOE intensities Chawner & Morikis, in preparation 1.8 Å: sum of van der Waals radii with some overlap

Torsion angles (°) Relative NOE intensities Type 22 22 H N (1)- H N (2) H N (2) - H N (3) H  (1) - H N (2) H  (2) - H N (3) H  (1) - H N (3) H  (1) - H  (2) H  (2) - H  (3) H N (1) - H  (2) H N (2) - H  (3) Direct70-60SWWWWN/OVWWN/O 70-70SWWWWN/OVWWN/O 85-60SWWWWN/OVWWN/O 85-70SWWWWN/OVWWN/O Inverse-7060SWWSWVW N/O -7070SWWSWVW N/O -8560SWWSWVW W -8570SWWSWVW W  -turns We classified the inter-proton distances: Å = strong Å = medium Å = weak Å = very weak Relative classification of NOE intensities

 2 (°) J 2 (Hz)  3 (°) J 3 (Hz) Type I Type I’ Type II Type II’ Type VIII Type  2 (°) J 2 (Hz) Direct707.1 Direct856.2 Inverse Inverse Solution of Karplus equation: calculations of characteristic 3 J(H N -H  )-coupling constants  -turns  -turns Chawner & Morikis, in preparation

 2 (°) J 2 (Hz)  3 (°) J 3 (Hz) Type I-60Weaker-90Stronger Type I’60Stronger90Weaker Type II-60Weaker80Stronger Type II’60Stronger-80Weaker Type VIII-60Weaker-120Stronger Type  2 (°) J 2 (Hz) Direct70S Direct85W Inverse-70W Inverse-85S We classified the turn’s 3 J(H N -H  )-coupling constants as stronger or weaker relative to itself, so that the different types can be differentiated comparatively  -turns  -turns Caution: small variations in  -torsion angles result to very large variations in j-coupling constants. In general, the use of j-coupling constants is not as helpful as NOE intensity patterns and connectivities.  -helix  -sheet Chawner & Morikis, in preparation

Conclusions NOE intensity patterns and connectivities can be used to distinguish turn type without a complete structure determination. We have created small NOE intensity databases that discriminate Type I, I’, II, II’, and VIII  -turns, and direct and inverse  -turns.  Caution: Classification of strong, medium, weak, and very weak NOEs is relative. Small variations of the characteristic  -torsion angles introduce very large variations in the 3 J(H N -H  )-coupling constant values, sometimes spanning the whole range of possible solutions for the Karplus equation and the whole allowed region of the Ramachandran plot.  Why? the small variations in  -torsion angles are owed to the dynamic character of turns in proteins and peptides and to conformational averaging. Overall, NOEs are more useful than J-coupling constants.

Acknowledgements Dr. Dimitrios Morikis Li Zhang Coordinators of BRITE Program Fellow BRITE students

3 J(H N -H  )  Cis  =0 o  =60 o  =90 o  =150 o Newman Projection N C=O C=O H H C  N C=O C=O H H C  N C=O C=O H H C  N C=O C=O H H C   =-90 o  =-30 o Trans  =180 o  =-120 o Solution of Karplus equation