konformační dynamika molekul velké molekulové reorganizace revoluční pokroky v laserové kontrole molekul nové & velké výzvy pro teorii

R-and (S)-thalidomid vykazuje rychlou interkonverzi in vivo Jak probíhá tato konverze?

Conformation instability strongly reflected by the state-dependence of the molecular properties Source of troubles Source of useful information Good for theorists Go Quantum Mechanics

Adenine Guanine Cytosine Thymine Tautomerizace a párování basí DNA James WATSON & Francis CRICK 1959 Co vede (či nutí) přírodu používat WC párování? Termodynamika to není !!!!

BAD NEWS ☺ ∩ !!!! Jak probíhá maligní párování, jak mu bránit?

EQUALLY BAD NEWS !!!! ☺

TAUTOMERY GUANINU 220 tautomerů D.N. & V.Š. : Studium adiabatické separovatelnosti N-H (O-H) vibrací Teoretické frekvence&intensity υ(NH) v rámci 1-d teorie ve “one-to-one” relaci s experimentalními hodnotami ↓↓↓ možnost kvalitativní i kvantitativní chemické analyzy smĕsi tautomerů Dost práce !

A peptide bond is a chemical bond formed between two molecules when the carboxyl group of one molecule reacts with the amino group of the other molecule, releasing a molecule of water ; usually occurs between amino acids The resulting OCNH group is called a peptide bond, and the resulting molecule is called an amide. Polypeptides and proteins are chains of amino acids held together by peptide bonds.

From Peptide Conformation to Protein Translocation in vivo Is it a player in the conformational dynamics of proteins? O-C-N-H Peptide Group : J.Ch., J.V., V.Š studium dynamické neplanarity peptidické vazby

The C-N bond has a partial double bond character, due to resonance which occurs with amides, and the molecule can normally not rotate around this bond. The whole arrangement of the four C,O,N,H atoms as well as the two attached carbons in a peptide bond is planar RiRi Points of rotation Large amplitude internal rotations/torsions cause conformational instabilities Usual assumption The peptide bond nearly always has the trans configuration

NH 3 + CH CH 3 C N O H CH CH 3 C O O - L-alanyl-L-alanine Relatively simple, but already hard for a correct quantum-mechanical treatment

Is the Peptide Bond Planar or Nonplanar ? The assumption under question : (perhaps the simplest possible one)

H.M.Sulzbach, P.v.R.Schleyer,H.F.Schaefer,III:J.Am.Chem.Soc. 117, 2632(1995) Influence of the Nonplanarity of the Amide Moiety on Computed Chemical Shifts in Peptide Analogs. Is the Amide Nitrogen Pyramidal? Theory M.W.MacArthur,J.M.Thornton:J.Mol.Biol.264,1180(1996) Cambridge Structural Database Deviations from Planarity of the Peptide Bond in Peptides and Proteins X-ray J-S.Hu,A.Bax:J.Am.Chem.Soc.119,6360(1997) NMR experiment Determination of ф and χ 1 Angles in Proteins from 13 C- 13 C Three-Bond J Couplings Measured by Three-Dimensional Heteronuclear NMR. How Planar Is the Peptide Bond? M.Ramek,C-H.Yu,J.Sakon,L.Schäfer : J.Phys.Chem.A 104, 9636(2000) Theory Ab Initio Study of the Conformational Dependence of the Nonplanarity of the Peptide Group A.K.Dasgupta,R.Majumdar,D.Bhattacharyya:Ind.J.Biochem&Biophys. 41,233(2004) Characterization of Non-planar Peptide Groups in Protein Crystal Structures Neutron diffraction Is the Peptide Bond Planar or Nonplanar ?

LIFETIMES v=11 10 miliseconds v=13 10 microseconds v=16 40 picoseconds L-alanyl-L-alanine Is geometry relaxation of the peptide bond of any consequence in the conformational dynamics? “Best localized” state

H N C O L-alanyl-L-alanine Equilibrium and vibrational average geometries r CN / Å r NH / Å α 1 o α 2 o α 3 o Σ o OCNH o OCNC 7 o State E inv / cm -1 Exp This study Equilibrium 1-d (T kin -exact) v= v= v= d (T kin -app) v= v= v=2 668 α1α1 C7C7 T kin -app and T kin -exact calculations are in a close harmony α2α2 α3α3

Scaling χ by F χ  χ * F τ 1 = 5.7 milisec τ 2 = 300 microsec τ 3 = 39 microsec τ 4 = 3.6 nanosec F=1.0,0.9,0.8,0.7 “Best localized” state:

ScalingV by F V -> V * F τ 1 = 5.7 milisec τ 2 = 1.0 microsec τ 3 = 364 nanosec τ 4 = 118 nanosec τ 5 = 41 nanosec F=1.0,0.9,0.8,0.7,0.6 Barrier height “Best localized” state:

Konformační nestabilita je vyvolána klasicky zakázaným tunelováním. Tunelovací transmisivita je silnĕ stavovĕ závislá. Rychlosti konformačních reorganizací jsou stavovĕ (silnĕ) závislé. Izomerační procesy jsou “ne-statistické” a jejich korektní popis vyžaduje kvantovĕ-mechanický přístup. Kvantové efekty dominují “nízko-teplotní” dynamiku kontrolovanou stavy “ležícími” pod konformačními barierami Obecné shrnutí !! Komerční výpočtové balíky nutné, ale naprosto nedostačující!!

Excited State Intramolecular Proton Transfer A typical, hot and still feasible task A plethora of equally interesting tasks “Inversion” motions in nonplanar systems (amide groups) Internal molecular rotations (rotation/torsion along the glycosidic bond in DNA) Electron transfer (dipole bound electrons in anions) From theoretical frontiers to an agonizing number crunching

BOA : A “Nondestructive” Tool for Separating High and Low –Frequency Motions

N-benzoyl-phenylalanine Think Big ! O C N H T kin -app and T kin -exact calculations are in a close harmony Equilibrium and vibrational average geometries r CN / Å r NH / Å α 1 o α 2 o α 3 o Σ o OCNH o OCNC 7 o State E inv / cm -1 Exp This study Equilibrium 1-d (T kin -exact) v= v= v= d (T kin -app) v= v= v=2 628 α1α1 α2α2 α3α3

L-alanyl-L-alanine NMR shifts C2C2 H 11 L-alanyl-L-alanine spin-spin coupling constants J(N 4 H 11 ) J(H 11 H 12 )

Proteins are essential to the structure and function of all living cells and viruses hemoglobin carries oxygen inzulin provides signals to store excess sugar actin and myosin allow muscles to contract antibodies fight infections collagen makes up tendons and ligaments

Formamide σ N /ppm J 13 /Hz J 14 /Hz J 15 /Hz J 34 /Hz J 35 /Hz J 45 /Hz State Equilibrium 3-d v= v= v=2 T=300 K Thermal average 1-d v= v= v=2 α1α1 α2α2 α3α3 Equilibrium and inversional NMR parameters 1-d calculations (T kin exact) verify SRB! Sizable vibrational effects !!!! Larger systems under study

SUMMARY The low-frequency “pyramidalization&torsion” motion of the peptide unit OCNH can be adiabatically separated from the remaining unit motions. The separation allows for a physically correct description of the effective (dynamical) “ground state” molecular geometry of the OCNH unit. Thus, the separation allows for reliable refining of the X-ray and NMR structural data of large molecular systems o ~ ∩ The “pyramidalization&torsion” motion of OCNH is profoundly curvilinear and nonharmonic. Thus, it cannot be described properly by means of the available “Force Field” programmes

Nascent polypeptide chain produced by a ribosome molecular factory Mature protein, ready to function Fluorescence quenching: a tool for single-molecule protein-folding study Protein Folding

When Proteins Misfold time to bother, challenging motivation