RESUMEN

Protein functional sites control most biological processes and are important targets for drug design and protein engineering. To characterize them, the evolutionary trace (ET) ranks the relative importance of residues according to their evolutionary variations. Generally, top-ranked residues cluster spatially to define evolutionary hotspots that predict functional sites in structures. Here, various functions that measure the physical continuity of ET ranks among neighboring residues in the structure, or in the sequence, are shown to inform sequence selection and to improve functional site resolution. This is shown first, in 110 proteins, for which the overlap between top-ranked residues and actual functional sites rose by 8% in significance. Then, on a structural proteomic scale, optimized ET led to better 3D structure-function motifs (3D templates) and, in turn, to enzyme function prediction by the Evolutionary Trace Annotation (ETA) method with better sensitivity of (40% to 53%) and positive predictive value (93% to 94%). This suggests that the similarity of evolutionary importance among neighboring residues in the sequence and in the structure is a universal feature of protein evolution. In practice, this yields a tool for optimizing sequence selections for comparative analysis and, via ET, for better predictions of functional site and function. This should prove useful for the efficient mutational redesign of protein function and for pharmaceutical targeting.

Asunto(s)

Proteínas/química , Proteínas/metabolismo , Algoritmos , Secuencia de Aminoácidos , Biología Computacional , Modelos Moleculares , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína

RESUMEN

We have used the Doppler-free, perturbation-facilitated optical-optical double-resonance technique to investigate the vibrational, rotational, and hyperfine structure of the 3 (3)Pi double minimum state of NaK. Since this electronic state arises from an avoided crossing with the nearby 4 (3)Pi state, we observe striking patterns in the data that provide a sensitive probe of the electronic wave function in the various regions of the double well potential. A single-mode cw dye laser excites 2(A) (1)Sigma(+)(v(A),J) approximately 1(b) (3)Pi(Omega=0)(v(b),J) mixed singlet-triplet "window" levels from thermally populated rovibrational ground state levels, 1(X) (1)Sigma(+)(v(X),J+/-1). Further excitation by a single-mode cw Ti:sapphire laser selects various 3 (3)Pi(0)(v(Pi),J(Pi)) rovibrational levels, which are detected by observing direct 3 (3)Pi(0)-->1(a) (3)Sigma(+) fluorescence in the green spectral region. Using the inverse perturbation approximation method, we have determined a 3 (3)Pi(0) potential curve that reproduces the measured energies to approximately 0.24 cm(-1). In addition, the hyperfine and spin-orbit constants, b(F) and A(v), have been determined for each region of the potential curve.

RESUMEN

We have measured the hyperfine structure of mutually perturbing rovibrational levels of the 1(b) 3Pi0 and 2(A) 1Sigma+ states of the NaK molecule, using the perturbation-facilitated optical-optical double resonance method with copropagating lasers. The unperturbed 1(b) 3Pi0 levels are split into four hyperfine components by the Fermi contact interaction bFIS. Mixing between the 1(b) 3Pi0 and 2(A) 1Sigma+ levels imparts hyperfine structure to the nominally singlet component of the perturbed levels and reduces the hyperfine splitting of the nominally triplet component. Theoretical analysis relates these observations to the hyperfine splitting that each 1(b) 3Pi0 level would have if it were not perturbed by a 2(A) 1Sigma+ level. Using this analysis, we demonstrate that significant hyperfine splitting arises because the 1(b) 3Pi0 state cannot be described as pure Hund's case (a). We determine bF for the 1(b) 3Pi0 levels and also a more accurate value for the magnitude of the singlet-triplet spin-orbit coupling HSO=[1(b) 3Pi0(vb,J)(H(SO))2(A) 1Sigma+(vA,J). Using the known spectroscopic constants of the 1(b) 3Pi state, we obtain bF=0.009 89+/-0.000 27 cm(-1). The values of (H(SO)) are found to be between 2 and 3 cm(-1), depending on vb, vA, and J. Dividing (H(SO)) by calculated vibrational overlap integrals, and taking account of the 1(b) 3Pi(Omega) rotational mixing, we can determine the magnitude of the electronic part H(el) of H(SO). Our results yield (H(el))=(16.33+/-0.15) cm(-1), consistent with our previous determinations using different techniques.

RESUMEN

Earlier high-resolution spectroscopic studies of the fine and hyperfine structure of rovibrational levels of the 1 3delta state of NaK have been extended to include high lying rovibrational levels with v < or = 59, of which the highest levels lie within approximately 4 cm(-1) of the dissociation limit. A potential curve is determined using the inverted perturbation approximation method that reproduces these levels to an accuracy of approximately 0.026 cm(-1). For the largest values of v, the outer turning points occur near R approximately 12.7 angstroms, which is sufficiently large to permit the estimation of the C6 coefficient for this state. The fine and hyperfine structure of the 1 3delta rovibrational levels has been fit using the matrix diagonalization method that has been applied to other states of NaK, leading to values of the spin-orbit coupling constant A(v) and the Fermi contact constant b(F). New values determined for v < or = 33 are consistent with values determined by a simpler method and reported earlier. The measured fine and hyperfine structure for v in the range 44 < or = v < or = 49 exhibits anomalous behavior whose origin is believed to be the mixing between the 1 3delta and 1 1delta states. The matrix diagonalization method has been extended to treat this interaction, and the results provide an accurate representation of the complicated patterns that arise. The analysis leads to accurate values for A(v) and b(F) for all values of v < or = 49. For higher v (50 < or = v < or = 59), several rovibrational levels have been assigned, but the pattern of fine and hyperfine structure is difficult to interpret. Some of the observed features may arise from effects not included in the current model.