Ramakrishna V. Hosur

Solution structure of d-GAATTCGAATTC by 2D NMR: A new approach to determination of sugar geometries in DNA segments

A new approach based on the correlated spectroscopy (COSY) in 2D NMR has been described for determination of sugar geometries in oligonucleotides. Under the usual low resolution conditions employed in COSY, the intensities of cross peaks depend on the magnitudes of coupling constants. There are five vicinal coupling constants in a deoxyribose ring which are sensitive to the sugar geometry. The presence, absence and rough comparison of relative intensities of COSY cross peaks arising from such coupling constants enable one to fix the sugar conformation to a fair degree of precision. The methodology has been applied to d‐GAATTCGAATTC. It is observed that ten out of the twelve nucleotide units in this sequence exhibit a rare Ol′‐endo geometry. The EcoRI cleavage sites (between G and A) in the dodecanucleotide show an interesting variation in the conformation with the two sugars attached to the Gs acquiring a geometry between C2′‐endo and C4′‐endo.

Curcumin and kaempferol prevent lysozyme fibril formation by modulating aggregation kinetic parameters

Interaction of small molecule inhibitors with protein aggregates has been studied extensively, but how these inhibitors modulate aggregation kinetic parameters is little understood. In this work, we investigated the ability of two potential aggregation inhibiting drugs, curcumin and kaempferol, to control the kinetic parameters of aggregation reaction. Using thioflavin T fluorescence and static light scattering, the kinetic parameters such as amplitude, elongation rate constant and lag time of guanidine hydrochloride-induced aggregation reactions of hen egg white lysozyme were studied. We observed a contrasting effect of inhibitors on the kinetic parameters when aggregation reactions were measured by these two probes. The interactions of these inhibitors with hen egg white lysozyme were investigated using fluorescence quench titration method and molecular dynamics simulations coupled with binding free energy calculations. We conclude that both the inhibitors prolong nucleation of amyloid aggregation through binding to region of the protein which is known to form the core of the protein fibril, but once the nucleus is formed the rate of elongation is not affected by the inhibitors. This work would provide insight into the mechanism of aggregation inhibition by these potential drug molecules.

Inhibition of insulin fibrillation by osmolytes: Mechanistic insights.

We have studied here using a number of biophysical tools the effects of osmolytes, betaine, citrulline, proline and sorbitol which differ significantly in terms of their physical characteristics such as, charge distribution, polarity, H-bonding abilities etc, on the fibrillation of insulin. Among these, betaine, citrulline, and proline are very effective in decreasing the extent of fibrillation. Proline also causes a substantial delay in the onset of fibrillation in the concentration range (50–250 mM) whereas such an effect is seen for citrulline only at 250 mM, and in case of betaine this effect is not seen at all in the whole concentration range. The enthalpies of interaction at various stages of fibrillation process have suggested that the preferential exclusion of the osmolyte and its polar interaction with the protein are important in inhibition. The results indicate that the osmolytes are most effective when added prior to the elongation stage of fibrillation. These observations have significant biological implications, since insulin fibrillation is known to cause injection amyloidosis and our data may help in designing lead drug molecules and development of potential therapeutic strategies.

J-scaling in two-dimensional homonuclear correlated spectroscopy for enhancement of cross peak intensities

New pulse schemes have been proposed for enhancement of cross peak intensities in two-dimensional homonuclear correlated spectra. These rely on non-selective scaling up of spin-spin coupling constant values and refocusing of the multiplet components. Experimental spectra illustrating the application of the new pulse schemes are presented.

A novel protocol based on HN(C)N for rapid resonance assignment in (15N, 13C) labeled proteins: implications to structural genomics

A novel protocol, based on the HN(C)N experiment, has been developed for rapid assignment of backbone H(N) and (15)N resonances in ((15)N, (13)C) labeled proteins. The protocol exploits the directly observable (15)N and H(N) sequential correlations and the distinctive peak patterns in the different planes of the HN(C)N spectrum, depending upon the nature of the residues displaying the correlations. Glycines and prolines, which are responsible for the distinctive features, provide many check/start points for the sequential walks. These features enhance the speed of data analysis and render side chain assignments less crucial for the success of the assignments. The application of the protocol has been demonstrated with FK506 binding protein (FKBP, molecular mass 12 kDa).

J tuning of SUPERCOSY as an aid to resonance assignments

Nuclear magnetic resonance spectroscopy has been extensively used for conformational analysis of biological molecules (I). With the advent of two-dimensional NMR techniques, it has become possible to obtain individual resonance assignments and detailed structural information on large molecules having molecular weights of the order of 6000-7000 (2-15). Recently a pulse sequence has been proposed (26) which is superior to the normal COSY (I 7, 18) scheme for observation of coupling correlations in complicated spin systems. The new pulse scheme has the sequence: 90-t,-A-180-A-90-A-180-At2, where A is a fixed delay and t, and t2, are, respectively, the usual evolution and detection periods of two-dimensional spectroscopy. The value of A depends upon the nature of the spin systems and the magnitudes of the coupling constants (J) between the spins. For an AX spin system, the optimum value of A is 1/4J and for an AX2 system, it is 1/2J. The main advantage of SUPERCOSY over the COSY scheme lies in the fact that the cross-peaks and the diagonal peaks in SUPERCOSY consist of in-phase and antiphase components respectively, while exactly the opposite is true in the COSY spectrum. Consequently, under conditions of overlap or poor digital resolution, cross-peaks build much faster in intensity, while the diagonal peaks tend to grow relatively slower. The fact that the fixed delay A in the SUPERCOSY pulse scheme depends on the value of J enables one to tune the experiment to a particular value of J which may be of special interest. In other words, cross-peaks arising from particular values of J can be selectively enhanced. We have used this principle here to observe selective proton couplings so as to arrive at a unique resonance assignment of the ring protons in the tryptophan residue of a decapeptide, leutinizing hormone releasing hormone (LHRH). Figure 1 shows SUPERCOSY spectra for two different values of A, 30 ms (A) and 40 ms (B). While the complete analysis will be published separately, the intention of this note is to demonstrate how J tuning of SUPERCOSY can be used as an aid in resonance assignment procedures. The two spectra shown in Fig. 1 depict variations in the intensities of several cross-peaks and diagonal peaks. Figure 2 shows expansions of the aromatic regions of the spectra (02 = 6.4-l 1 .O ppm; WI = 6.4-l 1 .O ppm). The peaks labeled A, B, C, D, etc, identify the J-coupling correlations between various ring protons. For example, in the spectrum of Fig. 2A,

Native and nonnative conformational preferences in the urea-unfolded state of barstar

The refolding of barstar from its urea‐unfolded state has been studied extensively using various spectroscopic probes and real‐time NMR, which provide global and residue‐specific information, respectively, about the folding process. Here, a preliminary structural characterization by NMR of barstar in 8 M urea has been carried out at pH 6.5 and 25°C. Complete backbone resonance assignments of the urea‐unfolded protein were obtained using the recently developed three‐dimensional NMR techniques of HNN and HN(C)N. The conformational propensities of the polypeptide backbone in the presence of 8 M urea have been estimated by examining deviations of secondary chemical shifts from random coil values. For some residues that belong to helices in native barstar, 13Cα and 13CO secondary shifts show positive deviations in the urea‐unfolded state, indicating that these residues have propensities toward helical conformations. These residues are, however, juxtaposed by residues that display negative deviations indicative of propensities toward extended conformations. Thus, segments that are helical in native barstar are unlikely to preferentially populate the helical conformation in the unfolded state. Similarly, residues belonging to β‐strands 1 and 2 of native barstar do not appear to show any conformational preferences in the unfolded state. On the other hand, residues belonging to the β‐strand 3 segment show weak nonnative helical conformational preferences in the unfolded state, indicating that this segment may possess a weak preference for populating a helical conformation in the unfolded state.

pH driven conformational dynamics and dimer‐to‐monomer transition in DLC8

Dynein light chain protein, a part of the cytoplasmic motor assembly, is a homodimer at physiological pH and dissociates below pH 4.5 to a monomer. The dimer binds to a variety of cargo, whereas the monomer does not bind any of the target proteins. We report here the pH induced stepwise structural and motional changes in the protein, as derived from line broadening and 15N transverse relaxation measurements. At pH 7 and below until 5, partial protonation and consequent interconversion between molecules carrying protonated and neutral histidines, causes conformational dynamics in the dimeric protein and this increases with decreasing pH. Enhanced dynamics in turn leads to partial loosening of the structure. This would have implications for different efficacies of binding by target proteins due to small variations in pH in different parts of the cell, and hence for cargo trafficking from one part to another. Below pH 5, enhanced charge repulsions, partial loss of hydrophobic interactions, and destabilization of H‐bonds across the dimer interface cause further loosening of the dimeric structure, leading eventually to the dissociation of the dimer.

Effects of remote mutation on the autolysis of HIV-1 PR: X-ray and NMR investigations.

Autolysis rates of the C95M and C95M/C1095A mutants of a HIV-1 protease tethered dimer have been determined by real time NMR and it is observed that the double mutant has approximately two times higher rate. X-ray structure of the C95M/C1095A double mutant has been solved and refined to 2.1 A resolution. Comparison of the double mutant structure with that of C95M single mutant reveals that there is a shift in the position of the catalytic aspartates and the bound catalytic water. The mutation also causes a loss of hydrophobic packing near the dimerization domain of the protein. These observations demonstrate that subtle changes are adequate to cause significant changes in the rate of autolysis of the double mutant. This provides a rationale for the effects of remote mutations on the activity and drug resistance of the enzyme.

NMR identification of local structural preferences in HIV‐1 protease tethered heterodimer in 6 M guanidine hydrochloride

Understanding protein folding requires complete characterization of all the states of the protein present along the folding pathways. For this purpose nuclear magnetic resonance (NMR) has proved to be a very powerful technique because of the great detail it can unravel regarding the structure and dynamics of protein molecules. We report here NMR identification of local structural preferences in human immunodeficiency virus‐1 protease in the ‘unfolded state’. Analyses of the chemical shifts revealed the presence of local structural preferences many of which are native‐like, and there are also some non‐native structural elements. Three‐bond HN–Hα coupling constants that could be measured for some of the N‐terminal and C‐terminal residues are consistent with the native‐like β‐structure. Unusually shifted 15N and amide proton chemical shifts of residues adjacent to some prolines and tryptophans also indicate the presence of some structural elements. These conclusions are supported by amide proton temperature coefficients and nuclear Overhauser enhancement data. The locations of the residues exhibiting preferred structural propensities on the crystal structure of the protein, give useful insights into the folding mechanism of this protein.