We have compared structures of 78 proteins determined by both NMR and X-ray methods. It is shown that X-ray and NMR structures of the same protein have more differences than various X-ray structures obtained for the protein, and even more than various NMR structures of the protein. X-ray and NMR structures of 18 of these 78 proteins have obvious large-scale structural differences that seem to reflect a difference of crystal and solution structures. The other 60 pairs of structures have only small-scale differences comparable with differences between various X-ray or various NMR structures of a protein; we have analyzed these structures more attentively. One of the main differences between NMR and X-ray structures concerns the number of contacts per residue: (1) NMR structures presented in PDB have more contacts than X-ray structures at distances below 3.0 A and 4.5-6.5 A, and fewer contacts at distances of 3.0-4.5 A and 6.5-8.0 A; (2) this difference in the number of contacts is greater for internal residues than for external ones, and it is larger for beta-containing proteins than for all-alpha proteins. Another significant difference is that the main-chain hydrogen bonds identified in X-ray and NMR structures often differ. Their correlation is 69% only. However, analogous difference is found for refined and rerefined NMR structures, allowing us to suggest that the observed difference in interresidue contacts of X-ray and NMR structures of the same proteins is due mainly to a difference in mathematical treatment of experimental results.

When considering protein folding with a transient intermediate, a difficulty arises as to determination of the rates of separate transitions. Here we overcome this problem, using the kinetic studies of the unfolding/refolding reactions of the three-state protein apomyoglobin as a model. Amplitudes of the protein refolding kinetic burst phase corresponding to the transition from the unfolded (U) to intermediate (I) state, that occurs prior to the native state (N) formation, allow us to estimate relative populations of the rapidly converting states at various final urea concentrations. On the basis of these proportions, a complicated experimental chevron plot has been deconvolved into the urea-dependent rates of the I$N and U$N transitions to give the dependence of free energies of the main transition state and of all three (N, I, and U) stable states on urea concentration.Keywords: protein folding; folding intermediates; tryptophan fluorescence; chevron plot; stoppedflow; apomyoglobin Apomyoglobin is a good object for protein folding studies because its thermodynamic (Griko et al. 1988;Hughson et al. 1990;Jennings and Wright 1993;Jamin et al. 2000) and structural (Barrick and Baldwin 1993a,b;Eliezer and Wright 1996;Eliezer et al. 1998;Jamin and Baldwin 1998;Jamin et al. 1999;Lecomte et al. 1999;Tcherkasskaya and Ptitsyn 1999;Tsui et al. 1999;Tcherkasskaya et al. 2000) properties in both the native and intermediate conformational states are well elucidated. At neutral pH, apomyoglobin structure is ''native'' globular, with seven of eight helices of holomyoglobin tightly packed (A-E and G, H; while F, involved in the heme binding, is disordered in apoprotein) (Eliezer and Wright 1996). At pH 4.2, the native structure undergoes the transition into a thermodynamically stable ''molten globule' ' (Dolgikh et al. 1981; Ptitsyn 1995) intermediate state (Griko et al. 1988) that contains three helices (Hughson et al. 1990), A, G, and H. This intermediate has two sub-states (stable at pH 4.2 and pH 3.9), which convert one into the other within a millisecond time range (Jamin and Baldwin 1998;Jamin et al. 1999). It was shown using the stopped-flow and quenchflow techniques that urea-induced apomyoglobin refolding goes via a kinetic intermediate that forms within 6 msec and is structurally similar to the equilibrium molten globule intermediate observed at pH 4.2 (Jennings and Wright 1993). Subsequent kinetic studies suggested that this intermediate is on-pathway (Jamin and Baldwin 1998). Quenchflow amide proton exchange combined with mass-spectrometry confirmed that apomyoglobin folds by a single pathway and that the intermediate is obligatory (Tsui et al. 1999). NMR analysis of mutant apomyoglobins also showed that some point mutation may change the folding pathway of apomyoglobin (Garcia et al. 2000).The presence of a kinetic intermediate on the apomyoglobin folding pathway makes this protein attractive for studies of three-state folding/unfolding reactions. Protein folding involves a transition state and, for the m...

We present here a simple approach to identify domain boundaries in proteins of an unknown threedimensional structure. Our method is based on the hypothesis that a high-side chain entropy of a region in a protein chain must be compensated by a high-residue interaction energy within the region, which could correlate with a well-structured part of the globule, that is, with a domain unit. For protein domains, this means that the domain boundary is conditioned by amino acid residues with a small value of side chain entropy, which correlates with the side chain size. On the one hand, relatively high Ala and Gly content on the domain boundary results in high conformational entropy of the backbone chain between the domains. On the other hand, the presence of Pro residues leads to the formation of hinges for a relative orientation of domains. The method was applied to 646 proteins with two contiguous domains extracted from the SCOP database with a success rate of 63%. We also report the prediction of domain boundaries for CASP5 targets obtained with the same method.

Kinetic investigation on the wild-type apomyoglobin and its 12 mutants with substitutions of hydrophobic residues by Ala was performed using stopped-flow fluorescence. Characteristics of the kinetic intermediate I and the folding nucleus were derived solely from kinetic data, namely, the slow-phase folding rate constants and the burst-phase amplitudes of Trp fluorescence intensity. This allowed us to pioneer the phi-analysis for apomyoglobin. As shown, these mutations drastically destabilized the native state N and produced minor (for conserved residues of G, H helices) or even negligible (for nonconserved residues of B, C, D, E helices) destabilizing effect on the state I. On the other hand, conserved residues of A, G, H helices made a smaller contribution to stability of the folding nucleus at the rate-limiting I-->N transition than nonconserved residues of B, D, E helices. Thus, conserved side chains of the A-, G-, H-residues become involved in the folding nucleus before crossing the main barrier, whereas nonconserved side chains of the B-, D-, E-residues join the nucleus in the course of the I-->N transition.

Background: The majority of experimentally determined crystal structures of Type II restriction endonucleases (REases) exhibit a common PD-(D/E)XK fold. Crystal structures have been also determined for single representatives of two other folds: PLD (R.BfiI) and half-pipe (R.PabI), and bioinformatics analyses supported by mutagenesis suggested that some REases belong to the HNH fold. Our previous bioinformatic analysis suggested that REase R.Eco29kI shares sequence similarities with one more unrelated nuclease superfamily, GIY-YIG, however so far no experimental data were available to support this prediction. The determination of a crystal structure of the GIY-YIG domain of homing endonuclease I-TevI provided a template for modeling of R.Eco29kI and prompted us to validate the model experimentally.