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PROTEIN FOLDING, DYNAMICS AND FUNCTION

Proteins have the remarkable ability to fold into unique three-dimensional structures that give rise to specific biological functions. In many cases, folding can occur spontaneously in vitro, indicating that the native structure of a protein is fully determined by its amino acid sequence. Although the successful folding of a polypeptide in the cell may require help from other factors to avoid nonproductive intermolecular interactions, there is no evidence that these molecular chaperones affect any of the intrinsic structural events. Thus, detailed knowledge of the basic structural aspects of folding is also a prerequisite for understanding the process in a larger biological context.

We use a variety of biophysical methods to investigate the kinetics and energetics of folding for several well-characterized proteins, including cytochrome c, ubiquitin and staphylococcal nuclease, as well as natural and engineered variants of these proteins. For the study of protein structure and dynamics, we mainly rely on multidimensional NMR spectroscopy often in conjunction with hydrogen exchange methods. To monitor the kinetics of folding or unfolding reactions, we make use of various optical probes, such as absorbance, static and time-resolved fluorescence, and circular dichroism, coupled with rapid mixing techniques. For a more detailed structural description of transient folding intermediates, we rely on pulsed hydrogen exchange methods. Finally, we often combine these structural and kinetic approaches with site-directed mutagenesis in order to investigate the role of individual residues and their interactions in stabilizing the various conformational states that participate in folding. Another area of interest is macromolecular interactions, especially those involved in the recognition of ligand proteins and peptides by antibodies and other proteins. This work also relies on hydrogen exchange and NMR.

PROTEIN-PEPTIDE INTERACTIONS PROBED BY QUENCHED HYDROGEN EXCHANGE AND NMR. ZHANG, RODER, in collaboration with PATERSON

In a previous study on the complex of cytochrome c with a monoclonal antibody, we showed that hydrogen exchange measurements can provide detailed information on protein-protein interactions. In this "epitope mapping" approach, hydrogen-deuterium exchange rates in the bound form are measured indirectly by exposing the complex to D2O, followed by dissociation of the ligand protein and 2D NMR analysis under slow exchange conditions. Amide protons that are protected against exchange in the complex thus define the parts of the protein involved in binding. This protocol makes it possible to investigate small or medium-sized proteins bound to substrates that are too large for direct NMR analysis.

One limitation is that only amide groups with sufficiently slow exchange rates in the dissociated ligand can be used as probes. In order to extend the method to rapidly exchanging surface protons or complexes with peptide ligands, we developed a more efficient method for quenching the exchange reaction during and after ligand dissociation, using organic solvent additives (2). We found that in a mixture containing 95% dimethyl sulfoxide and 5% D2O under optimal conditions (pH 5, 10 C), amide groups in model peptides became deuterated with time constants of 30 to 150 hours, which is about 100-fold longer than in D2O. This provides ample time to record a 2D NMR spectrum on a quenched hydrogen exchange sample without loss of amide proton labels. Addition of other cosolvents (toluene and cyclohexane) resulted in improved chemical shift dispersion. We used this solvent quenching method to measure NH exchange rates for mellitin (a 26 residue peptide found in bee venom), both in its free monomeric state in water at low pH, as well as in a high-affinity complex with calmodulin. Whereas exchange rates in the free peptide are characteristic for a disordered conformation, the pattern of protected amide protons in the bound form is consistent with a helical conformation of mellitin. The most highly protected amide protons are found for hydrophobic residues along one side of the helix, especially in the C-terminal half of the peptide.

THERMODYNAMIC AND KINETIC ROLE OF HELIX PACKING INTERACTIONS IN THE FOLDING OF CYTOCHROME C. COLÓN, ELÖVE, RODER, in collaboration with SHERMAN

The pairing of two alpha-helices at opposite ends of the polypeptide chain is a highly conserved structural motif found throughout the family of c-type cytochromes. We have shown that this interaction is a critical early event in the folding process and is responsible for the formation of a prominent folding intermediate, INC (4). To gain further insight into the structural and energetic basis of this helix packing interaction and its role in folding, we prepared a series of horse cytochrome c variants in which Leu 94, a central residue at the helix contact site (Figure 1), was replaced by other aliphatic side chains. Three variants containing L94I, L94V, and L94A mutations were expressed in yeast and then purified. For their thermodynamic and kinetic characterization, we used a conformational probe based on the intramolecular quenching of the fluorescence of Trp 59 by the covalently bound heme group.

The effect of amino acid changes on protein stability was determined from changes in the chemically induced unfolding transition. The Ile and Val substitutions resulted in only minor changes in unfolding free energy (±0.5 kcal/mol relative to a free energy of 9.3 kcal/mol for the wild-type), indicating that conservative mutations can be accommodated at the helix interface with only minor structural perturbations. In the case of the L94V variant, this conclusion was confirmed by the NMR solution structure analysis discussed below (1). In contrast, the L94A mutation resulted in a nearly 4 kcal/mol decrease in stability, suggesting that the smaller Ala side chain causes severe packing defects at the helix interface. These equilibrium results show that hydrophobic interactions between the N- and C-terminal helices of cytochrome c are critical for the stability of the native structure.

The effect of mutations at position 94 on the kinetics of folding and unfolding was studied in a systematic series of stopped-flow fluorescence measurements as a function of denaturant concentration and pH. In spite of their small equilibrium effects, the Ile and Val substitutions gave rise to substantial kinetic changes. With the wild-type protein, the folding reaction at low denaturant concentration is characterized by two major processes, a fast phase with a rate of ~50 s-1 that corresponds to the helix-helix association event (formation of INC), and an intermediate phase with a rate of ~2 s-1 that represents the formation of the native state following dissociation of a trapped histidine-iron ligand (4). In the case of the L94I and L94V variants, the fast phase has a much lower relative amplitude. These observations, as well as the denaturant-dependence of the rates and amplitudes, are consistent with a substantially reduced population of the INC intermediate compared to the wild-type protein. This effect is amplified in the case of the destabilizing L94A variant, which exhibits slower folding kinetics with even lower population of INC. These results represent our most direct evidence so far for an early folding event involving association of the N- and C-terminal helices. Since conservative mutations that have little effect on the stability of the native state can lead to major destabilization of the INC intermediate, the mutual structural recognition of the two alpha-helices during the initial stages of folding appears to involve rather specific side chain packing interactions.

PROTEIN SOLUTION STRUCTURE REFINEMENT USING PARAMAGNETIC NMR SHIFTS. GOCHIN, RODER

The effects of unpaired electrons on nuclear chemical shifts and relaxation times have long served as a valuable source of qualitative structural information for molecules containing paramagnetic centers. Dipolar paramagnetic (pseudocontact) shifts are theoretically well understood and, because of their range, can provide useful long-range distance information. However, previous applications have been limited to qualitative structure analysis of macromolecules in the vicinity of bound paramagnetic metals.

We introduced a new method for quantitative NMR solution structure refinement that makes use of dipolar paramagnetic shifts as geometric constraints in energy minimization or molecular dynamics calculations (1). The method was applied to horse cytochrome c and the L94V mutant mentioned above. Chemical shift differences between oxidized and reduced forms for more than 300 assigned proton resonances were used as paramagnetic shift constraints to calculate a refined solution structure for the wild-type protein and to define the conformational changes caused by the L94V mutation. Constrained energy minimization, using the wild-type x-ray coordinates as a starting point, resulted in low-energy structures and excellent agreement between calculated and measured pseudocontact shifts (8-fold improvement relative to the starting structure). For all except the most peripheral regions of the protein, the proton positions were highly constrained by the paramagnetic shift term (uncertainties of 0.1­0.5 Å out to a distance of 20 Å from the iron). Comparison of the solution structure for the L94V variant with that of wild-type cytochrome c shows small (<0.5 Å), but precisely defined, rearrangements for some of the side chain and main chain atoms in the vicinity of residue 94.

We anticipate that paramagnetic NMR shifts will find more widespread use in macromolecular structure determinations. Our approach is especially promising as a refinement tool in combination with conventional NMR distance and dihedral angle constraints (3). Systems amenable to this method include a large number of proteins with tightly bound paramagnetic metals, such as Fe3+, or Co2+. For other proteins, it may be possible to replace an intrinsic diamagnetic metal (e.g., Mg2+, Ca2+, or Zn2+) with a suitable paramagnetic analogue, such as a lanthanide.

PUBLICATIONS

GOCHIN, M. and H. RODER. Protein structure refinement based on paramagnetic NMR shifts: applications to wild-type and mutant forms of cytochrome c. Protein Sci. 4: 296­305, 1995.

ZHANG, Y. Z., Y. PATERSON, and H. RODER. Rapid amide proton exchange rates in peptides and proteins measured by solvent quenching and 2D NMR. Protein Sci. (in press).

LAUB, P. B., S. KHORASANIZADEH, and H. RODER. Localized solution structure refinement of an F45W variant of ubiquitin using stochastic boundary molecular dynamics and NMR distance restraints. Protein Sci. (in press).

Paper in press at time of previous report:

ELÖVE, G. A., A. K. BHUYAN, and H. RODER. Kinetic mechanism of cytochrome c folding: involvement of the heme and its ligands. Biochemistry 33: 6925­6935, 1994.

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