Kinetic Role of Helix Caps in Protein Folding Is Context-Dependent

Abstract

Secondary structure punctuation through specific backbone and side chain interactions at the beginning and end of R-helices has been proposed to play a key role in hierarchical protein folding mechanisms [Baldwin, R. L., and Rose, G. D. (1999) Trends Biochem. Sci. 24, 26-33;Presta, L. G., and Rose, G. D. (1988) Science 240, 1632-1641. We have made site-specific substitutions in the N-and C-cap motifs of the 5-helix protein monomeric λ repressor (λ 6-85 ) and have measured the rate constants for folding and unfolding of each variant. The consequences of C-cap changes are strongly contextdependent. When the C-cap was located at the chain terminus, changes had little energetic and no kinetic effect. However, substitutions in a C-cap at the boundary between helix 4 and the subsequent interhelical loop resulted in large changes to the stability and rate constants of the variant, showing a substantial kinetic role for this interior C-cap and suggesting a general kinetic role for interior helix C-caps. Statistical preferences tabulated separately for internal and terminal C-caps also show only weak residue preferences in terminal C-caps. This kinetic distinction between interior and terminal C-caps can explain the discrepancy between the near-absence of stability and kinetic effects seen for C-caps of isolated peptides versus the very strong C-cap effects seen for proteins in statistical sequence preferences and mutational energetics. Introduction of consensus, in-register N-capping motifs resulted in increased stability, accelerated folding, and slower unfolding. The kinetic measurements indicate that some of the new native-state capping interactions remain unformed in the transition state. The accelerated folding rates could result from helix stabilization without invoking a specific role for N-caps in the folding reaction.The native and denatured conformational ensembles involved in the process of protein folding are important in proper cellular function. If the native state is the functional species, then that function is lost during any time spent in the unfolded state or in the folding process. The denatured or unfolded ensemble of structures may be more susceptible to protein turnover through cellular degradation pathways. Additionally, proper cellular localization and protein trafficking is often coupled to the folding process. A detailed understanding of the medically important process of protein misfolding and aggregation requires better molecular characterization of kinetic folding mechanisms.Each residue in a protein does not have an equal role in the folding process. Several studies have shown that often only a small subset of residues attain nativelike structure in the transition state (3-6). In this paper, we consider the role of helix termini in protein folding by determining the kinetic consequences of site-specific substitutions in helix N-and C-cap structural motifs.Helix N-and C-cap residues are found at the beginning and end of helices, ending the regular i to i + 4 hydrogen bond pattern ...

Germ-Line Mutation of NKX3.1 Cosegregates with Hereditary Prostate Cancer and Alters the Homeodomain Structure and Function

“…Several studies have shown that so-called N-and C-capping structural motifs can stabilize a-helices through hydrogen-bonding or hydrophobic interactions (41)(42)(43). Thr 164 at homeodomain position 41 is located in the N-cap position of the N-capping motif, which initiates the third helix of the homeodomain.…”

“…Thr 41 is well conserved among other NK-like homeodomain sequences (35) and is located at the NH 2 -terminal cap position of the third helix in the NKX3.1 homeodomain. The NH 2 -terminal helix cap is important for maintenance of the helical structure and its alteration may affect helix formation (41,42). Although Thr 41 is not involved in direct hydrogen bonding with the NKX3.1 DNA recognition sequence, it is likely to affect helix III structure and both DNA and protein interactions.…”

“…The NH 2 -terminal helix cap is important for maintenance of the helical structure and its alteration may affect helix formation (41,42). Although Thr 41 is not involved in direct hydrogen bonding with the NKX3.1 DNA recognition sequence, it is likely to affect helix III structure and both DNA and protein interactions. The homologous Thr 41 in the human tinman homologue NKX2.5 has been mutated to methionine in a family with hereditary atrial septal defect and atrioventricular block (49).…”

“…Although Thr 41 is not involved in direct hydrogen bonding with the NKX3.1 DNA recognition sequence, it is likely to affect helix III structure and both DNA and protein interactions. The homologous Thr 41 in the human tinman homologue NKX2.5 has been mutated to methionine in a family with hereditary atrial septal defect and atrioventricular block (49). The NKX2.5 (T178M), homeodomain T41M, mutation causes marked reduction in DNA-binding affinity of NKX2.5 but does not affect complex formation either with wild-type NKX2.5 or with GATA4.…”

Introduction of Negative Charge Mimicking Protein Kinase C Phosphorylation of Cardiac Troponin I

“…4). Whereas metal binding at site III is primarily responsible for stabilizing the hydrophobic core (12), helix-capping interactions are known to increase domain stability and accelerate folding (28). Destabilization of N-cap interactions in helices C and G by mutation have been shown to decrease Ca 2ϩ -binding affinity in TnC (15,27).…”