Lynne Regan

Structure and function of KH domains

The hnRNP K homology (KH) domain was first identified in the protein human heterogeneous nuclear ribonucleoprotein K (hnRNP K) 14 years ago. Since then, KH domains have been identified as nucleic acid recognition motifs in proteins that perform a wide range of cellular functions. KH domains bind RNA or ssDNA, and are found in proteins associated with transcriptional and translational regulation, along with other cellular processes. Several diseases, e.g. fragile X mental retardation syndrome and paraneoplastic disease, are associated with the loss of function of a particular KH domain. Here we discuss the progress made towards understanding both general and specific features of the molecular recognition of nucleic acids by KH domains. The typical binding surface of KH domains is a cleft that is versatile but that can typically accommodate only four unpaired bases. Van der Waals forces and hydrophobic interactions and, to a lesser extent, electrostatic interactions, contribute to the nucleic acid binding affinity. ‘Augmented’ KH domains or multiple copies of KH domains within a protein are two strategies that are used to achieve greater affinity and specificity of nucleic acid binding. Isolated KH domains have been seen to crystallize as monomers, dimers and tetramers, but no published data support the formation of noncovalent higher‐order oligomers by KH domains in solution. Much attention has been given in the literature to a conserved hydrophobic residue (typically Ile or Leu) that is present in most KH domains. The interest derives from the observation that an individual with this Ile mutated to Asn, in the KH2 domain of fragile X mental retardation protein, exhibits a particularly severe form of the syndrome. The structural effects of this mutation in the fragile X mental retardation protein KH2 domain have recently been reported. We discuss the use of analogous point mutations at this position in other KH domains to dissect both structure and function.

Guidelines for Protein Design: The Energetics of β Sheet Side Chain Interactions

To determine the interaction energy between cross-strand pairs of side chains on an antiparallel β sheet, pairwise amino acid substitutions were made on the solvent-exposed face of the B1 domain of streptococcal protein G. The measured interaction energies were substantial (1.8 kilocalories per mole) and comparable to the magnitude of the β sheet propensities. The experimental results paralleled the statistical frequency with which the residue pairs are found in β sheets of known structure.

Design of stable alpha-helical arrays from an idealized TPR motif.

The tetratricopeptide repeat (TPR) is a 34-amino acid alpha-helical motif that occurs in over 300 different proteins. In the different proteins, three to sixteen or more TPR motifs occur in tandem arrays and function to mediate protein-protein interactions. The binding specificity of each TPR protein is different, although the underlying structural motif is the same. Here we describe a statistical approach to the design of an idealized TPR motif. We present the high-resolution X-ray crystal structures (to 1.55 and 1.6 A) of designed TPR proteins and describe their solution properties and stability. A detailed analysis of these structures provides an understanding of the TPR motif, how it is repeated to give helical arrays with different superhelical twists, and how a very stable framework may be constructed for future functional designs.

Protein alchemy: Changing β-sheet into α-helix

For most proteins the amino acid sequence determines the tertiary structure. The relative importance of the individual amino acids in specifying the fold, however, remains unclear. To highlight this. Creamer and Rose put forth the ‘Paracelsus challenge’: Design a protein with 50% sequence identity to a protein with a different fold. We have met this challenge by designing a sequence which retains 50% identity to a predominantly β-sheet protein, but which now adopts a four helix bundle conformation and possesses the attributes of a native protein. Our results emphasize that a subset of the amino acid sequence is sufficient to specify a fold, and have implications both for structure prediction and design.

An inverse correlation between loop length and stability in a four-helix-bundle protein

BACKGROUND The loops in proteins are less well characterized than the secondary structural elements that they connect. We have used the four-helix-bundle protein Rop as a model system in which to explore the role of loop length in protein folding and stability. RESULTS A natural two-residue loop was replaced with a series of glycine linkers up to 10 residues in length. All 10 mutants are highly helical dimers that retain wild-type RNA-binding activity. As loop length is increased, the stability of Rop toward thermal and chemical denaturation is progressively decreased. CONCLUSIONS All the mutants assume a wild-type-like structure, which suggests that the natural loop does not actively dictate the final protein fold. The strong inverse correlation observed between loop length and stability is well described by a simple polymer model in which the entropy of loop closure is the dominant energetic term. Our results emphasize the importance of optimization of loop length to successful protein design.

Protein design: novel metal-binding sites

In natural proteins, metal ions play a variety of roles, including nucleophilic catalysis, electron transfer and the stabilization of protein structure. The de novo design of metal-binding sites is therefore an attractive means by which to impart proteins with novel properties and activities.

The folding and design of repeat proteins: reaching a consensus

Although they are widely distributed across kingdoms and are involved in a myriad of essential processes, until recently, repeat proteins have received little attention in comparison to globular proteins. As the name indicates, repeat proteins contain strings of tandem repeats of a basic structural element. In this respect, their construction is quite different from that of globular proteins, in which sequentially distant elements coalesce to form the protein. The different families of repeat proteins use their diverse scaffolds to present highly specific binding surfaces through which protein-protein interactions are mediated. Recent studies seek to understand the stability, folding and design of this important class of proteins.

The interface of protein structure, protein biophysics, and molecular evolution

Abstract The interface of protein structural biology, protein biophysics, molecular evolution, and molecular population genetics forms the foundations for a mechanistic understanding of many aspects of protein biochemistry. Current efforts in interdisciplinary protein modeling are in their infancy and the state‐of‐the art of such models is described. Beyond the relationship between amino acid substitution and static protein structure, protein function, and corresponding organismal fitness, other considerations are also discussed. More complex mutational processes such as insertion and deletion and domain rearrangements and even circular permutations should be evaluated. The role of intrinsically disordered proteins is still controversial, but may be increasingly important to consider. Protein geometry and protein dynamics as a deviation from static considerations of protein structure are also important. Protein expression level is known to be a major determinant of evolutionary rate and several considerations including selection at the mRNA level and the role of interaction specificity are discussed. Lastly, the relationship between modeling and needed high‐throughput experimental data as well as experimental examination of protein evolution using ancestral sequence resurrection and in vitro biochemistry are presented, towards an aim of ultimately generating better models for biological inference and prediction.

Detecting protein-protein interactions with GFP-fragment reassembly

The detection of protein-protein interactions in vivo is of critical importance to our understanding of biological processes. The classic library approach has been to use the yeast two-hybrid screen, where an interaction between known bait and unknown prey proteins leads to restoration of transcription factor activity 1 . However, its use is limited by host organism and nuclear localization requirements, and a tendency to detect indirect interactions (false positives). Bacterial two-hybrid screens have eliminated localization requirements and simplified many technical aspects of the procedure 2 . An innovative approach has been the reassembly of protein fragments, which then directly report interactions. A suitable reporter protein is dissected at the genetic level, and the fragments are fused to bait and prey, which are then coexpressed in vivo. Bait and prey interaction brings the reporter fragments together, facilitating reassembly of the active reporter protein, giving a direct readout of the association. This method has been demonstrated for dihydrofolate reductase 3,4 , ubiquitin 5 and the green fluorescent protein6 (GFP) from Aequorea victoria. We recently described improvements to the original screen based on the reassembly of the GFP enhancedstability mutant sg100 in Escherichia coli 7 . Our system, presented in the protocol that follows, consists of two plasmid vectors for the independent expression of fusions with N- and C-terminal fragments of GFP, and allows for simple visual detection of protein-protein interactions with a K D as weak as 1 mM.

Amino-acid substitutions in a surface turn modulate protein stability.

A surface turn position in a four-helix bundle protein, Rop, was selected to investigate the role of turns in protein structure and stability. Although all twenty amino acids can be substituted at this position to generate a correctly folded protein, they produce an unusually large range of thermodynamic stabilities. Moreover, the majority of substitutions give rise to proteins with enhanced thermal stability compared to that of the wild type. By introducing the same twenty mutations at this position, but in a simplified context, we were able to deconvolute intrinsic preferences from local environmental effects. The intrinsic preferences can be explained on the basis of preferred backbone dihedral angles, but local environmental context can significantly modify these effects.